MASS SPECTROMETRIC INVESTIGATIONS OF MOSQUITO ATTRACTION TO HUMAN SKIN EMANATIONS By ULRICH R. BERNIER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1995 saavaan vQiaoi:! do kmrn^m ACKNOWLEDGMENTS I wish to express sincere appreciation and gratitude to my graduate research supervisor, Dr. Richard A. Yost, for his guidance, patience, and insightful discussions throughout the course of my graduate studies. I would like to also thank the members of my committee. Dr. James D. Winefordner, Dr. David H. Powell, Dr. J. Eric Enholm, and Dr. Daniel L. Kline, for their assistance during the course of these studies. I am indebted to Dr. Jodie V. Johnson and Dr. Anthony P. Annacchino, Jr. for their instructions and guidance in the operation of the TSQ70 triple quadrupole mass spectrometer. I wish to thank Mr. Carl E. Schreck, Dr. Daniel L. Kline, and Dr. Donald R. Barnard for their assistance and open communication, over the last three years, with respect to the entomological aspects of this research. I also wish to thank soon-to-be Dr. Matthew Booth for spending many hours analyzing samples for me (above and beyond the call of duty) on a purge and trap GC/MS. Dr. Brad Coopersmith deserves thanks for assistance with reaction mechanisms during the course of this work. One final thanks with respect to colleagues goes out to all former and present group members for their friendship and assistance throughout the time I spent at the University of Florida. 11 Special thanks go to Pamela Cannon for her love, patience, and assistance over the last year. Pam did an extraordinary job of typing in tables for hours at a time as I sifted through data looking for additional compounds to insert into those tables. I wish to also thank Jesse Cannon for spending hours learning the elements of the periodic table, including exact masses, during his summer vacation. I probably had more fun quizzing him than he did learning. Finally, my greatest thanks are extended to my mother, who has stood by me through everything I have done in my life. She always encouraged me to obtain more education; I am certainly appreciative of her effort, especially now that I am near the completion of this degree. HI TABLE OF CONTENTS ACKNOWLEDGMENTS ii Abstract viii CHAPTER PAGE 1 INTRODUCTION 1 Research Objectives 1 Entomological Overview 2 Mosquito Physiology 3 Characteristics of gender and species 3 Mosquito sensilla 4 Mosquito vision 6 Mosquito Repellents 7 Mosquito Attractants 8 Nature of concern for attractant identification 9 Vision 11 Heat 11 Moisture 12 Carbon dioxide 12 Sound 13 Chemical attractants 14 Relation to Semiochemical Studies 16 Overview of Analytical Methods and Detection 18 Overview of Mass Spectrometry 18 Sample introduction 19 Sample ionization 21 Triple quadrupole mass spectrometry 22 Analysis of Complex Environmental and Biological Samples . . 28 Organization of Dissertation 29 IV CHAPTER PAGE 2 SAMPLING METHODS 31 Introduction 31 Sampling Considerations 31 Entomological Sampling 32 Olfactometer 32 Field studies 36 Mass Spectrometric Methods of Sample Introduction 36 Experimental 39 Thermal Desorption from a Single Bead 39 Thermal Desorption from Multiple Beads 40 Thermal Desorption from Multiple Beads/Cryo-focused GC Separation 45 Purge and Trap/GC Separation 47 Results and Discussion 49 Thermal Desorption from a Single Bead 49 Thermal Desorption from Multiple Beads 64 Thermal Desorption from Multiple Beads/Cryo-focused GC Separation 72 Purge and Trap/GC Separation 82 Conclusions 87 3 STUDIES INVOLVING ATTRACTION 89 Introduction 89 Lactic Acid as a Model Compound 89 Reaction Studies 89 Altering Attraction 90 Analysis of Methanolic Perspiration Solution 91 Experimental 91 Reactions of Lactic Acid 91 Altering Attraction 94 Analysis of Methanolic Perspiration Solution 97 Results and Discussion 97 Reactions of Lactic Acid 97 Characteristic fragmentations 98 Oligomerization and attachment reactions 108 Altering Attraction 130 Addition of acid or base to lactic acid 131 Addition of acid or base to esters 142 Analysis of Methanolic Perspiration Solution 142 CHAPTER PAGE Phase differences 149 Implication to attractant origin 152 Conclusions 153 Lactic Acid Reactions 153 Altering Attraction 154 Origin of Attraction 154 4 APPLICATIONS OF TANDEM MASS SPECTROMETRY 156 Introduction 156 Analysis of Multiple Beads Without GC Separation 157 Daughter Library 157 Compound Class Screening 158 Experimental 159 Daughter Library 160 Compound Class Screening 162 Results and Discussion 163 Analysis of Multiple Beads Without GC Separation 163 Daughter Library 176 Compound Class Screening 187 Conclusions 202 5 IDENTIFICATION OF SKIN EMANATIONS 205 Introduction 205 Sample Introduction and Separation 205 Cryo-focusing GC 208 Purge and trap GC 208 Mass Spectrometry 209 PCI theory 209 NCI theory 212 Characteristic ion fragmentations 217 Experimental 220 Identification of Emanations by CI and EI MS 220 Cryo-focusing GC/MS 220 Purge and trap GC/MS 223 Case Study Comparison of Emanations between Subjects .... 224 Case Study Comparison of Bio-assay to GC/MS Assay 226 Cryo-focusing GC/MS 226 Olfactometer 227 Results and Discussion 228 Identification of Emanations by CI and EI MS 228 VI CHAPTER PAGE Cryo-focusing GC/MS 240 Purge and trap GC/MS 260 Case Study Comparison of Emanations between Subjects .... 270 Case Study Comparison of Bio-assay to GC/MS Assay 278 Conclusions 283 Identified Emanations 283 GC/MS Assay of Subjects with Different Attraction Levels . . 283 Bio-assay versus GC/MS Assay 284 6 CONCLUSIONS AND FUTURE WORK 285 Conclusions 285 Future Work 288 APPENDIX CARBON DIOXIDE AS A CI REAGENT GAS 294 Introduction 294 High Pressure Charge Exchange Mass Spectrometry 295 Electron Capture Negative Ion Chemical Ionization 297 ECNCI with Carbon Dioxide 298 Instrument Optimization and Background Ions 299 Selected Examples 314 Summary 324 REFERENCE LIST 325 BIOGRAPHICAL SKETCH 333 vn Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MASS SPECTROMETRIC INVESTIGATIONS OF MOSQUITO ATTRACTION TO HUMAN SKIN EMANATIONS By Ulrich R. Bernier December, 1995 Chairperson: Richard A. Yost Major Department: Chemistry Volatile compounds emanating from the host are the basis for chemical attraction of mosquitoes. This work is centered upon the identification of volatile emanations from the skin. The goal of this work is to provide the foundation for predicting relative host attraction by comparison of components and their relative abundance in samples. Altering the attraction of hosts (by changing the matrix conditions on the skin) may assist in understanding the factors which produce differences in attraction. The underlying premise to this work is that chemical analysis (conducted by mass spectrometry) should allow for sample detection in a fashion similar to that which mosquitoes encounter: a volatile sample in the gas phase. Therefore, the sampling methods studied in this dissertation reflect that criterion. Direct thermal vni desorption of volatiles from handled glass beads placed in the injection port of a gas chromatograph (GC) followed by cryo-focusing/GC analysis was determined to be the best sampling method with respect to sensitivity and selectivity. Other than carbon dioxide, lactic acid is the only previously known chemical attractant for the Aedes aegypti species of mosquito. The acid/base effects on attraction to lactic acid, esters of lactic acid, and methyl isovalerate were studied. The addition of acid enhanced attraction for all tested compounds, while the addition of base decreased attraction. Perspiration was analyzed to determine the skin gland origin of attractants. The major volatiles desorbed from handled glass beads have been identified through the use of positive and negative ion chemical ionization in conjunction with electron ionization. Mass spectrometric assays of two human subjects, differing markedly in their attraction to mosquitoes, have been conducted to determine differences in components present. Direct comparison of mass spectrometric assays to bio-assays over a five day period has been carried out and is presented herein. Additional identification of minor components present in skin emanations was accomplished by purge and trap GC/mass spectrometry (GC/MS). The utility of tandem mass spectrometry (MS/MS) as a tool for compound class screening is presented in the context of this work. IX CHAPTER 1 INTRODUCTION Research Objectives The primary goal of this work is to provide a better understanding of the chemical basis for the attraction of mosquitoes to human hosts. Novel studies and approaches were conducted in three areas to meet this goal. The first objective is to determine the best method of sample introduction for analysis. The decision involves balancing sensitivity, resolution, as well as selectivity. Here, selectivity implies sampling in a manner similar to that in which mosquitoes are exposed, i.e. a volatile sample in the gas phase. The second objective of this work is to determine methods to alter attraction. Studies involving changing the matrix conditions of a sample and the effect on attraction will be addressed. The third objective is to determine compounds which emanate from the skin. This objective is closely linked to sampling in that the sampling method ultimately determines how many and which compounds are detected. An important component of this objective is to differentiate between unattractive and attractive components which emanate from the skin. This has been approached by case studies involving the comparison of skin emanations between hosts who differ in attraction to mosquitoes and by monitoring 2 mass spectrometric assays concurrently with samples analyzed for mosquito attraction. Entomological Overview Mosquitoes are a vector for the transmission of more than 250 million new cases of viral diseases each year; these diseases include malaria, encephalitis, and filariasis [1,2]. One method to control the spread of disease is alteration of mosquito capability to carry diseases. The yellow fever mosquito, Aedes aegypti, is responsible for the transmission of both yellow fever and dengue fever [3]. This species has shown reduced disease-spreading ability by gene alteration [1]. This approach ultimately requires mapping of the genome for this species and each new species examined. Thus, this method is tedious and may require some time until a useful strategy can be implemented. A second approach to control the spread of disease is to develop and employ repellents; this is probably the standard approach to reducing biting from mosquitoes. A third approach, consistent with the work of this dissertation, is to develop an understanding of the basis of attraction. TTiis would allow the development of strategies to reduce or counteract the attraction of human hosts. This knowledge would then allow for manufactured traps, possibly containing both insecticides and attractants. The difficulty with this approach is that it requires a suitable knowledge of attractants for a variety of species. As will be discussed in the following text, many species exhibit marked differences in terms of responses to cues. 3 These differences most likely result from the differences in sensilla. For example, species which are more dependent on olfactory cues will contain a greater proportion of chemosensilla versus mechanosensilla than for a species dependent primarily on physical cues. Understanding mosquito attraction necessitates an understanding of the physiological function of mosquito sensory organs. Mosquito Physiology Much research has been conducted on mosquitoes with the primary focus affixed upon characterizing the finding and selection of hosts. The use of scanning and transmission electron microscopy as well as electrophysiology has greatly expanded the understanding of mosquito sensory physiology [4]. The mosquito nervous system consists of three systems: the central nervous system (CNS) (brain, ventral nerve cord, ganglia), the stomodaeal nervous system (various ganglia and nerves), and the peripheral nervous system (PNS) (motor and sensory neurons, sense organs). The system most directly pertaining to host attractant/repellent stimuli responses is the PNS; the fundamental components of the PNS are examined in the following sections. Characteristics of gender and species The gender differences between male and female mosquitoes, for most species, lie in the host-seeking behavior of females. Generally only females take blood-meals, which are necessary for egg production. Both genders will feed on nectar. Males will respond to wingbeat frequency of females to orient towards the 4 females for mating. Additionally, differences exist among species as to preference of hosts for blood-meals. Ciilex, spp., generally prefer avian hosts ^h\\t Anopheles, spp., feed on mammalian hosts (e.g. man) [5]. Therefore, there are some innate differences in the genetic make-up of different species. This difference will result in different preferences for various cues (attractants) as well as differences in sensilla between species. Mosquito sensilla Sensilla, or sense organs, are constituents of the PNS. The function of a sensillum is to transform a response from a stimulus into a viable means of response the mosquito can process, such as a nervous impulse [4]. There are a range of sensilla which can function to detect variations of thermal, chemical, mechanical, or visual stimuli, as well as changes in humidity. Visual detection will be addressed under its own section separate from this discussion of other sensilla. There are five types of chemosensilla found on the antennae o{ Ae. aeg\'pti and one type of chemosensillum on the palps (capitate pegs) [4,5]. The capitate pegs function in the detection of carbon dioxide. Grooved pegs, found on the antennae flagellar segments, respond to airborne vapors. These vapors may be water vapor or other airborne volatiles such as lactic acid, fatty acids, and essential oils in the case oiAe. aegypti [6]. The detection by grooved pegs and palps is processed by the CNS (see pg. 8); i.e., detection by each of these sensilla is specific and independent for different cues [7]. Anophelines contain large sensilla coeloconica which are thought to be similar to grooved pegs. The detection purpose of these sensilla is still 5 unknown, although they are presumed to provide information similar to that of the grooved pegs. Small sensilla coeloconica are at the tips of the antennae and respond to changes in air temperature. There are two sets of neural cells comprising the small coeloconica. One is activated by a temperature increase, whereas the second is inhibited by it. This allows a mosquito (i.e. Ae. aegypti) to sense temperature changes at short-range (within one meter of the host) [6]. The sensilla ampullaceae on the antennae are only suspected to function in temperature and humidity detection; no experiments as of yet confirm this. Sensilla trichodea are the most numerous sensilla found on the antennae. Fewer of these sensilla are present on male antennae compared to female antennae (for the same species). The responses of these sensilla vary widely in terms of olfactory detection, e.g. fatty acids, essential oils, oviposition compounds, and repellents. None has been found to give a response to lactic acid [6]. Sensilla found on the mosquito antennae may not be the sole location for chemical reception of odor cues. The mouth may also contribute some ability for chemical reception of odors and has been postulated to function in infrared detection [4]. The various setae found on the mosquito body are generally mechanosensilla, as are the chordotonal organs. In contrast to setae, chordotonal organs found in the cuticle provide information as to the position of mosquito body parts. Sound detection in mosquitoes involves frequency detection by the Johnston's organ in antennae [4]. Experiments involving antennae removal or impedance of the Johnston's organ resulted in a lack of response of mosquitoes to sound stimuli [8]. 6 The antennae of male mosquitoes is such that resonant vibration occurs with female wingbeat frequency; the shaft of the antennae transmit the vibration to the Johnston's organs. Directional information is most likely acquired from sound due to a triangulation method with the antennae. Depending on the phase offset of vibrations on each antennae, it is postulated that mosquitoes can determine whether the sound is originating within or outside of a 30 degree arc of their flight path line [4]. Thus, their approach to a stimulus follows a zig-zag pattern [9]. Additionally, mosquitoes fly at approximately one meter per second when in a controlled flight toward a host and up to eight meters per second maximum [2,9]. One final note on the sensory capacity of the PNS: in a study of Toxorhynchites brevipalpis, there were 622 neurons forming the PNS, not including photoreceptors. Mechanoreceptors attributed for 526 of the neurons, and chemoreceptors made up the final 96 neurons. Most of the mechanosensilla (500 of 526) were body setae. Mosquito vision The discussion in this section will focus mainly upon the eyes as sensilla pertaining to Ae. aegypti. In the larval stage, two types of eyes are present, the lateral ocelli and the developing adult compound eyes [4]. The ocelli are separated into two dorsal ocelli, a central ocellus, and a ventral ocellus. The ocelli contain cells to gather light and transmit the visual information to the brain via photosensitive nerve cells. Rhodopsin is the visual pigment in mosquito vision; the An,3x for absorption is 515 nm. TTiis corresponds to a visual range of 323-631 nm with 7 a maximum spectral sensitivity at 520 nm obtained from electroretinograms withAe. aegypti [4,10]. In adults, the lateral ocelli degenerate and compound eyes are present. The dorsal ocelli are absent. The compound eyes consist of ommatidia. Each ommatidium consists of a diotropic apparatus (cornea, lens, and four cone cells for light collection) and a retinal cell layer [4]. For Ae. aegypti, the interommatidial angle is 6.2°; this angle is much greater than, for example, for houseflies. Due to the greater angle, theAe. aegypti has relatively poor visual resolution and poor acuity [10]. However, mosquito vision does exhibit a high overall sensitivity to light [9, 10]. Mosquito Repellents Almost all compounds found to be chemical attractants (kairomones) for a specific species of mosquito are chemical repellents (allomones) for other specific species. Work conducted using l-octen-3-ol (see section on Emanations from animals including man) has shown that this is a very promising attractant. Octenol, in combination with carbon dioxide, attracted some of the 35 species in a field study by Kline [11]. However, this compound acted as an allomone in the case of Cidiseta melanura. The grooved pegs on Ae. aegypti respond to lactic acid and are inhibited by the popular repellent diethyl-meta-toluamide (deet) [3,6,12]. Although oxalic acid itself is not an attractant (i.e. the lactic acid excitatory neuron does not respond), it does interfere or inhibit lactic acid response [12]. This interference is also the basis 8 for the action of deet; it too inhibits the response of the lactic acid excitatory neuron. It has been observed that mosquitoes exhibit slower flight rates upon exposure to repellent; turn angles are greatly increased and the number of turn readjustments taken is also increased. Therefore, deet does not repel; rather, it inhibits favorable response to mosquito attractant(s). Mosquito Attractants The mechanism of attraction or repulsion from a potential host involves a behavioral response by the mosquito to one or more stimuli. This overall response has been characterized as a four-step process; detection of stimuli by the PNS, interpretation of the stimuli by the CNS, activation of the appropriate response to the stimuli, and mosquito response [4]. This response can be in the form of attraction, repulsion, or can be anosmic towards the source of the stimuli. Sound attracts male mosquitoes via the Johnston's organs in the antennae; however, this connotation is more specific for attraction of males to females, via wingbeat frequency, for purposes of procreation. The stimuli used by mosquitoes for host location are visual cues [4-6,9,10,13-15], moisture [4-6,16], heat [4-6,16], carbon dioxide [4-7,9, 1 1, 13, 15-24], and chemical (olfactory) attractants [4-6,9, 1 1, 13, 16, 18-24]. Pheromones are long or short range olfactory attractants among members of the same species for purposes of mating. In Ae. aegypti, the male tarsal chemosensilla has been found to detect a female contact pheromone. Attraction to bacteria has been examined [17]; although the results demonstrated some attraction resulting 9 from bacterial emanations, it should be noted that carbon dioxide is excreted during bacterial growth. Females are attracted to a host at a greater distance than males; this is likely due to the greater number of olfactory sensilla found in females [4]. In species where the female does partake in blood meals, the difference between the number of chemosensilla between male and females is much greater than for species where the female does not blood-feed from hosts [4,5]. Nature of concern for attractant identification One of the primary reasons for searching for mosquito attractants is the increasing number of restrictions placed upon suitable insecticides. This is attributable to increased costs incurred from Environmental Protection Agency registration (under the Federal Insecticide, Fungicide, and Rodenticide Act), greater costs to produce insecticides for a smaller market demand, and pressure from environmental groups [25,26]. In searching for a natural attractant, the risk of an airborne hazardous insecticide is alleviated. This would then allow for contained traps to be lined or filled with insecticide while minimizing contamination of the environment with the insecticide. Current state of mosquito research. Research in the area of attractants continues with attractants/repellents and with the physiology of the mosquito. Research methods which have been successful for other species of pests are being adapted and used to aid in understanding the behavior of mosquitoes. Takken summarized the state of mosquito research in his 1991 review [5, p. 293]: 10 In the light of recent developments in tsetse ecology, where a range of kairomones has been found, it is surprising that to-date only one chemical (lactic acid) has been demonstrated to be a mosquito kairomone, while several studies indicate that other human emanations are also attractive to mosquitoes. Most studies used Ae. aegypti as a target insect and much remains to be done on host-oriented behavior of medically important groups such as the anophelines. . . . It should be noted that studies in the last few years involving l-octen-3-ol as an attractant for Ae. taeniorhynchus , among other species, have shown great promise since the review by Takken. Attraction of mosquitoes to l-octen-3-ol will be addressed later in this chapter in the section covering emanations from animals including man. Trapping of mosquitoes. Mosquitoes generally fly less than 2.5 m above the ground with the 1.2 to 1.8 m range having the greatest number of mosquitoes collected [14]. Some studies show that 0.6 m is the average height for appetitive flight [2]. Additional concerns in trapping focus on shapes of the target as well as construction material. For example, attraction was found to be more prevalent for rectangular traps compared to pyramidal traps and most species were attracted to projecting parts of these traps [14]. Color is also of concern in trapping and will be addressed in the section on visual cues as attractants. Short- versus long-range attractants. The physiological state of the mosquito and the proportions of sensilla play a role in determining which stimuli will be employed by a species for long-range and short-range cues. Vision is generally employed for long-range attraction with respect to orientation for upwind flight, location of nectar, and location of oviposition sites [5]. Carbon dioxide also tends 11 to alert and/or attract from long-range as well as olfactory attractants [5,14]. Experiments with carbon dioxide show that near a source of carbon dioxide, mosquitoes behave abnormally; this may be due to lack of other stimuli for short- range attraction, or due to a tonic response of sensilla from non-intermittent release of carbon dioxide [7]. These airborne attractants are detected as odor plumes. Short-range attraction may be accomplished by visual cues, as by changes in temperature or humidity [5,7]. Additionally, sound or wingbeat frequency is used at short-range for mate location. Vision Visual patterns on the ground generally control appetitive flight. Mosquitoes will respond to dark shapes, movement, and colors preferably as visual cues; these will alter the mosquito flight path [9]. Darker colors attract to a greater extent that lighter colors [14]. One theory attributes vision to be used in orienting upwind flight. This theory describes mosquito ability to judge windspeed by measuring its own movement at specific heights [2]. For shelter-seeking mosquitoes, vision is the means of site location. Visual cues also tend to be preferred over olfactory cues for nectar and oviposition site location in mosquitoes. This implies a long-range use of vision for these conditions, and that olfactory cues are employed for short-range with respect to nectar feeding and oviposition [9]. Heat The thermal neurosensory response can be tonic (continuous) to ambient temperature or phasic (intermittent) during rapid temperature changes [4]. 12 Temperature changes of 0.05°C can be detected, allowing a 2 kg animal to be detected from a distance of 2 m, provided the change in temperature is rapid [12]. Mosquito activity has been found to be greatly reduced for temperatures below 52- 56"? [14,15]. Moisture Humidity may play a role in determination of suitable locations for oviposition by the female [4]. In contradiction to this is a theory that the reception may just be a reception of temperature changes rather than water vapor [16]. It has been established that mosquitoes are attracted preferentially to humid, warm air rather than dry, cold air. The attraction of mosquitoes to warm and damp areas may be explained by humid conditions carrying temperature information better than dry conditions [12]. The attraction to humidity alone is much less than attraction to volatile chemical attractants. Experiments show dry emanations attracted 48% of caged {ema\e Anopheles quadrimaciilatus versus 3% attraction to damp air alone [16]. Carbon dioxide A component of exhaled breath is carbon dioxide. Carbon dioxide is a stimulus for almost all species studied by Kline and others [3,7,11,12,15-18,20-24]. The actual role of carbon dioxide still remains a mystery as to whether or not it provides a cue for mosquitoes to alight [5]. Experiments have shown carbon dioxide to bring mosquitoes to within two meters of a wood trap; however, they turn away before being collected into this trap. Greater numbers were captured with plexiglass traps; therefore, vision most likely plays a range in short range attraction in this case 13 [15]. It is certain, however, that carbon dioxide activates flight in mosquitoes [7,17]. Experiments involving removal of carbon dioxide from exhaled breath showed reduction in mosquito attraction to a host; however, it is not certain that carbon dioxide was the only volatile compound removed in such a study [7]. The range of effectiveness of carbon dioxide has been shown to extend beyond 60 ft, possibly up to 120 ft [15]. Studies involving Ae. aegypti palpal sensilla showed logarithmic phasic response to carbon dioxide from 0.01% to approximately 0.5% and that these sensilla can detect changes in concentration of 0.01% carbon dioxide. Saturation occurs above 0.5%, with little or no additional response at higher carbon dioxide concentrations. Exhaled human breath contains approximately 4.5% carbon dioxide compared to 0.01-0.10% found in the surrounding air [7]. Therefore, detection of carbon dioxide plumes by the mosquito is possible after a two order-of-magnitude dilution of carbon dioxide in exhaled breath. An additional note is that upon palpectomy, mosquitoes showed little or no response to carbon dioxide at any concentration level [4,7,16]. Sound Sound (wingbeat frequency) is a short-range attractant for orientation of males to females for purposes of mating [4,8,27]. The males of almost all species of mosquito have a wingbeat frequency which is approximately double that of the female [8,27]. The few species not employing sound as a short-range cue have approximately equivalent wingbeat frequencies for both genders. Sound level is also 14 important, as loud sounds tend to repel mosquitoes in flight and will not activate mosquito flight. The benefit of sound attraction is that it can attract many male mosquitoes in a relatively short period of time. For example, 80% of caged male Ae. aegypti were attracted within 5 s [8]. Doppler frequency shifts have been shown to have little effect upon attraction. Chemical attractants The search for attractants may identify single attractants for specific species; however, a universal mixture to attract a wide range of species is sought. Certain combinations of chemicals may synergistically attract species more than others [5.20- 23]. As with heat, some evidence exists that responses to volatile attractants and carbon dioxide may be tonic or phasic. In a constant (tonic) emission of attractants and/or carbon dioxide, mosquito response was found to decrease within minutes [7,17]. Airborne chemical attractants are carried by wind producing a series of plumes of host odor [9]. These plumes are neither uniform in size or distribution, thus eliciting a phasic rather than tonic response by mosquitoes. As previously mentioned, mosquitoes fly upwind in a zig-zag pattern, constantly adjusting to fly upstream into the plumes. The turning readjustment increases as mosquitoes near the host or source due to increased plume rate as well as decreased plume size. Odor plumes alert mosquitoes; however, visual cues provide better means for long- range attraction. 15 Emanations from animals including man. The use of l-octen-3-ol and its effect on some species of mosquito has been examined [1 1,20-24]. Octenol is present in ox breath and has been found to be an attractant for the tsetse fly. Although the response to octenol alone is not as great as the response to carbon dioxide, synergism is present when both are employed for attraction in some species of mosquitoes [20-24]. Lactic acid and octenol provided an additive effect for Ae. taeniorhynchus [11]. An interesting note is that Kline suspects that octenol will not activate flight, nor directionally alight the mosquito to the host at short range; instead, octenol is suggested to play a role in upwind flight, involving odor plumes, towards the host [11]. Lactic acid, obtained from acetone washings of human skin, was first identified as a chemical attractant XoAe. aegypti by Acree et al. in 1968 [28]. Studies of structurally similar compounds to lactic acid have produced mixed results [19]: to-date, lactic acid is the only widely accepted attractant iorAe. aegypti. In females, the response to lactic acid has been found to elicit response from the grooved peg sensilla [12]. Attributed to the grooved pegs are neurons which either give an excitatory or an inhibitory response to lactic acid. Studies comparing mosquito attraction between host-seeking and non-host-seeking mosquitoes have demonstrated differing sensitivities to lactic acid. After a blood meal, a mosquito which is non- host-seeking has a suppressed excitatory neuron response [12]. Emanations from plants. Almost all mosquito species examined take sugar meals [13]. Nectar feeding is necessary for survival in both sexes. If any differences 16 exist in sugar meals taken, males may feed more often but in less amount per feeding, regardless of age. Location of plant nectar is accomplished possibly by visual and most likely by olfactory cues. It has been suggested that nectar feeding occurs more often than blood feeding in females; nectar feeding may occur as often as once per night [13]. In terms of priority, however, blood meals most likely take precedence over nectar sugar (from studies involving /It?, aegypti andAe. albopictus). Flower odors play a role in alighting a mosquito onto a plant. The nectar sugar itself is not an airborne cue; however, contact sensilla most likely detect the presence of sugar. Fruits, honey, milkweed and rose extracts attract mosquitoes; strawberry and lilac extracts are suggested repellents [13]. Male Ae. aegypti have been found to be attracted to honey odors. Honey fragrance consists of methylphenylacetate and ethylphenylacetate and these were found to attract Ae. aegypti. Ethyl lactate and methyl propionate function in finding suitable oviposition sites [4]. Synthetic fragrances, specifically apple and cherry, were found to be attractive for Ae. aegypti [13]. Relation to Semiochemical Studies Analysis of body secretions and excretions, particularly those focused on perspiration, yields knowledge of compounds present on the skin. Perspiration is a dilute solution of compounds containing salts and other involatile compounds as well as volatiles. Combined liquid chromatography/mass spectrometry (LC/MS) analysis has detected the presence of lactic acid (lactate is a by-product of exercise), urea, 17 and various amino acids (phenylalanine, leucine, valine, and alanine) [29]. Amino acids, e.g. alanine, are believed to have too low of a vapor pressure to be present at detectable levels by mosquito chemosensilla [12]. Direct analysis of perspiration differs from work conducted for purposes of identification of mosquito attractants in that direct analysis detects both involatiles and volatiles. A necessity for mosquito attraction is that the attractant is suitably volatile such that long-range detection by mosquito chemosensilla can occur. Analysis of human body odors satisfies the criterion of examining volatiles which emanate from the skin. Odor analyses are typically conducted with GC separation. The detection can be accomplished by mass spectrometry, or another suitable detector, such as a flame ionization detector (FID). Determination of odiferous compounds can be done by using GC/MS in conjunction with GC/organoleptic evaluation by humans [30,31]. This is analogous to the use of GC/MS and an olfactometer in the work of this dissertation; the olfactometer performs the function of determining attraction level analogous to the use of the human nose to determine fragrance. Performing GC/MS and olfactomer studies on-line was not feasible at this time due to the complexity involved in the relocation of either the mass spectrometer or olfactometer. Additionally, mosquitoes typically require time to re-settle after detection of an attractive odor stimulus. Work involving GC separation with electrophysiological responses from antennae would obviate the need for re-settling time. Combined GC- 18 electroantennograms (GC/EAG) would be a valuable extension to the work reported in this dissertation; this topic will be readdressed in Chapter 6. Overview of Analytical Methods and Detection The majority of the work in this dissertation consists of sample introduction methods and detection of compounds by mass spectrometry. This section will provide fundamental information about the techniques referred to throughout this dissertation. This initial overview is intended to be very general in scope. More specific consideration of sample introduction methods, ionization methods, and tandem mass spectrometry will be addressed appropriately in subsequent chapters. Overview of Mass Spectrometry The first reports of mass spectrometry, as recounted by Nier. occurred in 1918-1919 from the work of Aston and Dempster [32]. Mass spectrometry allows for the determination of abundances of specific masses (specifically mass-to-charge ratios) [33]. It is arguably one of the most powerful tools for identification and quantitation of compounds. Identification of compounds by GC/MS trace detection is likely the most common information derived from mass spectrometric detection. This dissertation employs GC/MS; however, the introduction method employed to sample components is modified and will be addressed later. The fundamental ionization process of mass spectrometry is that of electron ionization. This mode as well as chemical ionization will be addressed from a historical view in this chapter 19 and a practical view in Chapter 5. The combination of successive stages of mass spectrometry (MS/MS) allows for analysis of more complex mixtures with less need of prior clean-up of sample and matrix [33]. Reaction studies, compound class screening, and elimination of chemical noise via selection of the parent ion of interest are some of the advantages attributable to MS/MS. Sample introduction The sampling methods examined for this work were, for the most part, chosen according to a specific criterion, to allow for sample detection in a fashion similar to that which mosquitoes encounter, i.e. a volatilized sample in the gas phase. Handled glass beads allows attractants present on the skin to be transferred to the glass surface; volatile attractants can then be desorbed by heating the glass. Thermal desorption methods. Direct thermal desorption methods, without additional separation or processing, are simple and quick with respect to detection of compounds. The direct insertion probe (or solids probe) allows for samples to be placed through a vacuum lock, directly into the ion source of the mass spectrometer. Normally, a sample is placed in a crucible designed to fit in the end of this probe. For studies in this dissertation, a glass bead fitted onto a glass stem was placed onto the end of the probe. The probe, inserted into the mass spectrometer ion source, is heated to assist in volatilization of compounds off of the bead. The idea of heating a single glass bead was extended to multiple beads. Since it is not possible to insert multiple beads directly into the ion source, an alternate method of transferring desorbed volatiles was required. Two to two hundred 20 handled beads were placed in an enclosed glass container which was placed inside the GC oven; helium passed over the beads transferred sample to the ion source via a deactivated fused silica column (transfer line). This technique will be discussed in greater detail in Chapter 2. Separation methods. Complexity of the sample may dictate that a method of separation is necessary in order to adequately resolve compounds whose thermal desorption profiles overlap. The focus of this work is on volatiles; therefore, gas chromatographic separation was chosen. The initial phases of this work employed short columns for faster analyses. However, due to the number of components desorbed, longer columns were employed for identification and case studies in the final stages of this work. Volatiles desorbed from the glass beads have wide desorption profiles; thus, some method of reducing bandwidth before chromatographic separation can significantly improve chromatographic resolution. This was accomplished either by cryo-focusing or by purge and trap. Cryo-focusing involves immersing a portion of the column, just after the column exits the injection port, into liquid nitrogen to collect volatiles and focus them into a narrow band [33]. After focusing, the column is removed from the liquid nitrogen and GC separation is conducted. Purge and trap is slightly more complex in that it typically contains an additional trap [33]. Prior to GC analysis, the sample is collected onto a suitable trap (e.g. Tenax) to perform the focusing operation. The trap is then heated to desorb volatiles which are then focused onto a cyro-trap. 21 Sample ionization Mass spectrometric analysis requires that compounds of interest form gas- phase ions. Ionization can be accomplished in a number of ways. For the work in this dissertation, ionization of volatiles (already in the gas phase) is accomplished either by electron ionization (EI) or chemical ionization (CI). Electron ionization fEI). The first EI source is credited to work in 1921 by Dempster; however, the precursor to the modern EI source was pioneered by Nier in 1947 [33]. Electron ionization involves direct ionization and fragmentation of volatile sample molecules in an electron beam. The fragmentation pattern provides structural information about the original sample molecules. Chemical ionization fCI). The technique of chemical ionization (positive chemical ionization or PCI) is credited to Munson and Field; a series of papers from 1956-1966, including the work of Franklin and Lampe, cover the development of this technique [34-53]. Chemical ionization is accomplished via a reagent gas of choice. This reagent gas is in greater abundance in the ion source than the sample, and at a pressure high enough (approximately 1 torr) to favor ion-molecule reactions. The reagent gas is ionized by electron ionization; the resultant reagent ions ionize the sample molecules via ion-molecule reactions, typically this is by proton transfer. The result is that less fragmentation of the sample molecule will occur due to the transfer of less energy from ionized reagent ions than from electrons given off by the filament. The greater abundance of intact molecular species relative to fragmentation allows for molecular weight determination. In addition to positive ion 22 CI, negative ion chemical ionization (NCI) provides additional molecular weight information, and in some cases structural information. The concept of CI will be addressed in Chapter 5; informative structural information derived from NCI is examined in Chapter 3. Triple quadrupole mass spectrometry The first report of a triple quadrupole mass spectrometer for chemical analysis occurred in 1978 by Yost and Enke [54]. Figure 1-1 depicts a schematic of a triple quadrupole system. This figure is representative of the Finnigan MAT TSQ70 utilized throughout this dissertation. Although this instrument is still called a triple quadrupole, the second quadrupole (Q2) has been replaced by an RF-only octopole for better transmission of ions. The ion source allows for passage of an electron beam from the filament to the collector. The electron beam is orthogonal to the vacuum-lock entrance (allowing for probe insertion) and to the GC transfer line. The TSQ70 ion source contains removable ion volumes which can be chosen to give either CI or EI operating conditions. Ions generated in the source are extracted into the first quadrupole mass filter (Ql) by a set of three lenses. The collision cell (Q2) is housed in an assembly such that a suitable inert gas can be leaked into it. Tlie inert gas functions to fragment ions via collision-induced dissociation. Resultant ions are then passed into the second quadrupole mass filter (Q3). Subsequently, detection is accomplished via a conversion dynode (biased for either positive or negative ions) prior to amplification by the electron multiplier. B vs -a u •♦J a C/D S3 24 u o Q H o 25 Single-stage detection. The triple quadrupole mass spectrometer can be used for single-stage mass spectrometric analyses [55]. The corresponding modes and operation of the quadrupoles for single-stage operation are presented in figure 1-2. A full mass spectrum can be acquired by either of two modes. The first quadrupole can be scanned, acting as the mass filter, while Q2 and Q3 are held in RF-only mode to pass all ions (figure l-2(a)). Alternatively, Ql and Q2 can be held in RF-only mode while scanning Q3, as shown in figure l-2(b). Selected ion monitoring (SIM), shown in figure l-2(c), is a single-stage mode allowing passage of only selected m/z values through the mass filter; it can be performed with either Ql or Q3. Tlie advantage to SIM is the sensitivity gained by scanning the quadrupole over only one or a few m/z values of interest [33]. MS/MS scan modes. The benefits of tandem mass spectrometry lie in the use of various modes of operation derived from the two successive stages of mass filtering [56]. The four modes available for MS/MS operation are shown in figure 1-3. A daughter scan (figure l-3(a)) consists of setting Ql to a specific m/z value. This selected parent ion is then fragmented in Q2, the collision cell, via the presence of an inert gas for collision-induced dissociation. The third quadrupole (Q3) then scans the resultant daughter ions. Figure l-3(b) depicts a parent scan; in this mode, Q3 is set to pass a specific m/z value of daughter ion produced by collisions in Q2. As Ql scans and sequentially passes each m/z ion over the scan range, the data system records the intensity of the daughter ion and correlates back to the parent m/z value that was passed through Ql. If Ql and Q3 are both scanned with 26 Ql Q2 Q3 (a); 2 ^ ^ Ql Q2 Q3 (b); Ql Q2 Q3 (c); Figure 1-2 Single-stage scan modes with a tandem quadrupole mass spectrometer set for (a) Ql full mass spectrum, (b) Q3 full mass spectrum, and (c) selected ion monitoring using Ql. 27 Qi Q2 Q3 (a); ^ ^^ ! -^ J. ^^ ;. Qi Q2 Q3 CID GAS QI Q2 Q3 (d); Figure 1-3 MS/MS scan modes of a triple quadrupole mass spectrometer set for (a) daughter mass spectrum, (b) parent mass spectrum, (c) neutral loss spectrum, and (d) selected reaction monitoring. 28 a fixed mass offset, a neutral loss spectrum can be generated (figure l-3(c)). The scan m/z value, for each ion passed through Ql, is offset lower in Q3 by the mass of the neutral loss sought. The final scan mode, shown in figure l-3(d), is selected reaction monitoring (SRM). The first quadrupole mass filter is set to a selected parent ion and the second mass filter is set to a selected daughter ion instead of scanning the full mass range. Several such reactions can be monitored sequentially. Analysis of Complex Environmental and Biological Samples The nature of the work found in this dissertation is similar to some issues in the analysis of environmental and biological samples. TTie approach involving identification of components is similar to methods used for environmental analyses. Modern environmental mass spectrometry is predominantly conducted by GC/MS, with ionization usually accomplished by EI and PCI [57,58]. Although the use of PCI can yield molecular weight information, the use of pulsed positive ion negative ion chemical ionization (PPINICI) allows for greater certainty in identification of the molecular weight of a particular compound [59]. Much of the identification of compounds emanating from human skin reported in this dissertation has involved the use of PPINICI and EI. Screening of selected metabolites in a biological matrix, such as urine or blood, provides a rapid method to diagnose disorders in patients [60-62]. Compounds of interest include pesticides, steroids, or drug metabolites [63-66]. The use of MS/MS in the screening process allows for rapid determination with less 29 sample preparation and/or in some cases little or no prior separation [65,66]. It would be beneficial to have in place a rapid screening method for the identification of mosquito attractants. The work in this dissertation is the beginning stage to the development of a screening method for the analysis of human attraction to mosquitoes. Organization of Dissertation This dissertation is comprised of six chapters; the overall emphasis is on a combinatorial approach, involving both chemistry and entomology, to better understand the basis of chemical attraction of mosquitoes to hosts. The first chapter has presented the objectives of this work, an introductory overview of entomological fundamentals concerning the mosquito, the relation of this work to semiochemical studies, and an overview of the analytical methods of sampling and detection by mass spectrometry employed in this dissertation. Various methods of sampling emanations are possible; a comparison of techniques tested for this work is summarized in Chapter 2. Chapter 3 focuses on altering attraction with lactic acid as the model compound. Reactions with lactic acid are examined and analysis of solution-based perspiration is described with respect to origin of attractants from the skin. Chapter 4 focuses on the utility of MS/MS to this project and addresses compound class screening. The identification of compounds present on the skin is addressed in Chapter 5. This chapter contains studies comparing two subjects of differing attraction to mosquitoes as well as comparing subject bio-assay attraction to mass 30 spectrometric assay results. The conclusions and suggested future experiments are contained in the sixth and final chapter of this dissertation. The appendix to this dissertation is located after Chapter 6; this appendix covers preliminary work conducted with carbon dioxide as a reagent gas for chemical ionization. CHAPTER 2 SAMPLING METHODS Introduction This chapter is an overview of methods of sampling and sample introduction. It will introduce the rationale for the sampling criteria and illustratively examine the various methods of sampling for the identification of volatile emanations from the skin. Sampling Considerations Ideally, the end result of this work would be to achieve a sampling method which maintains the integrity of the sample. Little chemical modification of the skin emanations is desired such that detection of volatiles is as comparable as possible to that of the mosquito sensilla. The mosquito chemosensilla show response to airborne components which specifically cause activation. The components on the skin which are attractive to mosquitoes can be transferred to glass via handling the glass object. Glass petri dishes handled by humans are attractive in olfactometer experiments and indeed retain their attraction for up to 6 hours [67]. Furthermore, differences in attraction between human subjects are reflected in the attraction of the petri dishes handled by these subjects. 31 32 The glass can then attract mosquitoes due to desorption of volatiles from the surface. The mass spectrometric detection is then desired to sample in this manner. Storage of samples and duration of the attraction once the sample is deposited on glass are also concerns. Some experiments involved collection of samples at a remote site with subsequent cooling in an acetone/dry ice bath to minimize premature desorption of volatiles. Volatile skin emanations, containing compounds which are attractive to mosquitoes, are amenable to cold-trapping. Experiments involving cold-trapping of emanations in an air stream provided, after reconstitution of the sample, approximately 60% attraction compared to direct introduction of emanations into an olfactometer [16]. Entomological Sampling Entomological work consists of determining the response of mosquitoes to various cues. Samples may be pure compounds, mixtures, or skin emanations transferred to a specific surface. The surface used for these experiments is glass due to the ability to transfer attractants to it and subsequently desorb these attractants. Experiments of this nature can be done in a laboratory controlled setting via the use of an olfactometer or directly in the field. Olfactometer The olfactometer used for measuring attraction is shown in figure 2-1 [67]. The olfactometer used in these studies consists of three pairs of ports for sample introduction; the figure represents only one pair of these. Each pair of ports a o ■M a ■*-• U s T3 ■+^ U (U o- 3 S ,o u l-c ti CQ O '3 cr O Ri a 0) lU T3 ♦J o u O B re o CO I 4> c > a> O > Ui o Vi C/2 Q ^— ^ ^^ ■^^ ■o c a> 1> £ > C/2 C3 ^ 39 a number of various shapes and volumes of glass sample containers. The enclosed vials allowed for heating of the beads for subsequent analysis, usually by cryo- focusing or trapping. Cryo-focusing and trapping methods are covered in greater detail in Chapter 5. Additional experiments conducted with glass beads involved solvent washing of the handled beads with methanol. Methanolic solutions were also employed for the direct analysis of perspiration via GC and for particle beam/liquid chromatography (PB/LC). Work involving solutions is covered in Chapter 3. Experimental Thermal Desorption from a Single Bead Single bead thermal desorption was employed to produce the chromatograms and spectra found in some of the figures in this chapter. The 0.115" (2.9 mm) glass bead was fitted (glass-blown) to a glass stem for insertion into the mass spectrometer ion source. The dimensions of the stem (5 mm long x 1.4 mm diameter) were chosen to fit in the direct insertion probe in place of the typical aluminum crucible. The bead (attached to the stem) was handled for 3-10 min prior to analysis. Handling consisted of rubbing in the palms of the hands only. Subjects for these analyses included the author of this dissertation, Mr. Dan Smith of the USDA, and Dr. Anthony Annacchino, Jr., a former member of this research group. Prior to analyses of a handled bead, a blank bead was analyzed to determine the background components. The experimenter who handled the bead then placed 40 the bead onto the solids probe for analysis. The solids probe containing the bead was inserted into the mass spectrometer ion source. The probe ramp consisted of increasing the temperature from 50°C to 300°C over a six minute period. The mass spectrometer was a Finnigan MAT TSQ70 triple quadrupole mass spectrometer equipped with a Varian 3400 gas chromatograph. TTiis modified instrument contains an octopole for Q2 and a 20 kV dynode. The instrument was employed only for single-stage (MS) detection. The filament emission current was set to 200 p,A in all cases. The electron energy was 70 eV for EI analyses and 100 eV for CI analyses. The manifold temperature was set to 70°C while the ion source temperature was set to 150°C for CI and 170°C for EI. The mass ranges scanned were m/z 10-650 for EI and m/z 60-650 for CI. The reagent gas for CI analyses was methane; the indicated pressure of methane in the ion source was 1650 mtorr. The electron multiplier was set between -1000 and -1200 V and the conversion dynode set at -5 kV. Prior to data acquisition, the instrument was tuned for maximum transmission of characteristic ions from perfluorotributylamine (PFTBA). Thermal Desorption from Multiple Beads The sampling of multiple beads is not amenable to solids probe introduction via insertion through the probe lock of the mass spectrometer. Initial sampling of multiple beads was accomplished by the apparatus shown in figure 2-3. The beads were placed in a round bottom flask; a 100 mL or 250 mL round bottom flask modified for use with this apparatus. The modification consisted of removing the Figure 2-3 Apparatus constructed for the delivery, via transfer by a deactivated FSOT column, of desorbed skin emanations from glass beads contained in the round bottom flask. 42 To Mass Spectrometer Helium In Toggle for Helium - Introduction Capillary Column Vacuum Toggle for Chamber Evacuation Four Way Cross Cajon Fitting 100 mL to 250 mL Round Bottom Flask 43 original stem and replacing it with a 1/2" diameter glass tube. This allowed the round bottom flask to be inserted into a Cajon fitting connected to a four-way 1/4" Swagelok cross via 1/4" tubing. The four-way Swagelok cross was attached to two toggle valves. One allowed for evacuation of air in the chamber prior to analysis; the other allowed for helium to be passed into the chamber, mixing with desorbed volatiles from the beads. The Whitey toggle valves were connected via a 1/4" to 1/8" reducer to the 1/8" Swagelok fitting. The vacuum line was connected to the TSQ70 pumping system via a Swagelok fitting normally used to evacuate the calibration gas probe. TTie high purity helium was delivered to the apparatus by diverting the helium line out of the pressure regulator on the Varian 3400 GC and interfacing it with the toggle for helium introduction. The final port on the four-way cross consisted of reducing the 1/4" fitting to 1/16" and employing a 0.4 mm i.d. vespel ferrule (SIS, GVF16-004) for insertion of a 1.0 to 1.5 m x 0.10 to 0.25 i.d. deactivated fused silica open tubular (FSOT) column. One end of the column extended down into the round bottom flask, above the layer of beads. The other end of the column was interfaced to the ion source of the TSQ70 via the GC transfer line. The operation and sampling for the apparatus shown in figure 2-3 consisted of placing it in the Varian 3400 GC oven. The round bottom flask was removed from the Cajon fitting just prior to the transfer of glass beads into it. The flask was then re-inserted into the Cajon fitting. For some studies, the round bottom flask and Cajon fitting were replaced by a 3" x 1/4" o.d. glass tube swaged to the cross with a 44 1/4" glass filled teflon ferrule. The tube had an inner diameter slightly larger than the bead diameter (approximately 1/8"). Once the sample was placed in the apparatus contained in the GC oven, evacuation is conducted by opening the toggle to vacuum. After 2-3 s of evacuation, the toggle was closed and the second toggle, supplying helium, is opened. The helium pressure was set to 8 psig via the regulator normally used to control injection port pressure on the Varian 3400 GC. Data acquisition was started at this point via instrument automated control through an ICL program. The GC column was a 1.0 to 1.5 m .x 0.10 to 0.25 mm i.d. deactivated FSOT column. The heating parameters consisted of a GC oven ramp and a transfer line ramp. The GC oven ramp consisted of a 0.5 to 1.0 min hold at 25 to 30"C followed by a 10-15°C/min ramp up to a final temperature of 190-210°C and a final hold of 2.0 to 5.0 min. The transfer line was initially set to 50°C and ramped immediately at 207niin up to 200-2 10°C and held at that temperature for the duration of the experiment. The NCI experiments were conducted with 1650 mtorr methane as the electron moderating gas. The scan times were 0.5 to 3.0 s; scanning over Q3 (i.e. Q3MS) was employed with a mass range of m/z 10-650. The electron multiplier was set at -900 V, the conversion dynode at +5kV, and the filament emission current set at 200 ij,A with 100 eV electron energy. The ion source temperature was set at 150°C and the manifold set at 70°C. Prior to analysis, the mass spectrometer was 45 tuned for Q3MS NCI mode via maximizing transmission of characteristic negative ions from PFTBA. Thermal Desorption from Multiple Beads/Cryo-focused GC Separation Heating of beads to desorb volatiles for cryo-focusing was accomplished by loading the beads into the GC injection port. A Varian 3400 GC fritted glass injection insert was inserted reversed into the injection port. This allowed for up to 12 beads to be placed between the frit (located approximately halfway down the insert) and the GC septum sealing the injection port. The frit kept the beads from dropping down onto the column entrance and provided a means for volatile emanations to be loaded onto the column; the column entrance extends up into the injector insert just below the frit. Operating the injection port in a entirely splitless mode (i.e. it was not necessary to open the split valve due to the absence of solvent in samples) for the duration of the experiment provides essentially only one exit for volatiles, through the column. Beads were rubbed for 5 min prior to being placed into the injector insert. Experiments were typically performed with 5 to 12 beads. After loading the beads into the insert, the insert was placed into the injection port held at 25°C to minimize sample loss from evaporation. Repetitive analyses required a method of cooling the glass insert prior to subsequent analyses; Dust-Off (difluoroethane) was used for this purpose. The cap, septum, and needle guide were replaced. TTiroughout the loading process the head pressure of helium was set to 0 psig to allow for proper alignment 46 of the septum and to prevent premature migration of volatiles past the intended point of cryo-focusing on the column. Before increasing the helium head pressure, liquid nitrogen was placed in a 12 oz. styrofoam cup. The cup was placed in the oven such that approximately 8 cm of column could be looped in the cup about 15 cm below the point where the column passes from injection port to oven. The helium pressure was then increased to 20 psig and the initial desorption phase started. This entailed loading a program to ramp the injection port from 25°C to 250°C over 7.5 min and holding at 250°C for 2.5 min. Throughout the cryo-focusing phase, the GC oven was set at 25''C and the transfer line set 40°C. Liquid nitrogen was added to the cup as necessary during this 10 min cryo-focusing phase. Subsequent to the completion of the cryo-focusing, a new program was loaded from the TSQ70 to the Varian 3400 GC. Prior to running this program and concurrently acquiring data, the cup containing liquid nitrogen was removed. Tlie GC oven ramp consisted of an initial 1.0 min hold at 25°C, a 12 min ramp at 15°C/min up to 210°C, and then a hold at 210°C for 5.0 min. The transfer line was concurrently ramped, after a 1.0 min hold at 40''C, up to 225°C at 15°C/min, and held 5.0 min at 225°C. Experiments were performed with either an 18 m x 0.18 mm i.d. DB-5 column (df=0.25 p,m) or a 20 m x 0.25 mm i.d. Carbowax column (df=0.25 Jim). The mass spectrometer was operated in NCI mode, positive and negative ion mode (PPINICI), or EI mode. For NCI and PPINICI experiments the reagent gas 47 was methane at 1650 mtorr and 1660 mtorr (indicated) pressures, respectively. The ion source and manifold temperatures were 150°C and 70°C, respectively, for CI and 170°C and 70°C, respectively, for EI. The electron energy for CI experiments was 100 eV and for EI, 70 eV. The third quadrupole (Q3) was scanned with a scan time of 2 s for figures in this section. The filament emission current was set at 200 jiA. The conversion dynode was set at +5 kV for positive ion CI and EI, and -5 kV for negative ion CI. Tlie electron multiplier was set at -1000 V to -1200 V. Prior to analysis the instrument was tuned with PFTBA as previously mentioned and blanks (appropriate number of beads without sample) were analyzed. Purge and Trap/GC Separation Some analyses were performed using a microscale purge & trap system. Tlie sampling difference between figures shown in this section of the chapter was the method of collection of skin emanations. Some experiments involved handling 200- 250 glass beads 10 min. The beads were then transferred to the same 100 mL round bottom flask discussed previously. The round bottom flask was attached to the purge and trap system via a 1/2" Cajon fitting to 1/4" Swagelok, further reduced to a 1/8" Swagelok fitting. Skin emanations were also sampled by placing the left hand in a Tedlar bag and fastening the bag around the wrist with a rubber band. The Tedlar bag was attached to the purge and trap system by a 1/8" Swagelok fitting. The flask or bag was attached to a port on an ELA2010 canister manifold (Entech Laboratory Automation). 48 The canister manifold allowed for 70-100 mL of volatiles and residual air to be sampled by the ELA2000 concentrator. The concentrator consisted of three stages. The first stage employed a dryer with a gradient of large to small glass beads through the dryer. This served to remove most of the water in the sample. During concentration, this stage is set to -160°C for three minutes and heats up to -16°C as sample is transferred to the second stage. The second stage in these experiments was a Tenax trap. During concentration, the trap was set to -20°C and then heated to 156°C for desorption of trapped volatiles onto the focusing trap. Tlie cryo- focusing trap was set a -160''C for concentration and was heated ballistically (approximately 10 seconds) to 150°C to purge volatiles onto the head of the GC column. The GC employed was an HP5890 series I with a 30 m x 0.25 mm i.d. DB-1 column (df= 1 |j,m). For analyses involving glass beads, the column was initially cryo- cooled at -35°C and held for 3.0 min. Subsequently, the column was ramped at 12°C/min up to 180°C, then ramped at 25°C/min from 180°C to 225''C and held for 5.0 min at 225°C. For analyses from the Tedlar bag, the column was held 6.0 min at -35''C then ramped at 6°C/min to 180°C and 12°C/min from 180°C to 225°C, and held for 10.0 min at 225''C. The mass spectrometer used for these studies was a Finnigan MAT Incos 50 single-stage quadrupole. The scan rate employed was 0.75 s per scan. The filament emission current was set at 750 |j,A with an electron energy of 100 eV. The electron multiplier was set to -1200 V. The ion source temperature was set at 180°C; there 49 is no heater for the manifold. Prior to analysis, the instrument was tuned with PFTBA. Results and Discussion Thermal Desorption from a Single Bead The initial crucial phase of the work in this dissertation was demonstrating the ability of mass spectrometry to detect emanations found on the skin. This problem was approached by employing the knowledge gained from olfactometer experiments that glass which has been handled retains attraction to mosquitoes. Shown in figure 2-4 is the total ion current versus time trace for a single handled glass bead. This trace is typically called a reconstructed ion chromatogram (RIC), even though no chromatography is being performed. This figure represents one of the experiments from this beginning series where the single glass bead was introduced into the mass spectrometer ion source via the direct insertion (solids probe). It is evident from the shape of the RIC that separation from distillation off of the probe is minimal and more sophisticated analyses would be required to discern the components hidden below these peaks. This figure clearly illustrates the drawback to this probe method, i.e. that temporal resolution, although satisfactory for simple samples, is not capable of providing efficient resolution from so complex a sample as skin emanations. Matters are further complicated when searching for trace components. One such example, for cholesterol (cholest-5-en-3-ol), is presented in figure 2-5. Displayed in this figure are the mass chromatograms for m/z 386, the characteristic .S 5 .s on Q C "" ■ k, o .2 > x^ c^ 2 — oo O "^ =*- Q X5 ■£ o ci, "* -a Q OJ (U .2^2- C C tfl m o u .i: -^ x> -a ^- o »^ o ^^ "£ ^ 0) .2 c £ > re M 1/1 • • — aj n •^ ^ i: x^ -^ o re v^ on C O t, re t^ w U ^ (/I D-i re ^ ^C 5^ o o.S E ^ T3 "-^ (u -5 i/T <+- •5; O ro = c B IS ro I— I cu *- c I- "^ -2 N "O r" (/3 00 ^ N bO E ^ 03 ■a (U E re re X) o c re o (« ex C 4= o E CO m O T3 b-5 o "2 re o E - o 2 js re " E =« c u X) f •- O > m '^ ^ ^.E Q E . ^o O V re o P o P-i -a ^ U 53 in Tj- iS CD + '3 ■* IN. ■3 — I + OS 00 n N s r- tSl — IS> CO CvJ o .S B .S ^ re c ^< 9 Q -U 3 ^ = ^ "=5 re ^ (^ E o 2 ■S « 2 CL. . -73 2 ^ ^ t: >^ S r; ^ re in r- '^^ r^ re *- c > ■■ago o 0) re c r? ■> re H .S£ u c ^^ re C O ;_ re c/] cu n E ^ W — 1/3 a^ re §.£ E •-I _ '-S t) w c u X) re re X) o ^ 3 ^ "-I re CJ cc .^^ 5 XI ^ c re .2 60 -a o 73 a> *i too O C re Tr.y^ c o o (N • - lU re 3 re 60 C :— C T3 re '^ 2 o 56 CO o (%) /C)isn9)ni 3AIJBPH 57 Figure 2-7 is the RIC of a second bead rubbed by Dan Smith of the USD A. This experiment was run 30 min after the experiment which produced the RIC in figure 2-6. The differences in peak heights between these figures show that significant variations may exist between analyses from the same person. This difference could very well be a difference in the substances deposited on the beads, i.e. some substances may be retained on the skin more efficiently than others; the difference between figures 2-6 and 2-7 could reflect this. The difference between these two figures may also indicate the irreproducibility of this method of sample introduction. Analysis of repeated samples taken over an extended period of time will be addressed in Chapter 5 of this dissertation. Figure 2-8 shows mass chromatograms obtained from the analysis giving the RIC trace in figure 2-7. The ions shown are m/z values for the protonated aliphatic fatty acids. Specifically these are the [M-hH]^ ions for tetradecanoic acid (m/z 229), pentadecanoic acid (m/z 243), hexadecanoic acid (m/z 257), and octadecanoic acid (m/z 285). The profiles found below scan number 150 are clearly offset and depict the trend of this series of acids; however, without the knowledge acquired later in this work, the results would still be lacking in terms of identifying the full series. With this knowledge, some of the earlier mass spectra, such as the spectrum presented in figure 2-9, can be examined. Figure 2-9 is a mass spectrum from scans 151-200 for a single bead rubbed by Dr. Anthony Annacchino. Clearly shown is the molecular ion for cholesterol (m/z 386) and the loss of water from cholesterol (m/z 368). Although, the ions at m/z 200, 228, 256, and 284 correspond to the molecular c re o o U o O o . — ° (J C t/3 1 1/5 •-; re <^ .E E ^ ii; *;; o (u tL, -U 4i CX 59 (%) /Cjisnajni 3ai}B[3^ o Q o ^ o o c o t/3 )-l a. "a o .5 ^ > ^ 00 0-1 re o •^ -S C re o ■^ fc- re ■> T3 J= ^ re .-s _, 00 -a ^ j2 o re w) S in W)^ ^' 5 ;^ reC> _ E-S !^ o -n E .2 re c re E 3 a- o re .& o c o -^ t; c 2 ,5 S re 3 .2Q ^ S£ e =" ■"ere -a o > ^ >^^ re o W re o m o ♦- I C 3 re >- " -a 1^ ^ i o (L> 5 c "*- ■-2 o re g 3 ^ O re (SO c c s =« t3 E UJ o [/I on (^ C re o <^ re - re ^ o o <^ c o ^ o S-^ Ji^ re o\ c ^ E 3 N m o 63 (9 + . » T T co CM C9 X CO ■ CO JL CO II I IT) ■ CO CVJ . s . IS CM lO 03 r- in " SI -U) n '-' T " ' ' ON (M ■ 00 ■ 00 col CM ■* CO 1 r 00 r I 1 ■o Cvl — _ o "^ -^ re >> "- o (U t^ g ^ o re j= re <" -^ ■ ■ re o O U '^ rri ^ ^ ^^ ^ E 3 -a O 00 u ■i=- ?^ E-S a 2 T3 -a O -2 ra re o g £^ B re 1/5 c re u re -4— > ■^ iJ -a o E GO a 73 cr o re re Q c re o a. .2 re -a uo _c T3 re E CC <^ •^ 4= <*- " «> o -a ^ E S re '^ > T3 T3 .2 X> re t- re U Z u E o o E GO -a f^ re Q J3 C oA-t: 2 S ■2 ° re u ^ lU c re lU c/1 J^ ^ 68 3 c u (%) iCjISUajOI 3AI}BI3H 69 are not present. However, this figure also illustrates that the thermal desorption profile is still less distinct than from the solids probe. In contrast to the solids probe, where volatiles are desorbed directly into the ion source, the enclosed sample container permits greater head space above the sample. This space allows for volatiles to be desorbed and remain in the enclosed sample container, mixing with additional desorbed volatiles as the temperature increases. Furthermore, the beads in the vial are probably not at a uniform temperature. As a result, the distinction between desorption profiles for different compounds appears simply as differences in the initial appearance of masses, similar in appearance to frontal chromatography. This point is illustrated in figure 2-12. Figure 2-12 depicts the mass chromatograms for four ions associated with lactic acid. These representative ions shown are the loss of H, from the [M-H]~ ion at m/z 87, which may also correspond to the [M-H]~ ion of pyruvic acid, the [M-H]~ ion of lactic acid at m/z 89, the [M2-H]~ lactic acid dimer ion at m/z 179, and the [M4-3H]^ lactic acid tetramer ion at m/z 357. The profiles of these ions are clearly disbursed over most of the run once the temperature is sufficient for the desorption of lactic acid. The wide profile makes identification of molecular and fragment ions with identical m/z values virtually impossible without a reduction in the profile width by either more efficient loading of desorbed species onto the column or by re- focusing the sample bands either prior to or directly on the column. CO u ^^ .c d re ■g <^ re -a X) o o re T3 a; B o O c o re 73 re E d o >_ re m OJ o o -5 s ^ ■re ^ ^-re lu re c % o ^ dj O <^ tu c -a re ._; on' P ^ i oC2 °° S ^^ k- CO "^ ;^ OJ o ■^ c re -a M 0 re 1 « re di u § re t/3 re c re .2 Q u ^ E "H, o ;_4 E '^- re o t/3 C/3 00 to ^ re c — ll") 00 ® -a- o ^ + CO + K£, LU . LU . m ,u> mm CO 00 + Tf u B a i/3 ^0 10 CD CSJ "S" (33 ^Q |"SJ 1^1 (^ GD S> CT (S O 03 Q O ® GD S) 00 vo •«• C\J fl^ - T3 >- Q o — t^ (U 6 ,^ c " . re > C OJ > 0 0 ■55 in "a 0 >^'^^ V) re 0 -S 1 c 11 -t: re « ■a'^ (U U ^ ^ t/i Z E 2 c/l re ^ *-• • - =: « = 5 in 8 E ^ o o ^ o •> s o :. XI 3 E H ■C on c B '^ o O (U t^ lU CO >^ C JS c 5 H C (« (u vo' E S ci- CO 00 "re t^ E >o'S.E E o S C/3 c ^ =« o -o JS '^ ?^ • - t^ J^ ^ ^ « c/, 2 « & . ^ c- c o ^ 1 W)"^ *^ x: C E re t/2 ■P C t/2 n kH C/3 3 ^ \^ ' ^" .S£ '^? O 1) > "iJi o "^^ '^ ^ II "Z re re ^ ct W) re ^^ c 1— 1 c ■*— 1 I-. u s >. <£ !:t 3 £) Si ^C 1) Cl, ^ ,M re D- H c k. c ^s D r- _o O CO <£ P l-l o ramsf owax 1/) c >■ E re o o MX) o o O ^ OJ ■ii re ^ T— < X) reu o -d o 1 re X5 ^-1 Ii C/2 E _c ;d ion 25 mi re kH g OJ 4= •2^ [a. o ^ o ■*— ' -«— ' .« ^ X 3 3 ,^ ^ 3 • "^ E ^^ t: E -a u to lU c 0-^ c o Xi o Reco n a 2 X) 3 I-. o — to c o TJ (U T— C C re lU r^-2 fs -4— > t/3 (S ■ 00 VD ■V C«J (%) AlSUajUI 3AIJBPH - o "t: ^:° . p , re S bO E - -C o 3 -^ o "^-' ■> 9" 3 re .id" 5. OJ J= "S II >^ 5 "^ ^ re ^ « c _c U E c i| o -" i: " i-H -H <£ E O Vi re c/5 ■a feat o ^ c re g CQ <«^ c/3 mpera emana o (U re O- fe: ^ o -4-* « J c E 60 .22" =*• re X) 'i75 ^H >. 11 Uh — ^ _re re u C "O -a re ^^ — ' o X5 aj — ^ > o -t: U (/3 t/2 J^ oo j= -a ^ -^ o -^ re . -a o ^^ 2i - a EH 5 2 _^ <« . .2P E "H. CO E E •£ ^ c/5 C O 5 in -a ^ jj •t-t -t^ ^ ■" *- "re o B fi--: -a ^ c ;£ =« "*^ a ' 2 re n) c re re P- 103 X O U I -a- T o X JL "7 X ^■ u - in X 1 1 CO 1 "T- "T" (M X I X I s "CO IT) 00 N a _1S SI in X 1 IS 1 CO 1 I o u X X o u x" x I in ■ .SI (M (%) i(jlSU3jni 3AIJBI3H 104 from m/z 87, respectively. Additionally, there is presence of an ion at m/z 85 from the neutral loss of H,. A summary of the fragmentation pathways for negative ions of lactic acid examined by NCI MS/MS is presented in figure 3-3. There are two points about this figure which warrant mentioning. The first is that ions shown in the figure represent a selection according to threshold conditions for these experiments. This chosen threshold was that the abundance of these ions be greater than or equal to 1% of the base peak intensity in each daughter spectrum. The second point concerning figure 3-3 is that the structures listed are postulated from losses and reasonable fragmentations and rearrangements [75-78]. Additional measures, such as the use of high resolution mass spectrometry and isotopic labeling, were not employed to confirm these structures. The trend via losses of H, is apparent; this loss most likely occurs by cleavage of the hydrogen from the a-hydroxy group and a y-hydrogen from the terminal methyl group. This process occurs through a four-centered intermediate ring state [79]. This same cleavage most likely produces the species at m/z 85 from the m/z 87 ion. Due to the absence of sufficient hydrogens, the m/z 85 species does not undergo a further neutral loss of Hj. The availability of hydrogens limit the possible fragments resulting from precursors in other cases. Examination of the loss of water from the m/z 89 and m/z 87 species reinforce this. Both these ions will undergo a neutral loss of water, while m/z 85 will not. The fragments formed (m/z 71 and m/z 69) show this trend. u ^ ■♦-' S-( O to C o • ^^ •^-t c 4^ a bO ed i-i «4-l -a 53 C4 vt U ca CO ^ ^_l a ^ ■*-• 3 U s •a a> 4-> CO s w O cx •« CO >, nS ^ JS -t-t a u. c o • f-^ ^^ e« •*-• S (U a oo ce ;-! VM c '6 o 'o (U ca > o ■^-1 -*— ' re o W) JS ■*-» ^ o 3 )_ 60 CU (U £ ■a 106 o o °o 1 o o=u 1 _ u o III N ^ III o o r X 107 The species at m/z 71 will undergo a subsequent loss of H, via elimination resulting in the formation of a triple bond at the methyl terminal to produce the ion at m/z 69; this is identical to the fragment occurring from loss of HjO from the m/z 87 species. In addition, m/z 71 undergoes additional losses of CO and H2CO, whereas the m/z 69 ion can only further lose CO. The direct neutral losses of CO from m/z 89 and m/z 87 are similar. However, again, the m/z 61 ion formed from m/z 89 can undergo losses of H2, and HjO, whereas the ion at m/z 59 cannot. It is interesting to note that the m/z 59 ion will undergo homolytic cleavage to form an odd-electron ion at m/z 42, via the loss of a HO' radical. This is relatively uncommon and unfavorable for an even-electron precursor [79]. Since the charge is retained on the odd-electron species, this fragment arises from cleavage of a single bond, i.e. between the hydroxyl group and the carboxyl carbon. A fragmentation involving cleavage of two bonds would necessitate charge migration for production of an odd-electron fragment. The only cases where a direct loss of formaldehyde occurs are from m/z 89 directly, or from m/z 71, which resulted via the loss of H2O from the m/z 89 species. In contrast to this, the loss of CO2 occurs from all three major species (m/z 89, m/z 87, and m/z 85). In each case the products formed depict losses of hydrogens characteristic of the precursor species. If an additional two hydrogens are lost via loss of formic acid, then the required number of hydrogens to constitute these losses are only present when losses come from the m/z 89 and m/z 87 species. 108 Oligomerization and attachment reactions Lactic acid in the gas phase is also susceptible to oligomer formation as well as other associative ion-molecule reactions. Examining mass chromatograms (figure 3-4) for oligomer and attachment ions produced from lactic acid demonstrates the presence of a dimer ion at m/z 179, a trimer at m/z 268, and a tetramer at m/z 357, all of the form [M„-H]~ The time correlation of the peaks indicate that these ions are related. The formation of oligomers is most prevalent at high concentrations of neutral species in the ion source. Therefore, the oligomeric peaks have narrower peak widths than their precursor ion. The presence of oligomers or at least dimerization is predictable from knowledge of the solution-phase chemistry. Lactic acid is an a-hydroxy acid. Due to the inability of this species to form a lactone ring which is stable, it undergoes oligomerization and self-esterification [SO]. Further examination of oligomeric ions will be addressed later in this section. Two additional ions yield peaks which correlate to lactic acid. These peaks at m/z 125 and m/z 127 arise from chloride ion attachment to a neutral lactic acid molecule. Verification of this is presented in the daughter spectrum in figure 3-5. The m/z 125 ion was mass-selected and fragmented by CID to yield fragment ions at m/z 35 and m/z 89. The m/z 35 ion is clearly the "Cl~ ion. By virtue of MS/MS, this ion could only have arisen from the m/z 125 ion rather than being attributable to background. The ion at m/z 89 is the deprotonated molecular species of lactic acid (i.e. the lactate ion). This fragment is formed via the neutral loss of HCl from •^ c MI O -a c re c re o re re cu 5 Q ^ o r -a u re ^ o O CO re CO re E o E ^ re c o (/J c 60 X) •jH U u 3 Eu 1- re lo ^ c 2-2 re "O E g JS c W O c/1 "" cfl ■<-' re c ■"" re 6012 re ^5 c s _ re o f— ' CJ o .2 u ° J5 § (/) p ,.— ■S O J2 ^^ ^-» -<-» C fe 1^ CXZ t;^ •5 ^ (^ 1) c 3 -a 60-i= ^ .— ore Uh re j= CO 3 o O -a lO ■JJ ra CO CD o ■ + Tf + IS + o I to 5 U £ V re o o N M-r f^i re ai ^ c • K.1 N <4-l O E 3 " 9 t/3 -is 3 t/) re (u re o -a c ^ re re 00 O r-1 S « z o ^ ;/) ^ re o 1/3 re oo re^a Q lU u-^ J j= o OJ u E-B 'p-i re in ITj o (%) /Cjisnajni aAijup^ 113 the m/z 125 parent ion. The daughter spectrum of m/z 127 consisted of the expected daughter ions at m/z 37 and 89. Chloride ion attachment spectra are useful for applications involving detection of organic compounds with acidic protons [81]. This attachment is somewhat selective for specific classes of compounds which meet this criteria. Typically, methylene chloride is used as the reagent gas to effect the attachment in the ion source. In the instrument employed for this work, residual chloride ions are always present in the negative ion background and, as demonstrated, can undergo ion- molecule reactions even in the presence of additional reagent gas. This reaction itself is of secondary importance in the work for this dissertation rather than a primary mode of identification. Negative ions which undergo a neutral loss of 90 Da (i.e. lactic acid) are presented in figure 3-6. The chloride attachment ion at m/z 125 was previously shown (figure 3-5) to undergo a neutral loss of 90 Da. The other ions found in this neutral loss spectrum are at m/z 179 and m/z 177. The lactic acid-lactate dimer ion at m/z 179 was pointed out in figure 3-4. Additionally, dimer formation can result from the combination of a neutral lactic acid molecule and the hydrogen-eliminated lactate ion (m/z 87) to produce a dimer at m/z 177. Experimental data not shown indicate that the m/z 177 dimer can also undergo losses of 88 Da and 92 Da to form ions at m/z 89 and m/z 85, respectively. Additional data will be presented later in this chapter (figure 3-10). 3 O E re ■a ■4— • re c re r- lU *: E-s -5 (in -a bo^ o o 4= re o T3 oi 3 ■*-* ',»"' pC re re -^ fc. re re o. ^ aj .5 "3 "S T3 re .S -^ -C T3 a- c .0 S !U t/2 •'-' re re u ^ & 3 CO flj ifl 60 a. c s 1 3 ^ ^ X5 -a O B isioi ads c/2 re —- (U Cn •a T-^ o t _^ -4-1 'C re f 1 o f^ lacti( ands c/) 13 Z c re o c/1 re i/^ u-> ^ 3 ^ k< en re m/z eing _o ■4— » OJ S & re 0\ <4-l Xi i/1 o ^ re 1/1 i-i & E - ■a i-i re O >-v 3 1« Ol E >< B o 't^ daugh were 0) k. 3 m . in 'o 1^ X) J5 .. <" 3 ■a (/2 ■4-* re 13 Ki > re o : 3-7 Neg; 9eV. Fi 4" o.d. gl yed (indi c _o "o o ^ O u 3 ra re D. E J3 *-• £ ^ _c 118 (%) Aisuajuj aAijepH 60^ ^« . re re re j= > CO re re re o r^ ^ .2 V3 ■a re > t/1 Uh re 3 E ■>< « re 1) 5 o ■a cj — : -^^ ^^ re oj u o 3 ^ s '^ O T3 !^-^ o c c .2'S « L^ ^ P-JH CU C 5 > o ' — ^-a •— ' w re -" X! . CO J3 lU <^ s ^ ° E.E E m tJ ^ W •r: re js re cx-^ 2: "S -a OO C *-. .S^ re 5 o o 3 (/) -a re e re re D Q u H t/) re E C/3 re 60 a *-• re c u 120 X _l_ •r P -iSi i-S- in N -1^ CO 1^ 1^ 1^ (%) ^jisnajni aAiiBja^ CO re ^ E -. o ■ S o ■^-t O vo c ■-C T-H re H <*-' o •Si. o oo C (U D '^ ^ -C I-J 3 H ^ Si > o ^ (U o re •a .H t/5 o "O re c .2 c on re lact at a o-S c ^ >^^^l E re <4-l Ml C o O PJ on C/3 ^1 ^p -a 5l •4-t re _o 2 T3 an • — -u re "^ _c -a .o c __ *-• re o re re «^ hJ Z •-= .Sa w re -— re 60 O ^ d D C Z^ Es 60 1/2 9 s 2i JU ^ 3 i . en <^ Ph c H o 122 (%) /(jisnajni aAijcia^ 123 A summary of selected major fragmentations of dimers and associated attachments is presented in figure 3-10. This figure is arranged such that reactions involving lactate, or the lactate-lactic acid dimer are found towards the top left corner of the diagram; those involving the Hj-eliminated analogs of these species are found towards the bottom right corner. The similarity in fragmentations of m/z 179 and m/z 177 is clearly evident. Both species undergo neutral losses of CO2 to form ions which can then cleave the remainder of the neutral lactic acid molecule to form ions at m/z 89 and m/z 87 depending on the initial dimeric species. Both the m/z 179 and m/z 177 dimer can form ions at m/z 89 and m/z 87. Additionally, both neutral lactic acid (90 Da) and the H2-eliminated lactic acid neutral (88 Da) can form chloride attachment ions. Differences are found in the fragments of the dimer ions. The m/z 179 dimer ion can undergo a neutral loss of CO and a neutral loss of HjO. The intensities of fragment ions formed by identical losses from the m/z 177 dimer ion fall below the threshold percentage (1% base peak) chosen for presentation. An additional dimer ion at m/z 175 formed from the association of Hj-eliminated lactate and lactic acid fragments to yield only m/z 87; this is also not presented in the figure. The only significant fragment ion arising from the m/z 177 dimer (with exception of m/z 89 and m/z 87) is due to the neutral loss of CO2, forming the m/z 133 ion. Lastly the issue of dimer formation in the NCI mass spectrum of lactic acid just prior to and after saturation of the ion source can be addressed and illustrated by examining figures 3-11 and 3-12. Figure 3-11 depicts the m/z 87 ion in greater U3 U o .k-1 •♦-' »— < CO a> (A s •♦-* 60 B CO o > ■*-" ct bO c ■l-> a &o S .2 ^H O 007- Ph O 125 I en CM + -^ 00 n O N O oj + N 1.E 00 CM O o n (N m X N J. 1 E CN 00 ^ s 00 1 I o (N N 7 E 00 00 I t _ c^ O N in > CO t A ET in o CM in ^>» CO + N o s E o ^ X N I —*«• [n E _ £ =" cc • (U £ ■G ^ o re '^ ti — -a o v« re X) O flJ c -^ "g O i/i ^ t; iS o *^ T3 -J . 2 Si E .5 § o ^- JS 3 Q. O i« (U — J3 -O *- re ■w a; O J3 c re o re -. >- rsi ^ O - P ^ <*-' o« ; .5 5 <+-' ^ c « re « 3 h -fi <^ O ♦-■ ^JD ■n- 1.-0 (%) ^isua;ui 3Ai}BiaH C C 3 aj c 4-. t/3 "^ ca = i£ ■*-'■— C o -' O W <" J O ^ c = « E o X3 2 '-S « - C t/j 00 .2 "= J; • - ^ C •a ^ D '^ -^ o ^S 8 « <" o w ^ c re re o C^ E B o S^ IS 0) P3 S .2 c^ -«-t e ^ C T3 CO t^ 3 (U > (U > o n] .2 ^ m ^ 60 0) o ^ S;; on re 3 -a ^ 60-- *= tin re ? ■a o 133 o o 1/3 a p3pBj;;Y s9o;inbsoj\[ jo 98B;uaDJ3 j 3 H .2 fc, o O "2 := «! *^ o c -^ •— o "^ o (U *3 1 E cs O X *- S 5 -^ i^ ^ "^ TO -^ <.2 X • - rs '^ « o t2 = « T3 — cu •e o re t*H 'o <" O (1) IS *- -' .« ^ E 3 3 cr o" 0) U-i T3 135 + C3 1^ + X A 1/3 C/3 -X O- o I u I X U 1 CO X u 2 < 136 involved the volatilization of lactic acid to the gas phase via desorption. This is a necessity for subsequent mass spectrometric analysis, as well as for mosquito detection of lactic acid which has evaporated off of the skin. This is the equilibrium represented by the double arrow between the aqueous and gaseous forms of lactic acid. Addition of heat to this system favors the volatilization of lactic acid. Oligomerization reactions of lactic acid in the gas phase were discussed previously. This process can also occur in the condensed phase as represented by the double arrow leading from lactic acid to (HA)„ (s,l). This process occurs at high concentrations of sample in the ion source. There is an acid dissociation equilibrium in the condensed phase governed by this K^ between the associated lactic acid and the dissociated form (free hydrogen ion and lactate ion). In the dissociated form, the lactate ion is not susceptible to volatilization; thus, detection of lactate by mosquitoes via olfaction cannot exist. Recalling LeChatlier's principle, this system can be affected by the hydrogen ion concentration, [H^]. Upon acidification, the system will move towards formation of associated lactic acid, increasing volatility. The removal of free [H^] via addition of base shifts the equilibrium towards the production of dissociated lactate. This will decrease the free acid available for volatilization, thereby decreasing attraction via mosquito olfactory cues. There was an additional condensed-phase concern besides implications of acidAiase effects on mosquito attraction. This was the possibility of additional species produced by reactions occurring in the condensed phase. The possibility of 137 reaction between lactic acid and methanol via acid-catalyzed or base-catalyzed esterification was examined by mass spectrometry. There are two purposes behind the selection of mass chromatograms displayed in figure 3-15. The first purpose is the verification that masses corresponding to the protonated molecular species of lactic acid and methyl lactate are indeed present (in this case, under basic conditions). The additional mass chromatogram (m/z 73) displayed in the figure explains additional peaks found in the RIC which are not attributable to either lactic acid or methyl lactate. A fragment ion at m/z 73 is characteristic of siloxanes from GC analysis. The second purpose behind selecting these data was to illustrate a phenomenon which can occur for these compounds. The data were acquired under El conditions; however, a significant abundance of the protonated molecular species occurs rather than the molecular ion. It was previously mentioned that lactic acid does not form a molecular ion under EI conditions; however, the formation of [M-l-H]^ is relatively facile by self-induced chemical ionization due to high ion source concentrations and pressure. Reactions of similar compounds at high ion source pressure under EI conditions support this supposition [82]. Verification that the peak found at scan number 320 is indeed lactic acid can be found in figure 3-16. The data for this figure were acquired under NCI conditions; this clearly depicts the characteristic chloride ion attachment and oligomer ions formed by lactic acid. Confirmation of the presence of methyl lactate by NCI is not possible since it does not readily form an [M-H]~ ion. o ^ o 1^ -a re c -= o S " (U I—. c aj ^ "S aj c c ^ o 2i 2'i- N O C3 G) o o l^p O 01 kO T >-\J O ■» ID ^ r.j iS> w <-D T rj o CO l.i.1 (%) /(^isnajni aAijBpH c o ^ J ^ o ^ o ^ c 5- (1) t^ o in -a c 3 o ■a 'a re t/5 re o .Si, O ^ .E - iS re ^ 00 1.2- <= o ^ re c ^ =" J re -a i > o o &« "^ ^ 't^ (U re u <4-l C ^ o re fS Ol nj "" ^ > O t, o re -^ E "3 K 2 SO t/5 > O 2 ^ ^ c a> .t; ^|l r^ -c :^ CO ^ 3 C (U o o — cu'*: fr ° o 144 (%) .{jisnajni aAijep^ o c cd ,£3 ■M lU a J :± M C "* rH ts c« «4-l o n o ■ (-H •M o (U "h" • ^^ J dL m c> < ^ o a RJ X ^ (U s c •^H 0) ■*-» ct ♦J u ^ ^^ >. J2 -*-» (U s -o >-i c« -a a ee ■*-» W3 "O <4-l u o N _>> E "re 3 c ki re •♦-' O (/5 u re a, 1/5 ? (A on «3 '4-1 6 3 HH 0 U , 3 W) -a o a a 3 •a ~ 1^ p ^ *-' b " O re ^ -^ E E o "^ re ^ I— I ^ -a rj bo.x ^ -IS =« o p .ii r^ 5 -^ 1 o o f^ i3 re O t/) — on 5 -J t/5 g bo re o £ E -S (U 151 V) 00 "3 fv- o c '3 03 3 J: IS IS '^ u a o X! s 3 z c fl ■3 (J 5J 173 SI •■s o CT IS O o IS iS IS ■s S s 03 kO ■» r\) o OD ko T CxJ (%) AlSUa^UI 3AIJBPH 152 ions which normally dominate the RIC trace were significantly reduced in intensity. Shown in the bottom mass chromatogram is the trace for the m/z 89 [M-H]~ ion of lactic acid. It is evident that the intensity of the lactate ion is reduced when compared to mass chromatogram of m/z 89 analyzed from rubbed beads. Implication to attractant origin The relation of this work to semiochemical studies was discussed in the first chapter. The work with methanolic solutions of perspiration directly overlaps with analysis of perspiration for odor determination. Analyses for odors focus upon emanations from three types of glands found on the human body, the sebaceous glands, the eccrine glands, and the apocrine glands [30]. Sebaceous glands are mainly found on the face and scalp. These glands excrete sebum, which contains cholesterol, alcohol esters, and fatty acids [5]. Eccrine glands are located over most of the human body and are most concentrated on the forehead, the palms, and the soles of feet. Apocrine glands are found mainly in the armpits and in the genital area. Eccrine and apocrine excretions are sudoriferous in nature. These excretions are mainly water and sodium chloride. Traces of metabolites from blood plasma may also be present in this solution [5]. Excreted sudor initially has no odor; it is the interaction with bacteria on the skin which produces the acidic odor. The main contributor to this odor has been identified as isovaleric acid [30]. Additionally, two steroids (17-oxo-5a-androstan-3a:- yl sulfate and 17-oxo-5a-androsten-3j3-yl sulfate) as well as 2-18 carbon aliphatic acids were found to be present [30,31]. It should be noted that these results were 153 obtained with sample concentration, whereas analyses in this dissertation were conducted without sample concentration. The existence of lactic acid in the oily phase implies that it preferentially resides in this phase. Handling glass beads appears to favor the deposition of oily or waxy emanations over aqueous perspiration. Perspiration on the skin tends to be very dilute [29]. It also exists above the layer of sebaceous excretions in order to readily evaporate. The absence of lactic acid in this phase supports the assertion that excess perspiration may mask lactic acid attraction. Additional attractants may also be masked by excess perspiration, implying that they also exist in the oily phase on the skin. The origin of most oily excretions are sebaceous glands. Therefore, the deduction from this work is that attractants, whether sebaceous in origin or not, tend to preferentially reside in the sebaceous phase rather than aqueous perspiration phase. Conclusions Lactic Acid Reactions The negative ion fragmentations, attachments, and oligomerization were examined and rationalized. Analysis of negative ions from lactic acid provided informative fragments allowing for quick identification of the presence of this compound in a sample; lactic acid is difficult to identify by EI library searching. The oligomerization reactions in the gas phase were found to be similar to solution- phase reactions. TTie formation of these oligomers (in the form of dimers, trimers, 154 tetramers, and esterification products) produce ions which allow complementary confirmation of lactic acid presence. Additionally, the chloride attachment ions can be used for confirmation of lactic acid. Altering Attraction The addition of acid or base to lactic acid solutions was found to alter the attraction oiAe. aegypti to the sample. These effects presumably occur through the acid dissociation equilibrium. Addition of acid shifts the equilibrium towards the formation of the volatile associated acid form. Addition of base shifts this equilibrium towards the involatile dissociated lactate anion. Examination of similar solutions to those employed in the acid/base lactic acid study revealed that methyl lactate was formed while in methanolic solution. Subsequent testing of lactic acid esters and a similar ester revealed that lactic acid provides greater attraction than the esters. It was also found that addition of acid enhanced mosquito attraction in all cases. Origin of Attraction The data presented support the view that excess perspiration masks attraction by hindering volatility off the skin of lactic acid and other possible attractants. These data also show that the lactic acid signal from direct dissolution of perspiration in methanol is diminished compared to analyses with one or more glass beads. This confirms the very dilute nature of perspiration, and that handling glass transfers a 155 greater amount of components of interest in this study. Lactic acid preferentially resides in the oily sebaceous phase. This supports the assertion that other attractants, also susceptible to masking by excess perspiration, reside in this phase. CHAPTER 4 APPLICATIONS OF TANDEM MASS SPECTROMETRY Introduction Tandem mass spectrometers contain a second mass analyzer not found in conventional single-stage instruments. This extra stage adds an additional dimension to the selectivity of analysis via fragmentation by collision-induced dissociation (CID) [33,56,83]. Tandem mass spectrometry has been demonstrated as an effective tool in the determination of moth pheromones as well as shown to be beneficial in the continuous monitoring of volatiles via the use of a membrane inlet system [84,85]. These examples utilize the capability of the tandem mass spectrometer to perform selected reaction monitoring (SRM). The work in this dissertation differs from these in that MS/MS is applied for compound identification rather than monitoring specific compounds. Additionally, the volatile compounds to be identified are relatively low in relative molecular mass and thus are more amenable to CID than high molecular weight compounds [86]. The applications of MS/MS to this project have been categorized into three sections. The first section addresses the use of daughter spectra for compound identification, both by spectral interpretation and by comparison to standards analyzed under similar conditions. The second part concerns the formulation of a 156 157 daughter library to aid in spectral interpretation and in screening for compound classes via examination of specific neutral losses. The final section examines the use of selected neutral losses for rapid screening for compound classes present in the emanations of a human subject. Analysis of Multiple Beads Without GC Separation Tandem mass spectrometry was initially employed in this work for the identification of compounds desorbed from multiple beads in a glass chamber. The lack of GC separation and poor temporal resolution achieved by simple desorption of volatiles off of glass beads necessitated the use of some means of additional selectivity (see Chapter 2). The use of a second stage of mass spectrometry provided the means for mass analysis of one selected mass of interest; however, similar compounds which fragment to yield the selected mass, or compounds with identical parent mass to the selected ion, complicated the mass spectral interpretation. This made GC separation attractive for further identification of components emanated from the skin. Daughter Library The focus of the second part of this chapter is the compilation of a daughter library under a set of conditions chosen to be constant throughout this work. The collision cell offset, collision gas, and collision gas pressure affect the degree of fragmentation via collision energies, collision processes, and numbers of collisions 158 [83,87,88]. Therefore, the set of conditions for the daughter library and subsequent analyses need to remain constant throughout to obtain reproducible daughter spectra. The daughter library allowed for determining the specific daughters and neutral losses for particular compounds and compound classes. This information provided the basis for additional spectral interpretation of daughter spectra as well as provided the information on specific neutral losses associated with compound classes. An additional note of interest is the extensive examination of negative ion fragmentations, as well as those of more typical positive ions, used in this dissertation. Analysis of compound classes by positive ions remains more prevalent than the use of negative ions [89-91]. There are some cases, e.g. for ethers and carboxylic acids, where negative ion analysis yields better results than positive ion analysis [92-95]. Compound Class Screening The final part of this chapter is focused on applying the information on neutral losses obtained from the daughter library for rapid compound class screening of samples of human skin emanations which have been desorbed off of handled glass beads. The actual screening employs cryo-focusing of desorbed volatiles and GC separation. The sample introduction used for MS/MS screening by neutral losses is also employed for almost all work pertaining to compound identification by GC/MS (addressed in Chapter 5). 159 Experimental Analysis of Multiple Beads Without GC Separation Five beads handled 5 minutes by the author of this dissertation were placed in a 1/4" glass tube (maximum 25 beads), which was inserted into the apparatus described in Chapter 2 (figure 2-3). Helium, at 6-8 psig, was passed over the beads and used as the carrier gas through a 1.0 m x 0.10 mm i.d. deactivated FSOT column. The GC oven (holding the glass tube) was held initially at 28-30°C for 1.0 min, followed by a 12 min ramp at 15°C/min to 207-2 10°C, with a 5 min hold at the final temperature. The transfer line was concurrently ramped from 50°C to 210- 215°C at 20°C/min once the run was started. Sample ionization was effected by positive ion CI or negative ion CI with methane reagent gas at an indicated source pressure of 1687-1700 mtorr. The ion source and manifold temperatures were set at 150°C and 70°C, respectively. The electron energy was set at 100 e V with the filament emission current at 200 ^l A. The parent ion was selected to be passed by Ql to the collision cell for fragmentation and subsequent daughter spectrum analysis. The collision gas was argon at an indicated pressure of 1.4-1.5 mtorr for positive ions and nitrogen at 1.97 mtorr for negative ions; the collision energy was set at 15 eV for positive ions and 9 eV for negative ions. The electron multiplier setting was -1200 V with the conversion dynode at -5 kV for positive ions and 4-5 kV for negative ions. Data were acquired with Q3 scanning at one s per scan for positive ions and 3 s per scan for negative ions. 160 A standard of ethylene glycol (250 ^L) was diluted to 100 mL with methanol. A 0.5 p,L injection of the solution was made onto a 10.5 m x 0.178 mm i.d. DB-5 FSOT column (df=0.4 p.m). The GC injection port temperature was set at 250°C; the helium carrier gas head pressure was set to 4 psig. The column oven was ramped after an initial 1.0 min hold at 30°C, up to 210°C at 15°C/min, then held at 210°C for 5 min. The transfer line was concurrently ramped from 50°C to 215°C at 20°C/min once the run had been started, and held at 215°C for the remainder of the analysis. Sample ionization was effected by PCI with methane reagent gas at an indicated source pressure of 1700 mtorr. The ion source temperature was set at 150°C and the manifold temperature set at 70°C. The electron energy was 100 eV and the filament emission current was set at 200 p,A. The collision gas was argon at an indicated pressure of 1.5 mtorr in the collision cell; the collision energy was 15 eV. The electron multiplier setting was -1200 V with the conversion dynode at - 5 kV. The daughter spectrum was acquired at a scan rate of 1 s per scan. Daughter Library The data comprising the daughter library consist of data tabulated from analyses of 40 standard compounds. The compounds differed in functional groups (some containing more than one functional group) as well as including isomers for comparison within a specific class. For solid-phase standards, approximately 50 mg of the standard compound was dissolved in methanol in a 100 mL volumetric flask. 161 For liquid-phase standards, approximately 250 ^L was diluted in methanol in a 100 mL volumetric flask. The standards employed were acrolein, acetone, acetic acid, ethylene glycol, pyruvic acid (sodium salt), DL-a-alanine, oxalic acid, glycerol, benzaldehyde, anisole, malonic acid, acetophenone, sec-butylamine, 2-butenal, 1,3- butanediol, 1,4-butanediol, benzoic acid, cyanoacetic acid, benzyl alcohol, benzonitrile, benzamide, 1-butanol, 1-decene, diethylene glycol, Ai-decane, dodecane, L-leucine, diethylamine, glycine, tetradecanoic acid, 2-butanone, phenol, styrene, 1- pentanol, 2-pentanone, vinyl acetate, sebacic acid, octadecanoic acid, tartaric acid, and toluene. Each standard solution was injected (0.5 ^iL) onto a 10.5 m x 0.178 mm i.d. DB-5 FSOT column (df=0.4 ^m). The GC injection port was set at 250°C and the helium carrier gas head pressure was set at 4 psig. The column oven was ramped, after an initial 1.0 min hold at 30°C, up to 210°C at 15°C/min, then held at 210°C for 5 min. The transfer line was concurrently ramped from 50°C to 215°C at 20''C/min once the run was started, and held at 215°C for the remainder of the analysis. Sample ionization was effected by PCI and NCI with methane reagent gas at an indicated source pressure of 1700 mtorr. The ion source was set at 150°C and the manifold set at 70°C. The electron energy was set at 100 eV and the filament emission current at 200 ^lA. The collision gas was argon at an indicated pressure of 1.5 mtorr; the collision energy was 15 eV. The electron multiplier was set at -1200 V and the conversion dynode set at +5 kV for negative ion detection and -5 kV for positive ion detection. Daughter spectra were acquired at the rate of 1 s per scan. 162 Each standard was injected and analyzed three times in order to obtain daughter spectra for the [M+H]"^ and [M-H]"^ ions in positive ion mode, and the [M-H]~ ion in the negative ion mode. Compound Class Screening Volatiles desorbed from 8 glass beads rubbed in the palms of the hands for 5 minutes by the author of this dissertation were analyzed by cryo-focused GC/MS. The beads were placed in a reversed fritted injection port liner and replaced into the GC injection port (set at 25°C). The cap, septum, and needle guide were replaced on the injection port. Liquid nitrogen in a 12 oz. styrofoam cup was placed in the column oven and approximately 8 cm of column (starting approximately 15 cm into the oven) was placed into the cup, as described in the experimental section on cryo- focusing in Chapter 2. The helium pressure was increased from 0 to 20 psig before initiating the cryo-focusing of desorbed volatiles. The cryo-focusing consisted of a 20 min injection port ramping program downloaded from the TSQ70. The program consisted of a ramping the injection port from 25°C to 250°C over 7.5 min and then holding at 250°C for 12.5 min. Throughout cryo-focusing, the GC oven was set at 25°C and the transfer line was set at 40°C. Liquid nitrogen was added to the cup as needed to compensate for evaporation. After the cryo-focusing stage was completed, a new program was downloaded to the GC. The cup containing the liquid nitrogen was removed and the analysis started. The GC program consisted of an initial 1.0 min hold at 40°C, an 18 min 163 ramp at 10°C/min up to 220°C, and then a 16.0 min hold at 220°C. The transfer line was concurrently ramped, at the onset of the program, from 50°C to 220°C at 20°C/min for 8.5 min, then held at 220°C for the remaining 27.0 min of the analysis. The experiments were conducted with by PCI and NCI with methane reagent gas at an indicated pressure of 1680 mtorr. The column employed was a 25 m x 0.20 mm i.d. (df=0.33 p,m) HP-FFAP FSOT column. The scan rate for all neutral loss scans was 0.5 s per scan over m/z 50-650. Each analysis consisted of examining three different neutral losses, controlled by a program written in ICL language on the TSQ70. The program allowed for incrementing the neutral loss data acquired with each scan such that a specific neutral loss was sampled every 1.5 s. The electron multiplier was set at -1400 V. The filament emission current was 200 jiA with 100 eV electron energy. The ion source temperature was set at 150°C and the manifold temperature was set at 70°C. The instrument was tuned, at the beginning of each day, to optimize the transmission of daughter ions, in both the positive and negative mode, produced from the m/z 69, 219, 264, and 414 ions of PFTBA. Positive ion daughter spectra were acquired with -5 kV on the conversion dynode. Negative ion daughter spectra were acquired with -1-5 kV on the conversion dynode. Results and Discussion Analysis of Multiple Beads Without GC Separation The analysis of handled glass beads requires the analysis of a complex sample. It is estimated that there are typically over 350 distinct component peaks visible in 164 the cryo-focused GC analyses of 5-8 glass beads (as discussed in Chapter 5). The technique of simple thermal desorption both from a single glass bead mounted on a probe tip and desorption from multiple glass beads heated in a container in the GC oven was discussed in Chapter 2. The RIC profiles of components desorbed from multiple beads in the apparatus (figure 2-3) depict the leading edge of a selected m/z value when it appears; if the compound is volatile enough, the tailing side of the component peak can also be observed within the time frame of the experiment. The "peaks" appear similar to those observed in frontal chromatography; however, chromatographic separation is not employed for these analyses. Clearly, analysis in this manner will only allow for the possible identification of the more volatile components. The spectra of components desorbed at higher temperatures will include ions from components previously desorbed due to inefficient removal of compounds from the apparatus. The use of tandem mass spectrometry was necessary to isolate a particular mass for further fragmentation. The first example using this method is that of ethylene glycol (figure 4-1). Ethylene glycol desorbs off of the glass beads early in the analysis to produce an ion at m/z 63, the [M+H]"^ ion, under PCI conditions in the ion source of the mass spectrometer. Positive identification of this compound was achieved by comparison of the daughter spectrum from handled glass beads (top of figure 4-1) to the daughter spectrum produced by a standard solution of ethylene glycol (bottom of figure 4-1) acquired under identical conditions. u (u n > «! IT) e en t/3 -DO) ^ r-< Ui o .£ -a = 3 C CI, c o V C/2 t/3 00 c re « .2 E N re CO re 60 ^ o t_ o U + 60p — c ^-^ «1 "" ^ ■1-' fC _ *-' £ re re i' o <*-! (1) o a. c '^ re £ it ^ -a ■* (U 60 K o 6 o o r~ o ^ 3 i) ;« !X> 3 ^ O t/i i« re -a ^ re 60 ^ c re .2 ^-l en re ^ o u i-c 6o£ H 166 a. ^ ■a o 1> (U -^-* ■*-» JH (A 3 E DO (/I r- 3 re OS re Q t/i 1— t (4-1 1 re o -4-» 3 Urn - o C u •" 3 - i/J CO Cfl ° i - s s ^ 3 -a >. ^ « I s-^ *^ ^- c c o re kN re > o E X 2^ 7^ C CU 1/2 j= Q re Q re x; Q re < Q re < re V2 (N Q (N Q c/2 rj Q rsi Q fti ■4—1 1/2 O E and m/z 90-100% t/i o *^ re c c/3 a> e in C 1-H +1 C +1 CO B +1 ^ B 1— 1 +1 •i: E aJ 5 _(/2 re ^ re -* CO re ^ ^H re 'S- ly at m/z neutral 1 "re )-i 3 1) C lU > ■a B _o B N "e XJ ■a b* ^ ^7i c^ s usual f small 'to (4-1 ^ ^ 1*^ '!^ D- ^ 'iJ2 _o '*^ 1/2 Cu o Z "re E on "re 3 * > ' > * > ' ' 1 fc- o c re o Table 4 in the c Parent [M-H]- Comp Class Aiken (10 or Alkan (10 or (U T3 < 178 T3 U 3 _C ■*-» C o o re H c/3 C/3 r- '% re r~- CO CO -^ U o ce S c/5 re _c < .23 y. o j: Oi ^ ^.s i a re re C/3 1/3 o < Oi C/3 0) CO < .2 A -*,^ ^oi c ^ U omp lass ^ 8 (U O •a -t-" "^ u u < ^ 179 U 3 C o u 3 re r% 1— t (/3 ■4— > 3 I. kl re o C lo (A C/3 c in c CO t/5 CO re re re o\ ^ re en •E u re '"' 4= CO re E ^ Q Q i/~i ON ha •^ a\ -c ir ■♦^ l-l ^o \o o o O O en m N f^*^ N T—H E kH re 1 XJ 3 is locatei 0% RA) -5 -a c re 3 < o in O- co CO — o in re f 18 Da triols is N E re c _2 l-l ■*— » 00 3 re ■o o 1 m re f 18 Da triols is 1 at m/z W3 O ■*-» (/) ■*— » o re 10 or more se daughter i o E c re Q 1 o CO (U 00 3 re ■o re o c o o re gnificant neutral loss o ase peak for diols and resence of daughter ior re U or acids of with intens re t/3 "re 3 CO CO _o "re 3 <4-i o aj o c CO o CO "3. re o t/3 c •4—* •4— » in in in oC VO eutral loss eutral loss o re '^ = a. c c < o 1) < ^ re k, 0, ngin Liently 5 and a, o < re a. 3 U k. <4-i 3 O" < re X) 3 cr (U esulti ; freqi m/z 6 u re X) 1^ c 00%; R nt ions RA O re 00% R nt ions at 1-2: a: o o CO re X! OJ .4—1 u 0 3 0 c ^ 1/-) re • — ^i OJ re re . o o 3 t ion is at 1 Da from p 3 ion at 1-2 t ion is at 1 Da from p 3 daughter re 0 3 ■a 0 "re 3 c .0 k. N typic -2D; ghtei W3 1/3 C o o- re Q c o V ■*- 00 3 re -a oo 3 re •a 00 c 0. re Q ^■4 0,1 1 lU -*-» ^~' re 8 E •4-< re 1— 1 X re c lO «^ OJ ■I-' % ■a c c 1—1 On c 3 e re vn N E le base is due E re X < < E re X (U c« N "e re 73 en O Oh en V U U l-H Pi 189 uT in a r- a NO J J J ^ H a 3 w Z II O — - ■3 ^ _'3 j L| S 4J C C/5 IfS ■ 2 -•SI s ■3 0 0 a 0 .3 I IS ■7J ■D T ^J (%) ^JlSUajUI 3AIJB13H 190 displayed in figure 4-6. The mass spectrum contains the m/z of ions passed by Ql into Q2, which fragmented by collision-induced dissociation to form ions detected by Q3 as 18 Da lower in mass than the ions transmitted by Ql. The actual assignment of the m/z 199 to be the [M-H]^ ion of dodecanoic acid could only be speculated at the time these experiments were conducted. The confirmation, both by GC retention time and by PCI and EI spectra is located in Chapter 5. Note that in addition to the [M-H]"^ ion, PCI of dodecanoic acid produces a number of other ions which undergo a neutral loss of H^O, including the [M+H]+, [M-H-(CH2)„]- [M-H-HP]" and [M- H-H20-(CH2)J~ ions at m/z 201, the series of m/z 185,171,157, . . ., the m/z 181 ion, and the series of m/z 167, 153, 139, 125, . . ., respectively. The last peak in the neutral loss of 18 Da chromatogram (scan number 2780) is also presented in this chapter as figure 4-7. As with the previous case, knowledge of retention time as well as information from Chapter 5 aids in drawing the conclusion that this peak is due to octadecanoic acid. This is the last visible peak in the chromatogram; therefore, additional acids of higher m/z in this series are either absent or are at a trace level not detectable by these experiments. The identification of the peak corresponding to lactic acid was straightforward, even without confirmation from GC/MS. Lactic acid has been used throughout this dissertation as the model or target compound for various applications due mainly to its characteristic being the only previously known attractant to Aedes aegypti. Additionally, it is typically the most abundant compound found emanating from the skin throughout the work for this dissertation. The neutral loss spectra examined in a. a. ct cd Q 00 T-H VM o tf2 Q ^^ ^_( rt ;^ "^-t ■d 3 lU B re (rt o (/5 'o U c o re 60 o b 3 60 J £ c 192 in (%) yC^isnajni aAijBp^ «! J= -*-' uo B 3 ^ ■*-* O n o 1 o o 3 3 .£P O iC •^^ c + O X OO 1 1-1 X3 (U E o 3 •^j C T3 C S!2 re 3 Q .2 CO ■♦-» re <4-l 8 o •^M E re o X U ,^ re r- i-i •4-* "4 3 i-i 3 3 re OO P-i o 194 + — Si in iS -iS X I CO eg- • in CM in ;_j. CO 00 m —> cr>_ '•■I 00 IS) 'j3 1^ CM N a i5 -tSi : — cs (%) iCjisnajni aAijBia^ 195 this section exhibit the most abundant peaks to be those of fatty acids rather than lactic acid. It is known from previous work in Chapter 3 that lactic acid will undergo a neutral loss of 18 Da (H2O) from the [M+H]"^ ion. Therefore, the presence of an ion at m/z 91 in the neutral loss (of 18 Da) spectra from these analyses should be readily be observed. Figure 4-8 reveals the location of what is suspected to be lactic acid in these experiments. This can be confirmed by comparing the retention times to those acquired under the same GC conditions in Chapter 5. It is interesting to note (from the RIC trace in figure 4-8), that lactic acid appears to be less abundant than the fatty acids. The experiments for figures 4-5 through 4-7 were conducted in December, a relatively cold month. Therefore, lactic acid may not be as prevalent on the skin as it would be were the temperature warmer. These experiments were repeated a month later under warmer weather conditions as well as after physical exertion to produce a greater amount of perspiration on the skin for subsequent sampling. Comparing the RIC for a neutral loss of 18 Da in figure 4-9 (bottom trace) to that of figure 4-8 (bottom trace), it is quite evident that lactic acid is the most abundant compound in this second sample. Included at the top of figure 4-9 is the chromatogram for the neutral loss of 28 Da (CO) from the m/z 91 ion, additionally providing confirmation that this is lactic acid. The final figure (4-10) confirms this; this is the neutral loss of 44 Da mass spectrum in the negative ion mode from the peak centered around scan 1480. Present are the [M-H]~ ion of lactic acid at m/z 89, which loses CO2 to form m/z 45, as well as the u OS N s, a o + 3 'o -5 n o 9^ U) o u « Q 00 ■t-l t-l O :S o u Cl, u C3 E ^ CO ■^-» 10 U H l-l 3 ftS M (U E -4— > 197 T <0 + OS •jj in •S IS + >J5 a e W e s s z .A a 3 z a 58 o o O O (S IS s> o IS" o CO '£■ T Cxi o CO IX) T f>J (%) /Cjisnajni aAijcp^ o o re a o as + + 6 o Q oo t/5 O « 3 C D o lU CO .2 S > o ■*-• re o -a ^ 'o CI- «5 g •— c« ■*— ' o CT O O ■s o •3 O o o: 'XI •3- ■-J f^ CO ',D T '^J (%) i()isn3)ni dAi^epH u CO _c -3 a o l-l l-l o u -a o B c _o > c« MX~' associative resonance capture 5-10 e,h~ + MX ^» M + X~' dissociative resonance capture 5-11 ep~ -I- MX ^ M"" -I- X~' -I- e~ ion-pair production 5-12 The common denominator of these processes is the capture of an electron by a species (MX). Ion-pair production (Eq 5-12) is a special case and will be explained later. In order for electron capture to occur, the electron affinity (EA) of MX must be positive. Provided the species does not rapidly undergo autodetachment of an electron, it can proceed through one or more of the processes given in equations 5-10 through 5-12. Associative resonance capture (Eq 5-10) involves the capture of a thermal electron, forming an excited species in which the excess energy can be distributed over the molecule without bond rupture [72]. Studies have shown associative resonance capture to be observed when electron energies are in the range of 0 to 2 eV [72,76,108]. Dissociative electron capture (Eq 5-11) occurs when the electron captured species contains sufficient excess internal energy to cause the rupture of one of more 214 bonds. This can result if the excited MX species makes the transition to an attractive state (forming both MX~' and M+X~'), or makes the transition to a repulsive state (forming M+X only). The fragment retaining the negative charge in these equations is the fragment that has the greater EA. Dissociative electron capture is typically observed for electron energies of 2-15 eV. The extent of fragmentation for dissociative capture and ion-pair production (below) is dependent upon the internal energy of the ion and thus the incident electron energy. The internal energy can also be influenced by temperature; increased source temperature has been shown to produce an approximately linear increase in fragmentation [72,74]. Ion-pair production (Eq 5-12) occurs for electron energies greater than 15 eV. In this case, the intermediate is more often an excited molecule rather than an MX species [72]. The primary electron (Cp") functions to excite MX to (MX*) which then dissociates to form the ion pair. In the absence of reagent gas, a conventional 70 eV electron beam yields approximately 1000 times more positive ions than negative ions [76]. Additionally, the negative ions are typically only low mass fragment ions or "molecular debris." [72]. Normally, the sensitivity with respect to detection of informative fragments of negative ions under these conditions is poor. In cases where an MX~' is produced of sufficient intensity to be observed, it can be explained by the production of secondary thermal electrons. That is, the species captures the primary electron, undergoes autodetachment as well as releasing a low energy secondary electron of near-thermal energy to form and M"^' ion. The secondary near- thermal electron can 215 then react with a neutral sample molecule via associative or dissociative resonance capture. Therefore, it is necessary that a suitable reagent gas is used to produce thermal electrons for ECNCI to take place. It is estimated that the rate of capture of electrons by compounds amenable to EC is approximately 400 times greater than for CI ion/molecule reactions; thus, enhanced sensitivity is observed for these compounds [72]. The compounds amenable to ECNCI are generally oxidizing or alkylating agents [107]. The molecular anions of these compounds were found to be stable if a "low-lying" unoccupied molecular orbital is available, or if the molecule can be stabilized through TT-system resonance [72]. It is best stated that the more "molecular debris" and less MX or [M-H]~, the less resonance stabilization is available to the molecule. Generally, the best systems or molecules for EC are extended 7r-systems with strong electron withdrawing groups. There are additional constraints as well as differences between the characteristics of negative ions and positive ions in the gas phase [72]. Each positive ion fragment can carry a charge, whereas the EA must be greater than zero for negative ion fragments to be present as an anion. The probability of anion formation is approximately a linear function of the EA of the neutral species. In positive ion analysis, the fact that the filament does not produce monoenergetic electrons nor does it significantly alter the fragmentation within the standard operation regime of filament electron energies (70 eV). The lack of monoenergetic electrons in the negative mode allows for multiple reaction processes to occur. The autodetachment 216 process competes with fragmentation in the negative ion mode; therefore, simple bond cleavages will be favored over more complex rearrangements. Due to autodetachment, only ions with low energy and relatively long lifetimes will be recorded [72,107]. Positive ions can retain this charge for longer times and can only lose a positive charge via abstraction of an electron or proton from collision with a neutral [107]. Despite these aforementioned limitations, ECNCI can still provide information complementary to or not accessible with PCI. The CI reagent gas can function in three ways for negative ion analyses [72]. It can produce thermal electrons for EC processes described above, produce charge transfer reactions (Eq 5-13) provided the EA of the sample molecule is greater than the EA of the reagent gas molecule, and it can allow for true CI reactions (Eq 5-14 and Eq 5-15) between the reagent ions and sample molecules to occur: X-' + M ^ M-' + X charge transfer 5-13 [X-H]~ + M -> [M-H]~ + X proton abstraction 5-14 X~ + M ^ MX- adduct formation 5-15 The term NCI is commonly used to indicate ion/molecule reactions between the reagent gas and sample. Electron capture reactions are usually referred to as ECNCI since these involve a different process than NCI ion/molecule reactions [76]. The problem with many reagent gases, including methane and isobutane, is that the spectra produced are difficult to classify as either ECNCI or NCI [76]. When using lower energy electrons, an [M-H]~ ion is typically observed in place of MX . In 217 some cases, this can be explained by high EA of the resulting anions or high bond dissociation energies of the C-H and O-H bonds. In this dissertation, the distinction is not of particular importance. What is sought is the MX ion or the [M-H]^ ion in order to deduce the r^ of the compound. The analysis of NCI spectra consisted of determining the m/z of either the MX ion or the [M-H]~ ion, regardless of whether generated by ECNCI or true NCI, in the mass spectrum to aid in the determination of relative molecular mass. Additional confirmation came from the presence of the following ions: [M+CH3']~, [M-H+CH3]~, and [M+CjHs]^ [76]. Additionally, manual inspection of mass spectra for compounds suspected of being esters or alcohols was conducted; esters fragment to yield the carboxylate ion while alcohols yield an [M-H-Hj]" ion [72]. Characteristic ion fragmentations (EI) Electron ionization generally employs a distribution of electron energies centered around 70 eV emitted by the filament in a low-pressure ion source. These conditions maximize fragmentation and structural information. As described above, negative ions formed by EI are low molecular weight fragments of quite low intensity. In the positive ion mode, ions retain their charges longer and can distribute excess internal energy more efficiently, thus undergoing less fragmentation. The purpose of this introduction to EI is not to provide a complete set of rules for EI fragmentation; these may be found elsewhere [79,109]. For the most part, EI mass spectra acquired in this dissertation were evaluated by library searching on the TSQ70 with a NBS library database of over 49,000 compounds. In some 218 cases, library searching failed to provide a reasonable compound choice for the unknown mass spectrum. When this occurred, r.^ information obtained from CI analyses was relied upon to further restrict the search. If this still failed to produce a reasonable selection, the mass spectra were manually examined. This involved a quick inspection for characteristic compound class fragments or McLafferty rearrangement ions. This information was then employed and library re-searched with new constraints. Compounds which remained unidentified are not listed in the tables contained throughout this chapter, although speculative identifications are listed and noted. The remainder of this introduction will address the characteristic fragments and McLafferty rearrangement ions which were examined in the interpretation used in this dissertation. Aliphatic acids show a characteristic McLafferty rearrangement ion at m/z 60. This rearrangement can occur for compounds with an available y-hydrogen and unsubstituted a-carbon [79,109,110]. For EI GC/MS analysis, mass chromatograms for this ion were examined to determine the location of the carboxylic acid series from propenoic acid up to octadecanoic acid. Fragment ions corresponding to [M- IS]"^, [M-28]"^, and [M-45]'^ were fairly common in these mass spectra. The possible presence of amines was determined by examining the mass chromatogram for an ion at m/z 58. Esters produce an m/z 74 ion when methyl substituted and an m/z 88 ion when ethyl substituted. Mass chromatograms for the m/z 58 and 74 ions were also examined in almost every case. 219 Other characteristic ions or fragments were not examined unless an unknown mass spectrum failed the library search. In general, examination of isotope peaks for halogen-containing compounds was not necessary due to the library database readily identifying the halogenated compounds. Mass spectra which the library targeted as sulfur-containing compounds were visually inspected for the presence of a loss of 34 Da (characteristic of thiols) as well as the presence of the series m/z 47, 61, 75, 89, .... Mass spectra of suspected aldehydes, ketones, or aliphatics were examined when needed for the presence of the series m/z 15, 29, 43, 57, . . . as well as for losses of 1, 18 and 28 Da from aldehydes. Mass chromatograms were examined for the McLafferty rearrangement ion for aldehydes at m/z 44, as well as for ions at m/z 58 and m/z 72, which could be indicative of a methyl and ethyl ketones, respectively; however, it should be noted that the m/z 58 ion also results from amines. Alcohols of five or more carbons display [M-IS]"^ and [M-46]'*" ions; however, acids may yield these ions also. Instead, analysis of the corresponding peak in NCI for the presence of an [M-H-Hj]^, as described in the NCI section, was employed for suspected alcohols. Mass chromatograms for the m/z 77 ion or cluster of ions within 2 mass units were examined to determine the presence of aromatics. Mass spectra were examined for an [M-27]* ion when an amine was suspected, as well as the even ion series of m/z 44, 58, 72, 86, . . . which is characteristic of amines. 220 Experimental Identification of Emanations by CI and EI MS Cryo- focusing GC/MS The results from these analyses are tabulated later in this chapter. The list of components was derived from the analysis of cryo-focused volatiles from handled glass beads. The cryo-focusing process involved heating of beads in the GC injection port to desorb volatiles. A Varian 3400 GC fritted glass injection insert was inserted reversed into the injection port. This allowed for up to 12 beads to be placed between the frit (located approximately halfway down the insert) and the GC septum sealing the injection port. The frit kept the beads from dropping down onto the column entrance and provided a means for volatile emanations to be loaded onto the column; the column entrance extends up into the injector insert and is located below the frit. Additionally, the injection port was operated in an entirely splitless mode for the duration of the experiment providing essentially only one exit for volatiles, i.e. through the column. Beads were rubbed for 5-15 min prior to placing into the injector insert. The initial experiments employed eight beads handled by the author of this dissertation; later experiments (discussed in the comparison sections) involved from two to five beads handled by Mr. Dan Smith, Mr. Carl Schreck, and Mr. Ken Posey, all of the USDA. After loading the beads into the insert, the insert was placed into the injection port held at 25°C to minimize sample loss from evaporation. The cap, 221 septum, and needle guide were replaced. Throughout the loading process the head pressure of helium was set to 0 psig to allow for proper alignment of the septum and prevent premature migration of volatiles past the intended point of cryo-focusing on the column. Before increasing the helium head pressure, liquid nitrogen was placed in a 12 oz. styrofoam cup. The cup was placed in the oven such that the approximately 8 cm of column could be looped in the cup about 15 cm below the point where the column passes from injection port to oven. The helium pressure was then increased to 20 psig and the initial desorption phase started. This entailed loading a program to ramp the injection port from 25°C to 250°C over 7.5 min and holding at 250°C for 2.5 min. Throughout the cryo-focusing phase, the GC oven was set at 25°C and the transfer line set at 40°C. Liquid nitrogen was added to the cup as necessary during this 10 min cryo-focusing phase. Subsequent to the completion of the cryo-focusing, a new program was loaded from the TSQ70 to the Varian 3400 GC. Prior to running this program and concurrently acquiring data, the cup containing liquid nitrogen was removed. There were four different ramp programs used. The initial experiments employed a GC oven ramp used for the analysis of 8 beads handled by the author of this dissertation. The ramp consisted of an initial 1.0 min hold at 25°C, a 12 min ramp at 15°C/min up to 210°C, and then a hold at 210°C for 5.0 min. The transfer line was concurrently ramped, after a 1.0 min hold at 40°C, up to 225°C at 15°C/min, and held 5.0 min at 225°C (18 min analysis time). Separation was effected by a 20 m x 0.25 222 mm i.d. Carbowax column (df=0.25 jim). Analyses involving the comparison of subjects were performed with two ramp programs, depending upon the column employed. The columns were a 25 m x 0.20 mm i.d. HP5 FSOT column (df=0.33 ^im) or a HP-FFAP 25 m x 0.20 mm i.d. FSOT column (df=0.33 ^m). For experiments conducted with the HP5 column (35 min run time), the GC ramp consisted of a-1.0 min hold at 40°C followed by a 11 min ramp at 17°C/min, and then a hold at 220°C for 23 min. The transfer line was concurrently ramped from 50°C to 220°C at 20°C/min over 8.5 min and held at 220°C for the remainder of the analysis. The ramp program for the FFAP column (45 min run time) consisted of a 1.0 min hold at 40°C followed by an 18 minute ramp at ll°C/min to 236°C, and then a hold for 26 min at this temperature. The transfer line was ramped from 50°C to 236°C at 23°C/min for 8 min and held at 236°C for the remaining 37 min. The final ramp which was used is described in the experimental section covering the five- day comparison of bio-assay to GC/MS. Acquisition of PPINICI data employed methane reagent gas at 1660-1690 mtorr (indicated) pressure. The ion source and manifold temperatures were 150°C and 70°C, respectively for CI and HCC and 70''C, respectively for EI. The electron energy for CI experiments was 100 eV and for EI, 70 eV. The third quadrupole was typically scanned with a scan time of 1 s per scan for PPINCI and 2 s per scan for EI data (except for the five-day comparison study). The filament emission current was set at 200 ^lA. The conversion dynode was set at +5 kV for positive ion CI and EI, and -5 kV for negative ion CI. The electron multiplier was set between -1000 V 223 and -1200 V. Prior to analysis the instrument was tuned with PFTBA as previously mentioned and blanks (appropriate number of beads without sample) were recorded. Purge and trap GC/MS These experiments involving microscale purge & trap mainly employ samples from the author of this dissertation; two samples, one from handled beads and one involving placing the left hand in a Tedlar bag, were collected from Mr. Matt Booth of Environmental Science & Engineering (ESE). The bead method involved handling 200-250 glass beads for 10 min, and transferring them to a 100 mL round bottom flask, as discussed previously (Chapter 2). The round bottom flask was attached to the system via a 1/2" Cajon fitting to 1/4" Swagelok, further reduced to a 1/8" Swagelok fitting. Alternatively, sampling was accomplished by placing the left hand in a Tedlar bag and fastening the bag around the wrist with a rubber band; the Tedlar bag was simply attached by a 1/8" Swagelok fitting. The 1/8" Swagelok was connected to a port on an ELA2010 canister manifold (Entech Laboratory Automation). The canister manifold allowed for 70-100 mL of volatiles and residual air to be sampled by the ELA2000 concentrator. The concentrator consisted of three stages. The first stage employed a dryer with a gradient of large to small beads. This served to remove most of the water in the sample. During concentration, this stage is set to -160''C for three minutes and heats up to -16°C as sample is transferred to the second stage. The second stage in these experiments was a Tenax trap. During concentration, the trap was set to -20°C; it was then heated to 156°C 224 for desorption of trapped volatiles onto the cryo-focusing trap. The cryo-focusing trap was set a -160°C for concentration and heated ballistically (over 10 s) to 150°C to purge volatiles onto the head of the GC column. The GC employed was an HP5890 series I with a 30 m x 0.25 mm i.d. DB-1 column (df= 1 jim) for initial experiments. Experiments with the hand enclosed in a Tedlar bag were conducted on a 60 m x 0.25 mm i.d. DB-1 column (df=l ^m). The column was initially cryo-cooled at -35°C and held for 3.0 min. Subsequently, the column was either ramped at 127niin up to 180°C, then ramped at 257min from 180°C to 225°C and held for 5.0 min at 225''C, or in later experiments, the column was held 6.0 min at -35°C then ramped at 67min to 180°C and 127min from 180°C to 225°C, and held for 10.0 min. The mass spectrometer used for these studies was a Finnigan MAT Incos 50 single-stage quadrupole. The scan rate employed was 0.75 s per scan. The filament emission current was set at 750 ^A with an electron energy of 100 eV. The electron multiplier was set to -1200 V. The ion source temperature was set at 180°C; there is no heater for the manifold. Prior to analysis, the instrument was tuned with PFTBA and the appropriate blanks were analyzed. Case Study Comparison of Emanations between Subjects A series of experiments were conducted comparing emanations desorbed off of beads handled by Mr. Carl Schreck and Mr. Dan Smith. Mr Schreck, in entomological assays via an olfactometer, consistently provides relatively low 225 attraction of mosquitoes to handled glass (c. 25%). Mr. Smith provides the highest attraction percentage on a consistent basis (c. 70%). Samples were analyzed in sets of two similar analyses, each involving beads handled by the two different subjects. In the morning session, five blank beads were analyzed prior to the arrival of the subjects. After arrival, 10-12 beads were handled by the first subject. A chosen number of beads, from 2-5 beads, were sampled immediately after handling. The number used remained consistent throughout the day. The remainder of the rubbed beads were placed in a 1/4" diameter tube at room temperature and open to ambient air. Fifteen minutes before the completion of the first analysis, the second subject handled 10-12 beads. Between 2 and 5 beads, consistent with the previous analysis, were sampled immediately; the remainder were stored in a second tube under the same conditions as previously described. TTie morning session involved either PPINCI or EI as the mode of ionization. The mode chosen to analyze at the beginning of the day was alternated for subsequent days. The TSQ70 was switched over to the complementary mode for the afternoon session; i.e. if PPINICI data was acquired in the morning session, EI data were acquired in the afternoon session. An analysis of 5 blank beads was performed prior to the return of the subjects. The subjects repeated the handling procedures. The beads were sampled and stored as described above for the morning session. After these sessions, the acquired data consisted of PPINICI and EI analyses from each subject. The analyses were repeated in the evening, with the same ionization mode used in the afternoon session, but with beads handled in the morning session. This 226 allowed a 6-8 hour gap between handling time and analysis time. After each sample from the morning session was analyzed, the beads stored from the afternoon session were analyzed under the complementary ionization mode. The GC parameters were dependent upon the column employed. The columns and corresponding GC parameters were discussed previously (in the cryo- focused GC/MS section of this chapter). The mass spectrometer was operated in EI and PPINICI modes as previously discussed and reported in this chapter. A blank was not analyzed in the evening for studies involving beads which had been stored in tubes (open to ambient air) for over 6 hr. Case Study Comparison of Bio-assay to GC/MS Assay Cryo-focusing GC/MS Experiments were performed involving analyzing emanations collected from one subject, each day, for five consecutive days. The collection process was similar to that for the case study described for the comparison of subjects. In this case, Mr. Ken Posey of the USDA served as the test subject. On each of the five days, a blank was recorded in EI mode prior to analysis. The subject handled ten glass beads for 15 min; three beads were used for each analysis. The remaining beads were stored for analysis with the complementary ionization mode. The cryo-focusing stage for the first handled-bead analysis was initiated at approximately 8:40 am each day. Upon completion of the this analysis, the mass spectrometer ion volume was switched to allow for PPINICI. A blank was acquired and three beads were 227 analyzed. The time delay between rubbing and analysis of the beads in this mode was approximately 2 hr. The cryo-focusing parameters were identical to those described previously in the experimental section of this chapter. The GC oven ramp, however, was conducted at a lower rate than previously used in studies. This was done to provide additional resolution for co-eluting peaks found in previous studies with ramp programs employing a steeper ramp. The GC oven ramp consisted of an initial 1.0 min hold at 40°C, a 27 min ramp at 6°C/min up to 200°C, and then a hold at 200°C for 12 min. The transfer line was concurrently ramped from 50°C up to 210°C at 107min for 16 min, and held for 24 min at 210°C. GC Separation was effected by a 25 m X 0.20 mm i.d. HP5 FSOT column (df=0.33 tim). The mass spectrometer operation in EI and PPINCI modes is similar to that described previously in this chapter. Methane reagent gas at 1680 to 1700 mtorr (indicated) pressure was employed for PPINICI analyses. The third quadrupole was scanned at 0.5 s per scan for EI and PPINICI analyses. The conversion dynode was set at +5 kV for positive ion CI and EI, and -5 kV for negative ion CI. The electron multiplier was set to -1000 V for EI experiments and -1100 V for CI experiments. Prior to the analysis of blanks, the instrument was tuned with PFTBA. Olfactometer Bio-assays of a glass petri dish handled by Mr. Ken Posey of the USDA were conducted approximately one hour after sample collection for GC/MS analysis. These experiments were conducted at the USDA laboratories and involved analysis 228 in the olfactomer at that location. The petri dish was handled for 15 min prior to insertion into a port of the olfactometer. Testing typically occurred at approximately 10:00 a.m. for each of the days. The testing was conducted with 75 (six-to-eight-day- old) female ^e. aegypti mosquitoes. The relative humidity, air temperature, and gas flow were controlled throughout each analysis. Over the five day period, none of the 75 mosquitoes tested each day were collected in the control port. The lowest collection percentage (12%) occurred on the first day (Monday). The remainder of the week, from Tuesday through Friday, yielded collection percentages of 24%, 21%, 27%, and 20%, respectively. Results and Discussion Identification of Emanations by CI and EI MS Prior to reporting compounds observed in the analyses of this dissertation, a compilation of compounds previously reported in the literature as being present on the skin are presented in table 5-1. Compounds not expected to be amenable to the manner in which analyses were conducted for this dissertation will be noted. Literature sources pertaining to skin emanations are relatively scarce [102]. The data compiled in table 5-1 are extracted from semiochemical work (odor analysis) and government studies (space exploration) concerning the identification of human dermal emanations [30,31,111]. Approximately 260 compounds, spanning a range of classes, are contained in this table. These classifications will remain as consistent as 229 Table 5-1 Compounds previously reported in the literature as species which emanate from human skin. Listed with the compounds are the corresponding molecular formula and relative molecular mass (r^j). Explanation of remarks follows this table. Compound Acids/Carboxylic acids Formic acid Acetic acid Propenoic acid Propanoic acid Butanoic acid Pentanoic acid Hexenoic acid Hexanoic acid Heptenoic acid Heptanoic acid Octenoic acid Octanoic acid Nonenoic acid Nonanoic acid Decenoic acid Decanoic acid Undecenoic acid Undecanoic acid Dodecanoic acid Hexadecanoic acid Octadecanoic acid Eicosanoic acid Oxoacetic acid Glycolic acid Pyruvic acid Lactic acid Malic acid Oxalic acid Propanedioic acid Butanedioic acid Pentanedioic acid Benzoic acid Cinnamic acid Isophthalic acid Formula Remarks Tm CH,0, 46 c^H.d, 60 CsH.O^ 72 CbH^O, 74 C4H8O, 88 C5H10O2 102 QH10O2 114 CftHi^O^ 116 c,u,,o. 128 QH,,o, 130 QH1402 142 QHi^Ot 144 C9H1602 156 CgHigOj 158 CioHigOj 170 ^10^20^2 172 C11H20O2 D 184 C11H22O2 186 C12H24O2 200 C16H32O2 C 256 C18H34O2 C 284 ^20^3802 312 C2H2O3 74 C2H4O3 76 C3H4O3 88 C3H,03 90 C4H,03 134 C2H204 90 C3H40, 104 QH,04 118 C5H804 132 QH,02 122 C9H802 148 C«H,0, 166 Table 5-1 Continued 230 Compound Acids /Carboxy lie acids (continued) Lithocholic acid Deoxycholic acid Cholic acid Hydrocyanic acid Hydrosulfuric acid Hydrochloric acid Hydrobromic acid Cyanic acid Isocyanic acid Carbonic acid Chloroacetic acid Chlorocarbonic acid Formula Remarks Tm C24H40O3 376 C24H40O4 392 C24H40O5 408 HCN A 27 H^S A 34 HCl 36 HBr 80 CHNO 43 CHNO 43 CH^Oj E 62 C2H3O2CI 94 CHO3CI E 80 Aeyl Halides I Related Formyl chloride Acetyl chloride Propanoyl chloride Carbamoyl chloride CHOCl C2H3OCI C3H5OCI CH2NOCI 64 78 92 79 Alcohols Methanol Vinyl alcohol Ethanol l-propen-3-ol Propano! Isopropanol Pentanol Tetradecanol Hexadecanol Ethylene glycol Glycerol Phenol Benzyl alcohol 1,2-dihydroxybenzene CH4O C2H4O C^H^O C3H,0 C3H8O C3H8O C5H12O C14H30O C1.H34O C^H^O^ C3H3O3 C,H,0 C^HgO CfiH^O^ 32 44 46 58 60 60 88 214 242 62 92 94 108 110 231 Table 5-1 Continued Compound Formula Remarks r Alcohols (continued) 1,3-dihydroxybenzene 1,4-dihydroxybenzene Inositol Dehydrocholesterol Cholesterol Aldehydes Formaldehyde Acetaldehyde Propenal Propanal 2-methylpropanal Butanal 3-methylbutanal Ethanedial Glycolicaldehyde Glyceraldehyde Aliphatics/Aromatics Methane Acetylene Ethane Propyne Cyclopropane Propene 2-methyl- 1,3-butadiene 2-methylnonane Squalene Benzene Toluene Amides Formamide Acetamide M C.HeO^ 110 C,H,0, 110 QH,,o, 180 C27H,40 384 C27H46O 386 CH2O A 30 C2H4O 44 C3H4O 56 C,H,0 58 C,H,0 72 C^H^O 72 C^H.oO 86 C^H.O, 58 C,H,0, 60 C3He03 90 CH4 A 16 C,H, A 26 C2He A 30 C3H4 A 40 C3H, 42 C3H, 42 CsHg 68 C10H22 142 ^30^50 410 C^H, 78 CyHg 92 CH3NO 45 C2H5NO 59 Table 5-1 Continued 232 Compound Amides (continued) Methylacetamide Propanamide Oxamide Cyanamide Formula Remarks r.M C3H7NO 73 C3H7NO 73 C,H,N,0, B 88 CH2N2 A 42 Amino acids/Amines/Related Glycine Alanine Proline Valine Isoleucine Leucine Phenylalanine Serine Threonine Tyrosine Aspartic acid Glutamic acid Lysine Tryptophan Histidine Arginine Cysteine Methionine Cystine Sarcosine Hydroxyproline Carbamic acid Aminobutanoic acid Aminoisobutanoic acid Aminobenzoic acid Ornithine Pantothenic acid Ammonia Methylamine Dimethylamine C2H5N02 B 75 CjH.NO^ B 89 C5H9NO2 B 115 QH„NO, B 117 QH,3NO, B 131 QH,3NO, B 131 CgHnNO^ B 165 C3H7NO3 B 105 C4H,N03 B 119 C^HnNOj B 182 C4H7NO4 B 133 C5Hc,N04 B 147 QH.^N.O, B 146 C„H,,N,0, B 204 QH^NjO, B 155 QH,,N,0, B 174 C3H7NO2S B 122 C5H11NO2S B 149 C,H,,N,0,S2 B 240 C3H7NO, B 89 C5H<,N03 B 131 CH3NO2 61 C4H9NO2 103 C^HgNO^ 103 C7H7NO2 137 CsH.^N^O, 132 C9H17NO5 219 H3N A 17 CH5N A 31 C,H,N 45 Table 5-1 Continued 233 Compound Amino acids/Related (continued) Ethylamine Trimethylamine 2-aminopropane Aniline Benzylamine 2-aniinotoluene Diphenylamine Aminophenol Ethylenediamine Guanidine Choline Acetylcholine Formula Remarks ^M C2H7N 45 CHgN 59 C2H9N 59 QH,N 93 C7H9N 107 C7H5N 107 CnHuN 169 C^H^NO 109 C^H^N^ 60 CH5N3 59 CjHijNO^ B 104 C^H^.NO,- B 146 Acid Anhydrides Formic anhydride Acetic anhydride C2H2O3 C^H^Oj 74 102 DNA bases Adenine Guanine C5H5N5 C5H5N5O B B 135 151 Esters Formic acid, ethyl ester Formic acid, propyl ester Formic acid, butyl ester Acetic acid, methyl ester Acetic acid, ethyl ester Acetic acid, isopropyl ester Acetic acid, propyl ester Acetic acid, pentyl ester Acetic acid, phenyl ester Propanoic acid, ethyl ester C4H8O2 C5H10O2 C3H,02 C4H802 C5H10O2 C5H10O2 C7H1402 CgHg02 C5H10O2 74 88 102 74 88 102 102 130 136 102 234 Table 5-1 Continued Compound Foimula Remarks Tm Esters (continued) Butanoic acid, ethyl ester QH,,0, 116 Carbonic acid, methyl ester C3H,03 90 Carbonic acid, ethyl ester C5H10O3 118 tLiners Dimethyl ether C^H^O 46 Methyl vinyl ether C,H,0 58 Methyl ethyl ether C,H,0 60 Diethyl ether C4H10O 74 Methyl propyl ether C,H,oO 74 Ethyl propyl ether QH.p 88 Dipropyl ether QH.oO 102 Ethyl phenyl ether CgHioO 122 nuiCUt^/i\ctUlcCl Fluorine F2 A 38 Chlorine CI, A 70 Bromine Br, 158 Iodine I2 254 Methyl chloride CH3CI 50 Methyl bromide CH3Br 94 Methyl iodide CH3I 142 Vinyl chloride C2H3CI 62 Ethyl chloride C3H5CI 64 Ethyl bromide C^HsBr 108 Ethyl iodide C.HjI 152 2-chloro-l,3-butadiene C4H5CI 88 Chlorobenzene C^H^Cl 112 Benzyl chloride C7H7CI 126 Methylene chloride CH2CI2 84 Dichloroethylene C2H2CI2 96 235 Table 5-1 Continued Compound Heterocyclics Pyrrole Pyridine Nicotinic acid Pyridoxal Pyridoxine Creatinine Indole Skatole Purine Ethylene oxide Propylene oxide Furan Tetrahydropyran Pyrone Thiophene Thiazole Ketones Acetone 2-butanone 3-pentanone Acetophenone Benzalacetone l-chloro-2-propanone 2,3-butanedione 2,4-pentanedione Nitra tes/Nitro/Rela ted Nitrogen Nitrogen monoxide Nitrogen dioxide Nitrogen tetroxide Foimula Remarks r^ C4H5N 67 C5H5N 79 C,U,NO, 123 CgHgNOj 167 CgHuNOj B 169 C^H.NjO 113 CgH.N 117 C9H9N 131 C^H^N, 120 C2H4O 44 C3H,0 58 C4H4O 68 C5H10O 86 C,H,0, 96 C4H4S 84 C3H3NS 85 C,H,0 58 C4H3O 72 C5H10O 86 QHsO 120 CioHioO 146 C3H5OCI 92 C4H,0, 86 C,H«0, 100 Nz A 28 NO A 30 NO2 A 46 N,04 92 Table 5-1 Continued 236 Compound Formula Remarks 'M Nitra tes/Nitro/Rela ted (continued) Nitrogen oxychloride Nitromethane Nitrobenzene Nitrophenol Ethyl nitrite Ethyl nitrate Acetyl nitrate Acetonitrile Diazomethane Sugars/Related Arabinose Ribose Xylose Deoxygalactose Galactose Glucose Mannose Sucrose Glucosamine Sulfides IRelated Dimethyl sulfide Diethyl sulfide Carbon disulfide Sulfur dioxide NOCl 65 CH3N02 61 C^HsNO, 123 QH3NO3 139 C2H5NO2 75 CHjNOj 91 C2H3NO4 105 CH3CN 41 CH2N2 42 QHioO; B 150 QHioOj B 150 CH.oO, B 150 C,H,,05 B 164 QH„o, B 180 QH.A B 180 QH.,o, B 180 C12H22011 B 342 C6H13N03 B 179 C^H^S 62 C4H10S 90 CS2 so. 76 64 Thio/Thioesters/Sulfonyls Thiomethane Thioethane Thioaceticacid CH4S C^H^S C2H4OS 48 62 76 Table 5-1 Continued 237 Compound Formula Thio/Thioesters/Sulfonyls (continued) Taurine Ethane sulfonyl chloride Urea/Related C2H7NOS C2H5OSCI Remarks Tm B 125 128 Urea Methylurea Acetylurea Thiourea Uric acid Vita m ins /Re la ted Thiamine Riboflavin Folic acid Dehydroascorbic acid Miscellaneous Carbon monoxide Carbon dioxide Methyl thiocyanate Methyl isothiocyanate Epinephrine Phenolphthalein Dehydrobilirubin Bilirubin CH,N,0 60 C^HeN.O 74 C3H,N,0, 102 CH4N2S 76 C5H4N4O3 168 C,,H,,N,OS B 264 CnH^oN.O, B 376 Ci^Hi^N^O, B 441 C,H,0, B 174 CO A 28 C02 A 44 C2H3NS 73 C2H3NS 73 C9H13N03 183 C20H14O4 318 C33H34N40, B 582 C33H3aN,0, B 584 238 Table 5-1 Continued Explanation of remarks: A) Low r^ and/or highly volatile compound not suspected to be amenable to identification by cryo-focused methods employed in this chapter. B) Involatile compound, salt, or compound which decomposes in the temperature range of the analyses. This compound is not expected to be amenable to GC. C) A gap in the class series exists; however, the presence of the missing compound in the series is implicit in one or more of the literature sources. D) This compound is suspected to be present, but unconfirmed in the literature. E) This compound exists in solution only and is not expected to be detected by GC. 239 possible, as well as the order within each group, for additional tables in this chapter which report qualitative identification of components. A total of at least 23 methods, including GC/MS, were used in the literature sources to identify compounds listed in this table. The low r^ compounds listed in this table may be difficult to confirm by the GC/MS methods employed here. There are compounds in this table for which the volatility is extremely high; these compounds are not expected to be effectively collected on glass beads, nor are they likely to remain on the glass beads until desorption in the GC. Compounds which fall under this category are noted by an (A) in the remarks column of the table. The majority of compounds listed were determined using separation methods other than GC. Some compounds in the table were separated by, for example, liquid chromatography (LC). These compounds are involatile and well chromatographed by LC but may not be amenable to GC. Compounds which fall under this category, such as involatiles, salts, or compounds which thermally decompose in the temperature range of analyses, are not amenable to GC analysis; therefore they are noted by the remark code (B) in the Table 5-1. There is an implicit comment made in one of the literature sources that the presence of the complete series of saturated carboxylic acids from acetic acid up to octadecanoic acid is observed in human perspiration, without specifically providing the data to confirm this [30]. Therefore, completion of the series is noted by the presence of (C) in the remarks indicating the compound(s) of lower carbon number 240 previous to this compound are present. However, if a compound was only suspected as being present in the literature sources, then the compound is noted by a (D) in the remarks column. The final remark code (E) is used to denote species which are present in solution only. Since GC involves separation in the gas phase, compounds so noted are not expected to be observed in the analyses of this chapter. In summary, this first table provides a general basis from which to proceed. It should be noted that the analyses which follow (in the remainder of this chapter) are specifically designed to provide information on volatiles from the skin. More specifically, these analyses will only consist of volatiles which transfer to glass (beads) and can be thermally desorbed back off of glass to be detected by either cryo-focused GC/MS or purge and trap GC/MS. Cryo-focusing GC/MS A single stage of cryo-focusing combined with GC/MS was the primary method used for identification of volatiles in this work. Most of the analyses involve the identification of volatiles from the hands, arms, and forehead of the author of this dissertation. Additional identification of volatiles and repeated confirmation of compound identification came from the case studies presented later in this chapter. Therefore, the data found in this section are compilations of emanations observed from four different subjects. Studies involved the acquisition of PPINCI and EI mass spectral data. Typical RIG traces from a set of PCI, NCI, and EI analyses of the same subject appear in figure 5-1. f2 M «« •<^ c ^ o "(3 T3 c lU R3 ■*-• D O tM JU V|— n l-l u c re C3 CA ■a Q l-i 0) c« )-^ 4-W ID H-T S E u >-. re Pu X) re ^ C/5 ex U "re z l-l o 3 C 1 c .2 u-> "3 on o T3 < CC o ^ %4-l d ■*-» u 5 ^3 E _3 O o x: u m t/5 Ph c o T3 K H re a O C/2 6 J2 tlH o 'So U T-H « E m S E 3 "H. o W o re (/3 b X 242 s 4) S o ^ s> s< S> CO kO '<■ (%) /(jisnajni aAijBpH 243 This figure is included for several reasons. Upon first inspection, the sample may not appear to contain more than 100 discernible peaks; however, the typical analysis by the methods employed here contains over 300 peaks, and may contain up to 350 distinct components. Contained within the RIC trace are many low intensity peaks obscured by the intensity scale use to display the acid peaks. It is evident from this figure that most readily seen components provide observable peaks in CI and EI. There are many components in these analyses that provided interesting problems in terms of identification; however, at this time, only two chromatographic peaks in this figure will be addressed. The first component of interest is poorly chromatographed and located at a retention time of 6 min. This peak is noticeably abundant in the sample, and it is clearly seen that information in EI is scarce by the absence of significant peak. Upon examination in NCI mode, this peak was identified by its relative molecular mass and characteristic fragment ions of lactic acid (see chapter 3). The second abundant component, located at a retention time between 8 and 10 min, eluded identification for some time, despite being one of the most abundant peaks in analyses conducted with this subject. Due to the absence of a molecular ion in the EI mass spectrum for this compound, the shape of the peak as well as relative molecular mass information from PCI and NCI were employed to aid in identification. The shape of this peak is similar to that of lactic acid; therefore, it was expected that this compound may also be a hydroxy acid, or possibly similar in 244 structure. Visual inspection of PCI data revealed that a r^ of 92 was plausible due to the abundance of the apparent [M+H]^ ion at m/z 93. However, NCI mass spectra did not contain an abundant [M-H]~ ion at m/z 91. The NCI spectra did contain an ion at m/z 183 which was postulated to be the [Mj-H]" ion of this compound. When the library searching was restricted to compounds with a r^ of 92, this compound, 1,2,3-propanetriol (glycerol), was still not correctly identified. Final confirmation was achieved by comparing the EI mass spectra to that of a standard of glycerol. Glycerol was delivered into the mass spectrometer ion source via the use of the direct insertion probe. When the ion source was saturated under these conditions, it was revealed that self-CI had occurred upon elution of glycerol during previous sample analyses. The presence of Cl-produced fragments in the EI mass spectrum did not permit successful library searching. One final note merits mentioning with respect to the importance of identifying this component in the sample. The glycerol peak from all other subjects was miniscule in comparison to the abundance found in this subject. It was later determined that this high level of glycerol is attributable to the Sta-Sof-Fro™ hair and scalp spray used by this subject; glycerol is one of the primary ingredients in the product. These two components are two of approximately 310 components observed in these analyses; components identified and components suspected are listed in table 5-2. In general, the subjects studied are similar with respect to composition of emanations. There are some exceptions; further studies may reveal whether these 245 Table 5-2 Compounds present and compounds suspected of being present which emanate from human skin. This Hst was derived from cryo-focused GC/MS analyses of four human subjects. Listed with the compounds are the corresponding molecular formula and relative molecular mass (r^). Explanation of remarks follows this table. Compound Foimula Remarks Tm Carboxylic acids 2-propenoic acid CsH^O, G 72 Propanoic acid CbH^O, G 74 2-butenoic acid C4H,0, 86 2-methyl-2-butenoic acid QHsO, B,C 98 3-methyl-2-pentenoic acid QH10O2 C 114 3-methyl-pentanoic acid QH,,o, C 116 Hexanoic acid C,Hi,0, A,G 116 Heptanoic acid C7H14O2 G 130 Octanoic acid QHigOj A,G 144 Nonanoic acid CgHigOj G 158 Decanoic acid C10H20O2 G 172 Undecanoic acid C11H22O2 A,G 186 Dodecanoic acid C12H24O2 G 200 Methyldodecanoic acid C13H26O2 C 214 Tridecanoic acid C13H26O2 G 214 Tetradecenoic acid C14H26O2 C 226 Methyltridecanoic acid C14H2SO2 C 228 Tetradecanoic acid C14H2SO2 G 228 14-pentadecenoic acid Ci5H2g02 240 12-methyltetradecanoic acid C15H30O2 242 Methyltetradecanoic acid C15H30O2 C 242 Methyltetradecanoic acid C15H30O2 C 242 Pentadecanoic acid C15H30O2 G 242 9-hexadecenoic acid C16H30O2 254 Methylpentadecanoic acid C16H3202 C 256 Hexadecanoic acid C16H3202 G 256 Heptadecenoic acid C17H3202 C 268 Heptadecanoic acid C17H3402 C,G 270 11-phenoxyundecanoic acid C17H2603 A,E 278 9,12-octadienoic acid C18H3202 280 246 Table 5-2 Continued Compound Formula Remarks I'm Carboxylic acids (continued) 9-octadecenoic acid C18H34O2 282 Methylheptadecanoic acid C18H36O2 C 284 Octadecanoic acid CisHjgOj G 284 Docosanoic acid C22H44O2 340 Lactic acid CaH,03 G 90 Hexanedioic acid QH10O4 A,E 146 Heptanedioic acid C,H,204 A,E 160 Benzoic acid C,H,02 G 122 4-hydroxybenzoic acid C,H,03 138 4-hydroxy-3-methoxybenzoic acid C3H8O4 168 Alcohols 2-butanol 3-methyl-4-penten-2-ol 2-hexen-l-ol 4-hexen-l-ol l-hexen-3-ol 2-methyl-3-pentanol l-hepten-3-ol l-octen-3-ol 2-octen-l-ol 2-methyl-3-octenol Nonenol 3,7-dimethyl-6-octen-l-ol Decenol, substituted 2-decanol Dodecenol Tridecanol 1-tetradecanol 2-hexadecanol 2-heptadecanol Cholest-5-en-3-ol Phenol Benzyl alcohol C4H,oO C 74 QH,20 B,C 100 QHi20 C 100 QH,20 100 C,H,20 100 QH,40 B,C 102 QHi40 114 QH,,o A,D 128 QH,,o 128 QHisO C 142 QHisG B,C 142 CioHzoO C 156 C10H20O B,D 156 C10H22O 158 C12H24O A,B 184 Cl3H2gO A 200 C14H30O 214 C1.H340 B 242 CnH3aO A,B 256 C27H4aO G 386 C,H,0 G 94 C,H«0 G 108 247 Table 5-2 Continued Compound Formula Remarks 'M QHiqO A,E 122 CuHazO C 206 C14H22O 206 C14H22O 206 C15H24O 220 Ci3HnN30 A,D 225 C,H,0, G 62 C9H20O2 A,E 160 C3H8O3 G 92 QH,203 A,E 120 Alcohols (continued) Phenylethyl alcohol 2,4-bis(l,l-dimethylethyl)phenol 2,5-bis(l,l-dimethylethyl)phenol 2,4-bis( 1, l-methylpropyl)phenol 4,6-di( 1, l-dimethylethyl)-2- methylphenol 2-(2H-benzotriazol-2-yl)-4- methylphenol Ethylene glycol 1,9-nonanediol Glycerol 2-(hydroxymethyl)-2-methyl- 1,3- propanediol Aldehydes Propanal C3H5O G 58 2-methylpropanal C4H8O G 72 2-methyl-2-butenal C4H6O B,C 84 2-methylbutanal CjHiqO C,G 86 3-methylpentanal CgHjjO 100 Heptanal QH14O 114 2,2-dimethylhexanal CgHi^O 128 Octanal CgHigO B,C 128 2,4-nonadienal C9H14O 138 Nonanal CgHigO 142 3,7-dimethyl-2,6-octadienal CioHi^O B,C 152 Decanal CiqHjoO B,C 156 Dodecanal C12H24O B,C 184 2-methylhexadecanal C17H34O A,B 254 Benzaldehyde CyH^O 106 3-hydroxy-4-methylbenzaldehyde C8H8O2 136 4-phenylmethoxybenzaldehyde C14H12O2 212 248 Table 5-2 Continued Compound Foiniula Remarks Tm AliphaticslAromatics Pentene C5H10 B 70 4-methyl-2-pentene QH12 84 Hexane QHi4 C 86 Dimethylpentadiene C7H12 B,C 96 2-methyl-l-hexene C7H14 98 Heptane CvHie C 100 3-ethyl- 1,4-hexadiene ^8^14 A,B 110 5-methyl-l-heptene QHis C,F 112 2-octene QH16 F 112 3-octene QHig C,F 112 4-octene QHie C,F 112 2,4-dimethylhexane CgHis C 114 Octane ^s^is C 114 3,4-nonadiene CgHie 124 2,6-dimethyl- 1-heptene CgHig 126 4-ethyl-3-heptene CgHjg C 126 4-nonene C9H18 126 Nonane C9H20 128 N-menth-6-ene ^10"l8 138 Menthane ^10^20 A 140 3,3,6-trimethyl-l,5-heptadiene C10H18 D,E 138 2,7-dimethyl- 1-octene ^10"20 A,E 140 5-decene ^10"20 140 Decane ^10"22 C,G 142 Undecadiene C11H20 B 152 2-methyl-2-undecene C12H24 D,E 168 3-methyl-5-undecene C12H24 D,E 168 4-methyl-4-undecene C12H24 B,C 168 2-methyl-2-dodecene C13H26 182 Methyldodecene C13H26 C 182 5-tetradecene C14H28 196 Cyclotetradecane C14H28 196 Tetradecane ^uHso B,C 198 Pentadecane C15H32 212 7-hexadecene C16H32 D,E 224 249 Table 5-2 Continued Compound Formula Remarks 'M A lipha tics 1 Aroma tics (continued) Cyclohexadecane C16H32 224 Hexadecane C16H34 226 Heptadecane C17H36 C 240 9-octadecene C18H36 252 Octadecane ^18^38 c 254 7, 1 1, 15-trimethyl-3-methylene C20H32 c 272 hexadecane Heneicosane C21H34 296 Docosane C22H46 310 Tricosane C23H48 324 Tetracosane C24H50 A,E 338 Pentacosane C25H52 352 Cholesta-3,5-diene C27H44 A,B 392 Triacontene, branched C30H50 C 410 Squalene C30H50 G 410 Benzene CaH, G 78 Toluene CyHg G 92 Styrene QHg 104 1,4-dimethylbenzene CgHio C 106 Ethylbenzene CgHio 106 Propylbenzene C9H12 120 4,4'-dimethyl-l,r-biphenyl C14H14 C 182 Amides lAmines I Related Propanamide N,N-diethyl-3-methylbenzamide N,N-bis(2-hydroxyethyl)dodecanamide N,N-didodecyl formamide 1,3-butanediamine N,N-dimethyl-l,2-ethanediamine N-ethylcyclopentamine N,N-dimethyl-3-butoxypropylamine N,N-dimethyl-3-benzyloxypropylamine N-methyl-N-nitroso-1-dodecanamine C3H7NO 73 C,2Hi,NO 191 C13H33NO3 A,E 287 C25H51NO B 381 C4H.2N2 A 84 C5H,N2 D,E 116 C,Hi5N A 113 C9H21NO A 159 C,2H,,NO 193 C13H23N2O A 228 250 Table 5-2 Continued Compound Formula Amides I Amines IRelated (continued) N,N-dimethyl- 1-dodecanamine C14H31N N,N-dimethyl- 1-tridecanamine C15H33N N,N-dimethyl- l-tetradecanamine CigHjjN N,N-dimethyl- 1-pentadecanamine C17H37N N,N-dimethyl- l-hexadecanamine C18H39N N,N-dimethyl- l-heptadecanamine C19H41N N,N-dimethyl- 1-octadecanamine C20H43N Esters 2-butenoic acid, butyl ester Butanoic acid, Methyl ester Nonanoic acid, methyl ester Tridecanoic acid, methyl ester 13-methylpentadecanoic acid, methyl ester 14-methylpentadecanoic acid, methyl ester 2-methylhexadecanoic acid, methyl ester Heptadecanoic acid, methyl ester 6-octadecenoic acid, methyl ester Hexadecanoic acid, methyl ester 16-methylheptadecanoic acid, methyl ester Octadecanoic acid, methyl ester Hexadecanoic acid, butyl ester Octadecanoic acid, phenyl ester Tetradecanoic acid, undecyl ester Tetracosanoic acid, methyl ester Pentanedioic acid, ester Pentanedioic acid, mono(2-ethylhexyl)ester Hexanedioic acid, ester Hexanedioic acid, ester branched Remarks Tm F 213 F 227 F 241 F 255 F 269 F 283 F 297 QHigOj A 128 QHiqO, A,B 102 ^XQ^lQ^l 172 C14H2802 A 228 C17H3402 270 C17H3402 270 C18H3602 284 C18H3602 284 C19H3602 C 296 C19H3802 298 C,9H3g02 C 298 Ci9H3g02 B 298 ^20^40^2 312 C24H40O2 360 C25H50O2 A,B 382 C25H50O2 A,B 382 D,E >144 C13H24O4 244 D,E >160 D,E >160 251 Table 5-2 Continued Compound Esters (continued) Hexanedioic acid, Mono(2-ethylhexyl)ester Hexanedioic acid, Octyl ester Heptanedioic acid, Dibutyl ester Hydroxybutanedioic acid, Ethyl ester 3-hydroxybenzoic acid, Ethyl ester 4-hydroxybenzoic acid. Propyl ester 2-hydroxybenzoic acid, Phenylmethyl ester 2,4-dihydroxy-3,6-dimethylbenzoic acid, Methyl ester Halides/Related Methyl iodide 1-chlorohexane 1-chloroheptane 1-chlorononane 1-chlorododecane 1-chlorotetradecane 1-chloropentadecane 1-chlorohexadecane l,6-dichloro-l,5-cyclooctadiene Benzylchloride 3-chlorobenzeneamine 2-chloro- 1-methylethylbenzene l-chloro-4-(4-niethyl-4-pentyl) benzene 2,3-dichlorobenzeneamine Heterocyclics 2-methyl- 1 H-pyrrole 3-methyl- IH-pyrrole Formula Remarks l-M C14H26O4 258 Cl4H2604 B 258 C15H28O4 B 272 C8H14O5 A 190 QHgOj 152 C10H12O3 180 C14H12O3 228 C10H12O4 196 CH3I G 142 C,H,3C1 120 C7H15CI 134 CgHjgCl 162 C12H25CI 204 1^141129^^1 232 C15H31CI B 246 C16H33CI B 260 C8fliod2 B,D 176 C7H7CI G 126 C,H,NC1 127 C9H11CI D 154 C12H15CI D 194 C,H5NCl2 C 161 C5H7N 81 C5H7N 81 Table 5-2 Continued 252 Compound Heterocyclics (continued) Pyridine 2-pyridinamine 4-pyTidinamine 4-methyl-2-pyridinamine 2-methylpyridine 4(lH)-pyridinone 6-amino-3-pyridine carboxylic acid 3(l-methyl-2-pyrrolidinyl)pyridine 1,2,3-4-tetrahydroquinoline 2,3,4-trimethylquinoline 2-ethylpiperidine 1-piperidineethanol 4-piperidinemethanamine l-phenyl-3-(l-piperidinyl) 2-buten-l-one IH-indole Indole, substituted 4,5-dihydro-2-methyl-lH-imidazole 1,5-dimethyl-lH-pyrazole Pyrazine 2,6-dimethylpyrazine Trimethylpyrazine 3-ethyl-2,5-dimethylpyrazine 5-methyl-2-methylthio-4(lH)- pyrimidinone 1-methylpiperazine 2,5-diniethylpiperazine Oxazole 2-methylfuran 3-methylfuran 2,3-dihydro-4-methylfuran 2-furanmethanol Benzofuran Dihydro-5-tetradecyl-2(3H)furanone Formula Remarks Tm C5H5N G 79 CsH^N, 94 CsH^N, 94 c,n,n. 108 QH,N 93 C5H5NO 95 QH^NP, B,F 138 C:oHhN, F 162 C9H11N 133 CnH„N A 171 QH.^N 113 C^HijNO D,E 129 QH,,N, A 114 Ci5H,<,NO 229 CsH.N G 117 D >117 c,w,n. 84 C^H^N, . C 96 C4H4N2 80 CaH^N, B 108 C7H10N, A 122 CgHi.N, C 136 C^HgNpS A 156 QH,,N, B 100 QH.4N, B 114 C3H3NO 69 C^H^O 82 CjH^O 82 CjHgO B 84 CsH^O, 98 CsH^O 118 C18H34O2 B 282 Table 5-2 Continued Compound Heterocyclics (continued) Formula Remarks 253 'M 2,3-dihydro-3,5-dihydroxy-6-methyl- 4H-pyran-4-one 2-methoxy-6-methyl-4H-pyran-4-one 2H- l-benzopyran-2-one 3-acetyl-6-methyl-2H-pyran- 2,4(3H)dione Thiazolidine 2-methylisothiazole 5-methyl-2(5H)thiophenone 2-methoxy-5-methylthiophene C5H3O, 144 QH803 B 140 C,H,0, 146 ^8^304 A 168 CjH.NS 89 C4H5NS A 99 CsH^OS 114 CeHgOS 128 Ketones 2-butanone 2-pentanone 3-pentanone 2-hexanone 6-methyl-3,5-heptadien-2-one 6-methyl-5-hepten-2-one 2-nonen-4-one 2-decanone 2-methoxy-2-octen-4-one 6,10-dimethyl-5,9-undecadien-2-one 3-hydroxy-androstan- 11, 17-dione C^HgO G 72 C5H10O 86 C^H.oO G 86 QH13O C 100 CsH.p C 124 CsH.^O 126 QHi.O B 140 CioH2oO 156 ^9^,502 162 CijH^^O A 194 C19H28O3 304 Sulfides Carbon disulfide Dimethyl disulfide CS2 C2H6S2 76 94 Table 5-2 Continued 254 Compound Thio/Thioesters/Sulfonyls Thiomethane 2-thiopropane 1-methyl thiobutane 1-thiododecane 3-methylthietane 3(methylthio)- 1,2-propanediol o-(2-butenylthio)phenol Thiocarbamic acid, Butyl ester Acetylthiocarbamic acid, Methyl ester Methanesulfonylchloride l,r-sulfonylbis[4-chlorobenzene] Urea/Related Urea Methylurea Thiourea N,N-dimethylthiourea Miscellaneous l-isocyanato-3-methylbenzene 1,3-dimethoxybutane 1,2,3-trimethoxypropane 1,4-benzenedicarbonitrile Compounds present in background /Blank analyses Methylchloride Tetrachloroethene 1,1-difluroethane Fluoroethene l,l,2-trichloro-l,2,2-trifluoroethene l-silacyclo-3-pentene Silacyclopentane Foiniula Remarks Tm CH4S G 48 CjHgS A 76 QHi2S 104 C12H26S D 202 C4H8S A 88 C4H10O2S D 122 C10H12OS 180 CjHiiNOS A,E 127 C4H7NO2 B 130 CH3OSCI 114 C12HSO2SCI2 286 CH^N.O G 60 C2H,N20 G 74 CH4N2S G 76 C3H8N2S 104 CgH^NO B 133 QH.4O2 A,E 118 QH14O3 A 134 QH14N2 A 128 inalyses CH3CI G 50 C2CI4 164 C2H4F2 F 66 C2H3F 46 C2CI3F2 167 C4H8Si 84 C4HioSi 86 Table 5-2 Continued 255 Compound Foimula Remarks Tm Compounds present in background/Blank analyses (continued) Trimethylsilanol CjHioOSi 90 Hexamethylcyclotrisiloxane QHigOjSij 222 Octamethylcyclotetrasiloxane CgH2404Si4 296 Decamethylcyclopentasiloxane CioHjoOjSij 370 Dodecamethylcyclohexasiloxane CnHjsO^Sig 444 Tetradecamethylhexasilioxane CuHizOgSig 458 Hexadecamethylcycloheptasiloxane Ci6H4g06Si7 532 3-isopropoxy-l,l,l,7,7-hexamethyI- CisHjjOySi, 576 3,5,5-tris(trimethylsiloxy) Tetrasiloxane 1,2-benzenedicarboxylic acid, C12H14O4 222 Diethyl ester 1,2-benzenedicarboxylic acid. C19H20O4 312 Butylphenylmethyl ester 1,2-benzenedicarboxylic acid C22H34O4 362 Diheptyl ester 1,2-benzenedicarboxylic acid, C24H38O4 390 Diisooctyl ester 1,2-benzenedicarboxylic acid. C24H38O4 390 Bis(2-ethylhexyl ester) 1,2-benznedicarboxylic acid, C26H42O4 418 Diisononyl ester 1,2-benzenedicarboxylic acid. D,E >415 Undeterniinded Explanation of remarks: A) Identity of the compound i s questionnable. This denotes a compound for which the most reasonable identity is listed; however, the EI library search purity value is low owing to either the absence of characteristic masses in the sample mass spectrum or the presence of additional masses in the sample spectrum versus the library spectrum. 256 Table 5-2 Continued Explanation of remarks (continued): B) The r^i of this compound is known from CI analysis. The actual structure given is questionnable. C) The structure is known to a reasonable degree of certainty. Uncertainty exists in the location of the substituted functional group and/or in the double bond location. D) The class or base structure of this compound is characterized by a specific EI fragmentation pattern. The information from CI may or may not be available to assist in determining the identity of this compound. E) The r^ from CI analysis for this compound is questionnable. F) Refer to the text for further explanation about this compound. G) This compound was previously reported in the literature as one having dermal origin. 257 originate naturally from the body, or are due to deposition on the skin from an external source. There are approximately 40 component peaks found present in each sample for which the identity could not be determined. These are included in this table. The remark codes used for this table appear at the end of the table. Absence of remark codes A-E indicates that this compound was identified with a relatively high purity (greater than approximately 800 of 1000 maximum possible score) in some cases. The purity value is generated by the library search program and reflects a weighted average of the forward fit and reverse fit values. The forward fit value is decreased when the sample mass spectrum contains additional masses not found in the reference library mass spectrum. The reverse fit value is decreased when the sample mass spectrum does not contain masses found in the reference library mass spectrum. If the compound had a lower purity, but was confirmed in numerous samples as well as a high reverse fit value, then the compound is reasonably certain to be that which is listed. In most complex analyses, additional masses will be present from background which will reduce the forward fit value but not affect the reverse fit as greatly. A remark code of (A) denotes those compounds for which the identity is questionnable; these compounds may even belong to a different class. If the molecular weight is known, but the structure given is still questionnable, the compound will be denoted with a (B) code. In some cases the r^ is known and the 258 EI fragmentation pattern is consistent with the structure. However, due to similarity between isomers, the exact structure can't be postulated with reasonable certainty. Compounds fitting this category are noted by a (C) code. If the EI pattern is consistent with a particular class, but the substitution identity is not known, a code of (D) is used. A code of (E) is given when the CI analysis for the compound did not yield information about the (r^)- A code of F listed in the table refers the reader to the text following this explanation of codes for additional discussion about specific compounds. The final code (G) denotes compounds previously identified in the literature, which are listed in table 5-1. There are four points of interest to be addressed concerning the compounds listed in this table. The first concerns the four octene isomers listed in the table and denoted by the remark code (F). These are listed in this fashion because in one or more analyses, there were in fact four peaks attributable to octene. This holds true for all multiple listings of isomers of the same compound. Any uncertainty as to the possible structure is treated by listing the best reverse fit structure from the library and using the remark code (C) to denote that this compound may in fact be an isomer of the compound listed. Substituted benzenes, for which two isomeric peaks are often detected, will include the meta- isomer, and either the ortho- or para- listed with a (C) code. This is due to the similarity in the fragmentation of two or more of these isomers which cannot be distinguished by visual inspection or by library search of the mass spectra. 259 The tertiary amines running from N,N-dimethyl-l-dodecanamine to N,N- dimethyl-l-octadecanaminewere not identified until relatively late in the work of this dissertation. Some of these amines elute at almost identical retention times as the carboxylic acids, and their peaks are sometimes hidden under more abundant sample components. When EI mass spectra were examined, there are only two significant ions present, that of the base beak at m/z 58 (due to ^-cleavage of the chain) and that of the corresponding odd-mass molecular ion. Library identification typically fails to identify these compounds correctly. Upon manual inspection of library mass spectra for suspected amines, it was determined that in addition to the 12 carbon or greater chain, substitutions of anything larger than but methyl groups would result in additional characteristic ions in the spectra. Once the proper restrictions were imposed upon the library search, the identification of these amines were achieved readily provided they were contained within the library. The final component of interest to be discussed in the text is one which was introduced unintentionally into the system but later used to aid in comparisons. The compound 1,1-difluoroethene is found in Dust-Off. Dust-Off was employed to cool down the glass injection port liner between runs prior to introducing a sample. When the glass injection port liner was reinserted and the analysis conducted, latent difluoroethene was cryo-focused with the sample. Normally, this was the first peak which eluted off of the column. This retention time of this component was later used in correlating analyses acquired with the same GC and MS parameters. 260 Purge and trap GC/MS Limited studies microscale purge and trap were some of the last experiments conducted for this dissertation. Although replicate analyses were conducted, the results are somewhat preliminary. The results from these analyses are reported in table 5-3. Complementary CI analysis was not conducted in these experiments. The compounds listed in the table were derived from EI analyses only. Due to the absence of information on compound r^ by CI, the identification process relied more heavily upon EI library search purity values. The remark codes used in the table reflect this. A remark code of (A) denotes a questionnable identity of a compound. If the mass spectrum displayed a characteristic EI fragmentation pattern attributable to a specific class, or if there was uncertainty in the structure due to isomers, a code of (B) was used. If the compound was previously identified in the literature (reported in table 5-1), the code (C) appears in the remark column. If the compound identified is also reported as observed in cryo-focused analyses (table 5-2), a code of (D) is present. Finally, a code of (E) in the table indicates that this compound will be addressed further in the following text. The first designated component to be discussed is ethanol. This was only identified to be present one time in the analyses of emanations from the author of this dissertation. The particular analysis was conducted approximately 15 hours after consumption of two glasses of wine for dinner the previous evening. This may have 261 Table 5-3 Compounds present and compounds suspected of being present which emanate from human skin. This Hst was derived from microscale purge and trap GC/MS analyses of two human subjects. Listed with the compounds are the corresponding molecular formula and relative molecular mass (r^^). Explanation of remarks follows this table. Compound Formula Alcohols Ethanol C^HgO Isopropyl alcohol CjHgO Butenol C4H8O 2-methyl-2-propanol C4H10O 2-butanol C4H10O 2-methylbutenol CgHigO Pentanol CsHjjO Hexanol CgH^O 3-methylcyclohexanol C7H,4 Heptanol, branched CyHjgO 1-heptanol CyHj^O Octenol CgHieO 2-propyl-l-pentanol CgHjgO 2-methyl- 1-heptanol CgHigO Octanol CgHigO 2-nonen-l-ol CgHjgO 2-decen-l-ol CjgHjoO 2-propyl- 1-heptanol C10H22O 2-undecen-l-ol C„H220 2-tridecen-l-ol C13H26 Eucalyptol CjoHigO 2-methyl- l,5-heptadien-3,4-diol C8H,402 Phenol CfiHgO 3,5-dimethylphenol CgHjgO 2-ethyl-5-propylphenol CnHi^O Aldehydes 2-methyl-2-propenal C4HgO 2-methylpropanal C4H8O Butanal C4H8O Kemarks Tm C,E 46 C 60 B 72 74 D 74 B 86 C 88 D 102 A 114 B,D 116 116 A,D 128 A 130 130 130 B,D 142 D 156 B 158 A 170 B,D 198 A 154 A 142 CD 94 122 A 164 70 CD 72 C 72 262 Table 5-3 Continued Compound Formula Remarks Tm Aldehydes (continued) 2-ethyl-2-propenal C5H3O 84 2-pentenal C^H^O 84 2,2-dimethylpropanal C^H.oO B 86 2-methylbutanal QHioO D 86 3-methylbutanal QH.oO C 86 Pentanal QH.oO 86 2-hexadienal QHsO 96 4-methyl-3-pentanal QHioO 98 2-hexenal C,H,oO 98 2-ethylbutanal QHj.O A 100 3-methylpentanal QH^O D 100 Hexanal CaHi.O 100 2,4-heptadienal C7H10O A 110 2-heptenal C,H,,0 112 Heptanal C,H„0 D 114 2-octenal QH„0 B 126 3,3-dimethylhexanaI QH„0 A,D 128 Octanal QH,,0 D 128 2-nonenal C,H,,0 140 Nonenal C,H,,0 B 140 Nonanal CgHjgO D 142 2-decenaI CioHigO B 154 Decanal C10H20O D 156 Undecanal C„H,,0 170 2-dodecenal Ci^H^.O B 182 Benzaldehyde C,H,6 D 106 Methylbenzaldehyde QH^O B 120 Benzeneacetaldehyde C3H3O 120 AliphaticslAromatics Propene C3H, A,C 42 Propane C3H3 44 2-butene C4H8 56 2-methylpropane C4H10 A 58 263 Table 5-3 Continued Compound Formula Remarks Tm AliphaticslAromatics (continued) 2-methyl-l,3-butadiene CjHg C 68 1,3-pentadiene CsHg 68 3-methyl-l-butene C5H10 70 1-pentene CsHio D 70 2-methylbutane C5H12 72 Pentane C5H12 72 2-methyl- 1,3-pentadiene QHjo 82 2-methylpentene QH12 84 4-methyl-2-pentene QH12 D 84 1-hexene C6H12 84 2,3-dimethylbutane QHi4 B 86 2-methylpentane C6H14 86 Hexane QH14 D 86 1,6-heptadiene C7H12 96 1-heptene C7H14 98 1-methylcyclohexane C7H14 98 2-methylhexane C7H16 B 100 3-methyhexane C7H16 B 100 Heptane C7H16 D 100 1,3-octadiene QH14 110 5,5-dimethyl- 1-hexene QH16 B 112 1-octene QHjg D 112 2-methylheptane QHjg 114 Octane QHjg D 114 3-ethyl-2-methyl-l,6-hexadiene CgHig B 124 3,6-dimethyl-l,5-heptadiene QHjg B 124 1-nonene C9H18 126 2,2,4, 4-tetramethylpentane C9H20 B 128 2,3,5-triniethylhexane C9H20 B 128 Nonane C9H20 D 128 Menthene C10H18 A,D 138 2,7-dimethyl-3,5-octadiene CioHig B 138 3-methylenenonane C10H20 B 140 3,5-dimethyloctane C10H22 B 142 3-ethyloctane C 10^22 B 142 264 Table 5-3 Continued Compound AliphaticslAromatics (continued) 3-methyInonane Decane 2,8-diniethyl-l,8-nonadiene 3-undecane Undecane 1,8, lO-dodecatriene 4-dodecene 2,2,7,7-tetramethyloctane Dodecane 2,2,6-trimethyldecane 2,6,7-trimethyldecane 2,5-dimethylundecane 3-methyldodecane 4-methyldodecane Tridecane Tetradecane 2,7, lO-trimethyldodecane Benzene Toluene Styrene 1,2-dimethylbenzene 1,3-dimethylbenzene Ethylbenzene Trimethylbenzne l-ethyl-2-methylbenzene l-ethyl-3-niethylbenzene l-ethyl-4-methylbenzene 1-methylethylbenzene Propylbenzene 1,2,3,4-tetramethylbenzene l-ethyl-3,5-dimethylbenzene 2-ethyl-l,4-dimethylbenzene Fonnula Remarks I'm C10H22 B,C 142 C10H22 D 142 ^n^io A,D 152 C11H22 B 154 C11H24 156 C12H20 A 164 C12H24 B 168 C12H26 170 C12H26 B 170 C13H28 B 184 ^n^2& B 184 ^n^zs B 184 C13H28 B 184 C13H28 B 184 ^uHzs 184 ^14"30 A,D 198 C15H32 A 212 CaH, CD 78 C7H8 CD 92 CsHs D 104 ^8" 10 B,D 106 ^8"l0 106 QHio D 106 Cc,Hi2 B 120 ^9^,2 120 C9H12 120 C9H,2 120 C9H12 120 C9H12 D 120 ^10^14 134 ^10^^14 B 134 ^ioHi4 134 265 Table 5-3 Continued Compound A lipha tics I A romatics (continued) 4-ethyl-l,2-dimethylbenzene 2,3-dihydro-5-methylindene Octahydro-2,2,4,4,7,7-hexamethyl- IH-indene Esters Formic acid, Propyl ester Formic acid, Butyl ester Formic acid, Octyl ester Acetic acid, ethyl ester Acetic acid, phenylmethyl ester Butanoic acid, ethyl ester 3-methylbutanoic acid, ethyl ester Halides Ethylchloride 1-chloropentane 1-chlorohexane 1-chlorooctane 1-chlorodecane Heterocyclics Pyrrole 2-methyl- IH-pyrrole 3-methyl-lH-pyrrole 2,3,6-dimethylpyridine 1,2,3,4-tetrahydroquinoline 2,3-dihydro- IH-indole l-methyl-3-nitropyrazole 2,3-dihydrofuran 2-methylfuran 3-methylfuran Formula Remarks I'm C10H12 C15H28 A A 134 132 208 C4H3O2 C 88 C5H10O2 C 102 C9Hig02 A 158 C4H802 C 88 C9H10O2 150 QH,202 C 116 QH,,02 130 CHjCl C 64 C5H11CI 106 QH13CI D 120 CgHj^Cl B 148 C10H21CI 176 C4H5N C 67 C5H7N D 81 C5H7N D 81 QH,,N B 121 C9H11N C 133 CgH^N 119 C4H5N3O2 A 127 C4H,0 70 CsH^O D 82 CsH^O D 82 Table 5-3 Continued 266 Compound Heterocyclics (continued) 2,3-dihydro-5-methylfuran 2-pentylfuran 2-octylfuran 3-furaldehyde 4-(5-methyl-2-furanyl)-2-butanone Ketones Acetone 2-butanone Bu tan one 2-pentanone 3-methyl-2-cyclopenten- 1-one 2-methyI-l-penten-3-one 4-methyl-2-pentanone 2-hexanone 3-methyl-2-hexanone 2-heptanone 6-methyl-3,5-heptadien-2-one 6-methyl-5-hepten-2-one 6-methyl-5-hepten-3-one 4-methyl-2-heptanone 6-methyl-2-heptanone 2-octanone 4-isopropyl-2-cyclohexen- 1-one 2-nonanone 2-decanone Acetophenone 2,3-dihydro-lH-inden-l-one Octahydro-2H-inden-2-one 2,3-butanedione 2,3-dimethylquinone Formula Remarks I'm QHgO D 84 C5H14O 138 C12H20O A 180 C5H4O2 96 CgHjjOj B 152 CjH^O C 58 C4H3O CD 72 QH^O B 72 C^H.oO CD 86 QHsO 96 QHjoO B 98 CaH^^O B 100 QH.^O D 100 C7H14O B 114 CyH,,0 114 QH12O D 124 QHi.O D 126 QH.^O 126 QH,,0 128 CsH,,0 128 C«H,,0 128 C9H,40 A 138 CgHigO 142 C10H20O D 156 CgHeO C 120 CpHgO B 132 C9H,40 A 138 C4H,02 C 86 QHiqOj A 138 Table 5-3 Continued 267 Compound Sulfides Carbondisulfide Dimethylsulfide Dimethyldisulfide Thio/Thioesters/Sulfonyls Thiomethane 3-methylthiopropanal Formula Remarks Tm CS2 CD 76 C,H,S C 62 CzHgSz D 94 CH4S C,D 48 C^HgOS B 104 Ureas/Related Urea CH,N,0 41^2^ CD 60 Miscellaneous 2-methoxy-l-propene C,H,0 B 72 l-methoxy-4( l-propenyl)benzene C10H12O A,E 148 Compounds present in background/Blank analyses Methylsilane CHjSi 46 Hexamethylcyclotrisiloxane CeHjgOjSij D 222 Octamethylcyclotetrasiloxane C8H2404Si4 D 296 Decamethylcyclopentasiloxane CioHjoOjSis D 370 Methylchloride CH3CI CD 50 Methylenechloride CH2CI2 C 84 Chlorofoim CHCI3 118 Carbontetrachloride CCI4 152 Trichloroethylene C2HCI3 130 1, 1, 1-trichloroethane C2H3CI3 132 Tetrachloroethylene C2CI4 D 164 1,2-dichlorobenzene C,H4Cl2 146 l,4-dichlorobenzne-D4 C,Cl2D4 150 268 Table 5-3 Continued Compound Formula Remarks r.M Compounds present in background/Blank analyses (continued) l-chloro-4-(l-chloroethenyl) CgHioClj 176 cyclohexene l-chloro-5-( 1-chloroethenyl) CgHioClj 176 cyclohexene 1,6-dichloro- 1,5-cyclooctadiene CgHjoClj 176 Dichlorofluoromethane CHCUF 102 Chlorodifluoromethane CHCIF2 86 Dichlorodifluoromethane CCI2F2 120 Trichlorofluoromethane CCI3F 136 l,l,2-trichloro-l,2,2-trifluoroethane C2CI3F3 D 186 Difluorobenzene CaH^F^ B 114 Bromodichloromethane CHCl^Br 162 Dibromochloromethane CHClBr^ 206 Bromochlorodifluoromethane CClBrF^ 164 Naphthalene ^loHg 128 Carene C10H16 A 136 3-carene ^ioHi6 B 136 4-carene C10H15 B 136 Limonene ^ioHi6 136 a-pinene ^ioHi6 136 /3-pinene ^10"l6 136 a-phellandrene ^10"l6 136 j3-phellandrene ^loHie 136 Explanation of remarks: A) Exact identification of this compound is questionnable c lue to low purity value. B) The class or base structure of this compound is characterized by a specific EI fragmentation pattern. Location of bonds and/or substitution may be uncertain. 269 Table 5-3 Continued Explanation of remarks (continued): C) Compound was previously reported in the literature as a compound having dermal origin (table 5-1). D) Reported as being observed in the cryo-focused GC/MS analyses (table 5-2). E) See text for further explanation. 270 further implications with respect to diet and mosquito attraction, where the consumption of foods enhancing mosquito attraction may increase potential attractants on the skin. Further studies involving alcohol or food consumption and emanation from the skin were not conducted for this dissertation. The second component of interest is l-methoxy-4(l-propenyl)benzene (anethole). The reason this compound was noted is due to previous field studies in which methoxybenzene (anisole) gave favorable response to the Culex nigripalpus species of mosquito. Should the chemosensilla of this mosquito be compound specific with some class dependency, then it would be beneficial to test similar structures which are present on the skin. Anisole was not observed in emanations analyzed for this work; however, anethole is similar in base structure. Case Study Comparison of Emanations between Subjects The comparison studies between subjects were performed not only for comparative purposes; the data were also examined for compounds which decreased in relative abundance over a period of time, typically 6-8 hours. This was accomplished by placing handled beads in a tube open to air. The subjects were chosen based upon their relative attraction of Ae. aegypti in an olfactometer bio- assays. The subjects were Mr. Dan Smith, who has been found to be at the high extreme with respect to attraction percentage (c. 70%), and Mr. Carl Schreck, who has been found to be at the low end (c. 20%). The RIC traces for one such 271 comparative study are presented in figure 5-2. The top trace, produced from beads handled by Mr. Schreck contains, for the most part, the same peaks as that of Mr. Smith (bottom trace). The glycerol peak, located at a retention time of approximately 18.8 min, is not only chromatographed better on the FFAP column employed here (versus the HP5 column used in figure 5-1), but it is present in much greater amount in Mr. Smith's sample. As mentioned earlier, this discrepancy is due to residual glycerol on the hands from the Sta-Sof-Fro~ hair and scalp spray. Peak heights were measured and normalized to the peak height of tetradecane. The relative peak height ratios of component peaks were then compared to determine specific components which were increased in one subject compared to the other. Sample analyses were also compared with respect to amounts of components present on the glass beads analyzed immediately and on glass beads which were analyzed 6-8 hours later. The results of these comparative studies are presented in table 5-4. This table contains only two remark codes. A code of (A) denotes compounds for which the exact structure is not known versus other isomers of the same compound. A remark code of (B) denotes that discussion of this compound is found in the following text. The designation of significantly or slightly combined with increased or decreased employed in the table merits some explanation. It was found that the relative abundances of present components in each sample vary between analyses. 'o 5 o .2 c« ^ c -a o c u o o i: E « « U ^ ^ >A o o E:S o c 9 ^ Ui . W .CO 273 e E u w w Oi « 9 01 B cs E s B S - CD - tt 1 Oi 3 1/1 e a E 3 S r «• s> - IS" UT 09 (S CD ® C' (S O) S) o C I'M (9 CO •■£> •T- CO (%) i()isna)aT 3aijbi3H 274 Table 5-4 Comparison of compounds present on the skin which are observed to be relatively increased from one host with respect to the other. Compounds which decrease markedly after an eight hour period from the more attractive host are also reported. This table is categorically divided into five sections. A significant change reflects a relative difference or change by a factor of 5 or greater between hosts, or between host emanations and emanations detected after 8 hours. A slight change refers to compounds with a relative difference of a factor range of 2-5. Compound Remarks Compounds significantly increased in more attractive host: Lactic acid Methyltridecanoic acid A Pentadecanoic acid 9-hexadecenoic acid Octadecanoic acid 4-hydroxy-3-methoxybenzoic acid l-hepten-3-ol B Glycerol Squalene Toluene N,N-dimethyl-l-dodecanamine B N,N-dimethyl-l-tridecanamine B N,N-dimethyl-l-tetradecanamine B N,N-dimethyl-l-hexadecanamine B N,N-dimethyl-l-octadecanamine B 14-methylhexadecanoic acid, methyl ester 6-Octadecenoic acid, methyl ester 2-hydroxybenzoic acid, phenylmethyl ester Pentanedioic acid, ester A Hexanedioic acid, mono(2-ethylhexyl ester) Pyridine 3-( l-methyl-2-pyrrolidinyl)pyridine B Oxazole 2,3-dihydro-3,5-methoxy-6-methyl-4H-pyran-4-one 2-methylisothiazole 2-butanone 2-pentanone 3-pentanone 2-decanone 275 Table 5-4 Continued Compound Remarks Compounds slightly increased in the more attractive host 12-methyltetradecanoic acid Methylpentadecanoic acid A Heptadecanoic acid 4-hexen-l-ol B 2,5-bis( 1, l-dimethylethyl)phenol 2-methylpropanal 7,ll,15-trimethyl-3-methylene hexadecane Docosane 2-butenoic acid, Butyl ester 4-hydroxybenzoic acid, Propyl ester 2-hydroxybenzoic acid, Phenylmethyl ester 1-chlorotetradecane 4-pyridinamine 2,3-dihydro-4-methylfuran A 4H-pyran-4-one, substituted A 6-methyl-5-hepten-2-one Urea Compounds significantly increased in the less attractive host Dodecanoic acid Cholesterol 3-methylpentanol Heptane Methyliodide 1,3-butanediamine A 14-methylpentadecanoic acid, Methyl ester Compounds slightly increased in the less attractive host Decanoic acid Methyltetradecanoic acid Heptanal Nonanal 2,4-nonadienal 276 Table 5-4 Continued Compound Remarks Compounds slightly increased in the less attractive host (continued) 4-nonene Nonane 3-dodecene Hexacosane Compounds significantly decreased after 8 hours (in the more attractive host) Hexanedioic acid, Mono(2-ethylhexyl ester) 2-methylpropanal 3-methyIpentanal N,N-dimethyl-l-dodecanamine B N,N-dimethyl-l-tridecanamine B N,N-dimethyl-l-tetradecanamine B N,N-dimethyl-l-hexadecanamine B N,N-dimethyl-l-octadecanamine B 2-octene 4-nonene Benzene Toluene Styrene Pyridine Oxazole IH-indole 2-butanone 2-Pentanone Explanation of remarks: A) Location of substitution or double bond position is uncertain. B) See text for discussion. 277 Examples of this follow. Lactic acid was found to vary from 5 times greater in the more attractive host to 1.3 times greater in the lesser attractive host. In the same analyses, 12-methyltetradecanoic acid was found to remain fairly consistent at 1.9-2.2 times greater relative abundance in the more attractive host. Therefore, the tables reflect not necessarily an average value; they reflect what is found in most of the analyses. However, the variation of components from the same host should be kept in mind when examining the results presented here. These compounds may not necessarily yield the answer to what components are attractants. These compounds are still the more likely candidates for the attractants and should be examined by olfactometer and field studies. The presence of 4-hexen-l-ol and l-hepten-3-ol are of particular interest. The use of l-octen-3-ol, as stated in Chapter 1, has been found to attract various species of mosquito. These similar, but lower molecular weight alcohols (and their isomers) may provide better attraction than l-octen-3-ol, again treating mosquito detection as class/structure dependent. The other components of interest were the tertiary amines previously discussed in the section corresponding to table 5-2. These amines were found to be significantly increased in the more attractive host and found to decrease significantly after 8 hours. These two findings fit the profile of attractants sought after. That is, compounds present in greater abundance in the more attractive host and decreased markedly after 6 hours (as noted in olfactometer tests). Care should be taken with this class of compounds; it is unknown whether or not these 278 compounds originate from humans or may are deposited on the skin by external sources. The final compound to be addressed from this table is 3-(l-methyl-2- pyrrolidinyl)pyridine (nicotine). This compound was found in much greater abundance on the skin for subjects who use tobacco products. Case Study Comparison of Bio-assay to GC/MS Assay The final study of this chapter involves the analysis of emanations from a single host, collected once per day for GC/MS and bio-assay, over a five-day period. The advantage to this comparison is thought to be that a single host provides a more stable and consistent matrix on the skin, with less chance of markedly different trace emanations. The drawback is that the subject variation in attraction to mosquitoes will not vary as much as it would for persons chosen at the extremes of attraction (as in the previous case study). The range of attraction in the olfactometer, for the five-day period, fell between 12 and 27%. The actual comparison employed only the first two days. The RIC traces from samples analyzed on these two days are shown in Figure 5-3. The results from these analyses are reported in table 5-5. The remark codes used in this table are identical to those for table 5-4. The two compounds of interest from this table are the unsaturated alcohols identified as 4-hexen-l-ol and l-hepten-3-ol. Both of these compounds were present at increased relative abundance on the day the human subject was more attractive -a .22 X) re CO c iS o aj O r-i D '^ +-i (U 3 c 1) -a (/I -a re c/j o c O re iX 1 s . SI CB R • 5 ^ : >. >% • Si «s CO •— (S 1 •o r IP B O 1/1 — ^ j 3 - u S S SI 00 ® Qd ® St O G kD ■«■ C\J «i 00 vc s- o c :i (%) iCjisnajuT aAiiBpH 281 Table 5-5 Comparison of emanations present on the skin on consecutive days from a single human subject. Bio-assays conducted on those days showed 12% and 24% attraction of mosquitoes in an olfactometer. An increase in the table reflects a compound found to be present in a greater relative amount on the second day (24% attraction) with respect to the previous day (12% attraction). A significant change reflects a relative difference by a factor of 5 or greater between days. A slight change refers to compounds with a relative difference of a factor range of 2-5. Compound Remarks Compounds significantly increased on more attractive day: Pentadecanoic acid Methylpentadecanoic acid A Hexadecanoic acid l-hepten-3-ol B Compounds slightly increased on more attractive day 9-octadecenoic acid 4-hexen-l-ol B 2,4-nonadienal Butanamine A Nonene Menthane A Tricosane 13-methylpentadecanoic acid, Methyl ester Methylhexadecanoic acid, Methyl ester A Compounds significantly decreased on more attractive day Benzoic acid, substituted A Heptadienal A 2-methyl-l-heptene Nonane, branched A 1-chlorohexane A 1-chlorononane Pyridine Urea 282 Table 5-5 Continued Compound Remarks Compounds slightly decreased on more attractive day 3-hexadecanol A Nonanal Benzaldehyde Dodecene A 2-methyl-2-dodecene 5-tetradecene Methyltridecanoic acid, ethyl ester A Octenone, branched A 3-methylthietane Explanation of remarks: A) Location of substitution or double bond position is uncertain. B) Discussed in text. 283 to mosquitoes. Again these compounds, as well as other compounds should be referred to as good candidates for attractants. In general, the comparison study conducted here, involving the same subject on consecutive days, showed less overall variation (compared to the previous study with different subjects) in relative abundances of sample components present. Conclusions Identified Emanations Approximately 310 compounds were detected in cryo- focused GC/MS analyses and 200 compounds from purge and trap GC/MS experiments. The components present on the skin were found to be very similar among subjects; however, relative abundances of components vary whether comparing two different subjects, or the same subject at different times. Cryo-focused GC/MS analysis allowed for identification of a wide range of classes whereas microscale purge and trap discriminated against polar compounds, giving slightly different information as a result. GC/MS Assay of Subjects with Different Attraction Levels There were 46 compounds which were increased in relative abundance in the more attractive host to mosquitoes. Only 9 compounds were found (with reasonable certainty) to be relatively more abundant in the lesser attractive of the hosts. Among 284 the 46 compounds are lactic acid, unsaturated alcohols, and tertiary amines. Lactic acid is a known attractant, and was observed present in greater abundance (in most cases) for the more attractive host. This may partially explain the attraction oiAedes aegypti to this host as well as fluctuations in mosquito attraction. The unsaturated alcohols are of interest due to the similarity in structure to l-octen-3-ol, a compound which has been found to attract various species of mosquitoes. The tertiary amines were not only found in increased abundance on the more attractive host; they also decreased significantly in abundance when analyzed for 8 hours later. This profile is similar to observations concerning attraction to handled glass in an olfactometer. Bio-assay versus GC/MS Assay The most difficult and potentially most informative study involved comparison of the same subject on consecutive days. TTie reason for this difficulty stems from the similarity of the skin matrix for the same host, only minor variations in relative abundances of most components were observed. Thirteen compounds, including the unsaturated alcohols mentioned above, were found to increase on the more attractive day; seventeen were found to decrease. This discrepancy may simply be due to normal variations in abundances from day to day, as was seen throughout many of the analyses conducted for the previous case study. Additionally, imprecision exists in bio-assays such that the attraction percentage may not exactly reflect differences between days reported in this study. CHAPTER 6 CONCLUSIONS AND FUTURE WORK Conclusions This dissertation has covered various facets of applying mass spectrometry to elucidating the chemical basis for mosquito attraction to human hosts. The initial concern was the utilization of a sampling method which minimally biased the compounds to be detected while providing some discrimination against compounds which are not of interest, i.e. non-volatile components. This was accomplished by desorbing volatiles off handled glass beads. Host attraction is known to be transferrable to glass and it is believed that the glass actually concentrates the attractant(s), as well as many other compounds. The sampling method chosen for the majority of work was cryo-focusing, with some studies employing microscale purge and trap. Simple cryo-focusing provides the benefit of narrowing sample bands on the GC column and the detection of gas- phase emanations with minimal sample discrimination. The additional step of employing Tenax and cryo-focusing traps (in microscale purge and trap) provided benefits with respect to the removal of the abundant carboxylic acids (as well as other highly polar compounds) by the metal in the system; however, it is not known at this time if this also removes potential attractants as well. The data from 285 286 microscale purge and trap GC/MS applied to skin emanations for this project are preliminary; microscale purge and trap experiments have shown that the composition of components desorbed from beads is similar to that from the enclosed hand in a Tedlar bag. The difference between these two sample introduction methods lies mainly in the markedly different relative intensities of some components. Further studies involving quantitation will be required to confidently report the differences. The negative ion fragmentations, attachments, and oligomerization (polymerization) of lactic acid, the only previously known attractant for Ae. aegypti, were examined in Chapter 3. Negative ions are typically not employed for structural information; however, lactic acid can be readily identified via NCI. Oligomerization reactions, as well as chloride ion attachment, provide easily detectable ions in the NCI mass spectrum of lactic acid. Although lactic acid is one of the major components found emanating from the skin, the determination of its presence by EI is not achievable due to the absence of the M"^' ion and the resultant low mass fragments. Altering the matrix conditions by adding acid or base to lactic acid affected the attraction oiAe. aegypti. The observed effects have been rationalized to result from simple acid-base dissociation equilibria. The addition of acid enhanced the attraction of mosquitoes to the sample; the addition of base decreased the attraction. The addition of acid to various lactic acid esters and methyl isovalerate 287 was also examined. Increased attraction of mosquitoes occurred in all cases for the sample containing acid relative to the unmodified sample. Skin emanations collected on three 2.9 mm glass beads were found to contain a greater amount of sample than is present from the dissolution of 0.15 mL perspiration in 1 mL methanol. Due to the absence of components in the aqueous phase, it is suspected that lactic acid (and other attractants) preferentially reside in the oily/waxy phase which emanates from the sebaceous glands. Decreased attraction to mosquitoes has been observed in cases of heavy perspiration. If attractants preferentially reside closer to the skin, below the evaporating aqueous phase, then the reduced attraction which is observed can be explained by a masking effect from heavy perspiration. Tandem mass spectrometry was employed in initial experiments of this dissertation project to compensate for short-column chromatography. The detailed fragmentation studies of lactic acid also necessitated the use of MS/MS modes. A daughter library of characteristic fragmentations was compiled and used to rapidly assess the compound classes present. The results from the rapid screening yielded a conservative estimate number of compound classes as well as number of components present within each class (or similar classes). Over 350 GC peaks are observed in the work of Chapter 5. There are 310 components identified in the cryo-focused GC/MS analyses; approximately 20 of these have been attributed to background. Human subjects observed throughout this 288 work appear, for the most part, to be similar with respect to composition of emanations present on the skin. The components present vary in relative abundance among subjects. This was the basis for the comparison studies conducted in this chapter. Components found increased or deceased between hosts are reported in this dissertation. Additional studies involved the comparison of GC/MS results to bio-assay results; compounds observed to increase or decrease between days were reported. The findings from these case comparison studies may not provide the ultimate answer to solving the problem of mosquito attraction; however, the work contained herein should bring the solution closer. Future Work Continued use of a single-stage cryo-focusing or single cryo-focusing trap may be beneficial when combined with the use of Tedlar bags described for purge and trap analyses. Although the preferential discrimination of samples which do not transfer and subsequently volatilize from glass is lost, preliminary work comparing the observed components from glass beads and from the hand enclosed in a Tedlar bag show that the presence of components is very similar. Additional studies need to be conducted to verify this. Continued use of a cryo-focusing GC/MS could be conducted with less steep ramp programs and longer columns. This would effect better separation of components which co-eluted during analyses in this work. The 289 real problem to be overcome will be the high concentration of acids present when analyzing for additional trace components. Continued use of microscale purge and trap with direct sampling of the hand in a Tedlar bag will most likely provide, in the near future, a useful method of removing acids. Though this method is subject to compound discrimination of polar compounds, concentration of sampled air is a desirable attribute. Air trapped in the bag was used for the experiments contained in this dissertation; however, it is not necessary that air be used. Once the hand is enclosed in the bag, the air could be purged from the bag by an inert gas, such as helium. Purge and trap analyses allowed for concentration of highly volatile materials and for removal of the high concentrations of carboxylic acids which interfere with determination of trace components present. Quantitation is a dilemma with respect to the bead sampling methods. The actual quantitation by mass spectrometry is not the problem; however, sampling in a consistent manner is. The concentration and presence of components on the skin is not constant and is affected by many variables that have been observed to vary from day to day. One possibility for quantitating known emanations from the skin with direct sampling employing a Tedlar bag might be to coat an area of the skin with a known non-toxic volatile to semi-volatile compound. This may yield some insight about collection efficiency of emanations. Attempts could be made to spike the beads with known concentrations of, for example, lactic acid and additional 290 carboxylic acids. In particular, since preliminary attempts to measure quantitatively (by weight) sample deposition onto beads were not successful, a study could be conducted to attempt to do this. The protocol may consist of using a steep temperature ramp to cut down on analysis time and focus only upon the carboxylic acids. Beads could be quantitatively analyzed after handling by comparing peak areas of target compounds from one bead, two beads, three beads, etc. The results from Chapter 3 demonstrated an enhancement in attraction of mosquitoes to samples which had been spiked with acidic solution. An experiment of interest is to spray a fine mist of acidic solution or basic solution onto handled petri dishes. Various comparisons could be made between the attraction of mosquitoes to the untreated handled dish, the dish sprayed with acidic solution, and the dish sprayed with basic solution. An enhancement in attraction towards acidified dishes for one or more species of mosquito may implicate an acid or acids as the potential attractants. This knowledge could then be applicable to GC/MS assays. The dietary intake of a host influences compounds on the skin. It is believed by some that certain foods enhance attraction. This matter should be examined by comparison studies involving both bio-assay and GC/MS assay. Combining any extraneous knowledge with the knowledge of what is present on the skin should assist in targeting potential attractants. If a connection is observed between dietary intake and mosquito attraction, then the cryo-focused GC/MS or purge and trap GC/MS procedures described in Chapter 5 could be applied to this difference. If specific 291 components are found to increase (or decrease), these should be targeted for testing by bio-assay. Another case study related to that described above involves exercise. Attraction of mosquitoes is enhanced (up to a point) during physical exertion and perspiration. Sampling at the proper time interval may yield samples with increased attractant(s) relative to other components in the matrix on the skin. This is speculative; however, should attractants be found in greater concentration on the skin, glass beads could be handled at that time. The bead method would then allow for greater concentration of the attractant(s) while keeping to a minimum the water (from perspiration) adsorbed to the glass, which in turn minimizes water subsequently introduced into the system. The case studies examined and discussed thus far have focused on human subjects. Humans are not the sole hosts of mosquitoes. Many species of mosquito feed on multiple hosts. Examining emanations from humans and animals known to attract the same mosquito species may aid in determining the attractants by reducing the components of interest to those present in both sets of emanations. This again is somewhat speculative in that there is no guarantee that specific mosquitoes have only a few types of chemosensilla. There is a possibility that the mosquito may detect different volatile components for each host. Tandem mass spectrometry was used only briefly in this work. The studies for chapter 5 yielded many identified components from PPINICI and EI analyses 292 alone. There were, however, components that failed to give useful fragmentation patterns in one or more of these modes, preventing confirmation of presence of a compound or in some cases (at least 37) prevented even a speculative estimate of compound identity. There were also cases where compounds were identifiable by PPINICI and/or by EI analysis; however, upon background subtraction, valuable fragmentation information was lost due to coelution of sample components. Tandem mass spectrometry could be employed to obtain daughter spectra of the selected molecular ion, protonated molecular species or deprotonated molecular species to assist in the identification process. There were cases where both the molecular ion (EI) and protonated or deprotonated species (CI) were absent; the use of a reagent gas with lower proton affinity is recommended for PCI to obtain "softer" ionization. Attempts at employing CO, as a reagent gas were not successful in this case; however, should this be of interest in the future as a potential reagent gas, the background is covered in the appendix to this dissertation. The final two studies to be discussed involve the most complex and intricate arrangements. On-line monitoring by mass spectrometry and olfactometer would be advantageous (analogous to organoleptic evaluations used in the fragrance industry). This application was discussed briefly in Chapter 1. The problems to overcome with respect to analyses of this type would be location of both the mass spectrometer and olfactomer, and more importantly, the ability of mosquitoes to respond to successive stimuli should that occur. Mosquitoes may be desensitized by certain compounds. 293 or high concentrations of compounds in general. In addition, the response of mosquitoes to a stimuli is not an immediate process, such as the detection of column eluents by a mass spectrometer. One way to achieve immediate mosquito response would be to conduct on-line tests with mosquito antennae. This requires a knowledge of mosquito antennae such that specific receptors can be correlated to give responses to specific stimuli. On-line mass spectrometry, with for example, a column spHt, could be used to identify the compound eliciting a response. In summary, work conducted in the future in any of the aforementioned areas will provide knowledge that may identify the attractant(s) that have eluded identification thus far; at the least, this information will move humankind one step closer to the answer. Care should be taken with respect to the sampling method such that it answers the question that is being asked. Knowledge of discriminatory effects of the sampling and detection method is of importance. One final issue, applicable to all analyses conducted, is washing of the skin prior to handling beads of sampling of the hand in a Tedlar bag. This methodology should be examined with respect to solvents and time required to allow the skin to dry and additional emanations to concentrate on the skin. Handling of objects should be restricted after washings and until the sample collection procedure is completed. APPENDIX CARBON DIOXIDE AS A CI REAGENT GAS Introduction This appendix is a compilation of background information and preliminary experiments involving carbon dioxide as a reagent gas for CI. Coverage in this appendbc is mainly directed towards the use of COj for production of thermal electrons for electron capture negative ion chemical ionization (ECNCI); however, discussion of charge exchange (CE) for positive ions is also included due to the predominant use of CO2 as a charge exchange reagent gas. The examples included in this appendix pertain to optimization of CO2/ECNCI on the TSQ70, background ions present in positive CO2/CE and negative CO^/ECNCI mass spectra, and illustrations of difficulties encountered in these experiments. Due to time constraints, it was not possible to conduct a complete exploration of CO2 as an electron moderator for ECNCI experiments related to the analysis of volatile skin emanations. Attempts to replace CH4 with CO2 as the reagent gas were not successful with the sampling method and detection scheme used for analyses in Chapter 5. The abundance of acid eluents in the ion source led to self-CI reactions. Removal of acids by the purge and trap system described in Chapters 2 and 5 would be beneficial for subsequent attempts at 294 295 employing this novel reagent gas. This appendix provides the fundamental background for future work of this nature with optimization related to that on the TSQ70 triple quadrupole mass spectrometer. High Pressure Charge Exchange Mass Spectrometry The main use of CO2 as a reagent gas has been in the analysis of positive ions [33,74]. This reagent gas functions in positive ion mode as a medium for charge exchange. Unlike reagent gases employed for work in this dissertation (i.e. methane and isobutane), carbon dioxide does not contain a hydrogen for proton transfer reactions. Therefore, only hydride (or heavier ion) transfer, electron transfer, or adduct formation are viable routes for the production of positively charged sample ions [33,74,112]. Charge exchange (see Chapter 5 for additional information) occurs by electron ionization of the reagent gas, with subsequent transfer of the positive charge from reagent gas to sample molecules via collisions. The resultant deposition of internal energy (Ej) from these collisions is given by: E; = RE(X*') - IE(M) A-1 where RE (X*') is the recombination energy of the reagent gas ion, X"^', and IE (M) is the ionization energy of the sample molecule, M. The recombination energy for COz"^' is 13.8 eV [74,113]. Extensive fragmentation occurs for a high value of E^. If RE (X"^*) is only slightly greater than IE (M), then the mass spectrum is expected to contain predominantly the M*' ion. 296 Charge exchange reagent gases provide a means for controHing the extent of fragmentation. The mass spectra produced are similar to EI spectra; however, due to the average energy deposition being lower for CE than for EI, the abundance of M^* will be greater. In the case of carbon dioxide, there are several species which can interact with sample molecules. The COj^* ion at m/z 44 is the most obvious charge exchange ion. The (C02)2** cluster ion (RE unlisted) at m/z 88 and the 02"^' ion (RE of 9.7-17.0 eV) at m/z 32 may also react. These species, however, will only have a minor impact on the overall fragmentation pattern due to the low abundance of these ions with respect to COj"^'. One concern involved with the choice of a charge exchange gas is the production of secondary ion species which may also be involved in the charge exchange process. There is also a greater variation, compared to conventional CI, in major ion abundances of CE reagent gases as a function of ion source pressure. Therefore, optimization and more precise control of the pressure is necessary such that the predominant charge exchange ion is the reagent ion of interest [112]. Water is one of the most detrimental impurities. The presence of [M+H]"^ ions from a reagent gas incapable of proton transfer may be due to self-CI or from reaction with ions produced from water, e.g. (H30)'^ at m/z 19, (CHO)^ at m/z 29, and (C0-H20)* at m/z 46. 297 Electron Capture Negative Ion Chemical Ionization Electron capture NCI was addressed in Chapter 5; a brief overview is included here to provide continuity of this appendix. Negative ions are produced from electron capture by three processes [33,72,74,108]. The least common process is that of ion-pair production, whereby the electron is captured, exciting the sample molecule, and then re-ejected at a lower kinetic energy, leaving the molecule dissociated into a positive ion and a negative ion. The more common processes are those of associative resonance electron capture (equation A-2) and dissociative resonance electron capture (equation A-3) of thermal electrons (Ct^"): e,h~ + MX ^ MX— A-2 e^h" + MX ^ M + X-' A-3 In order for a molecule to capture an electron, the electron affinity (EA) of the molecule must be positive. Additionally, the electron attached species must be long- lived enough for collisional quenching (MX~) or dissociation (M-f X~) to occur [72,74,108,114-116]. The appeal of ECNCI, for compounds amenable to this process, is due to the higher efficiency of electron/molecule reactions compared to conventional CI ion/molecule reactions [72,74,108,117]. Unfortunately, ECNCI is not applicable to a majority of compounds; however, this selectivity is generally considered an advantage rather than a disadvantage. Although the electron capture processes are fairly straightforward, mass spectra obtained by ECNCI are not always simple to 298 interpret. Halogenated species are excellent candidates for ECNCI; however, adduct formation (e.g. [M+Cl]") may occur [72,81,107,108,114,118-120]. Ion source surface or wall reactions may take place, ultimately complicating mass spectra [114,119,121]. Tetracyanoethylene (TCNE), having a relatively high (positive) EA, readily undergoes EC reactions. It is also particularly susceptible to unexpected ion formation from electron capture [119,121,122]. This compound, when examined by ECNCI with methane and with carbon dioxide reagent gases revealed the absence of unexpected ions for COj/ECNCI [121]. ECNCI with Carbon Dioxide Carbon dioxide has been examined previously as a moderator for thermal electron production for ECNCI of TCNE [121]. It has been shown that when mixed with argon for NCI work, it will enhance the production of the [M-H]~ ion [123]. The allure of CO2 to work contained in this dissertation is from the potentially simplistic mass spectra for the determination of volatile skin emanations which can undergo electron capture. Carbon dioxide is efficient at relaxing electron energy distributions, i.e. forming thermal electrons [124,125]. The physical reason for this is the ability of the CO2 molecule to form a short-lived ion (COj"), which autodetaches a lower energy electron, leaving CO2 in a vibrationally excited state [126]. By virtue of the size of a CO2 molecule, it is also efficient at collisional quenching (compared to other inert 299 and relatively inert gases) [115]. One additional feature of this reagent gas merits mentioning. Due to the absence of hydrogen in the system and the low proton affinity of CO2 (548 kJ/mol), formation of the [M-H]"" ion from samples would be a rare occurrence with this reagent gas. As with CO2/CE, impurities present in the ion source need to be minimized to prevent unexpected or unwanted reaction pathways. Production of an [M-H]~ ion from this reagent gas is a likely indication that an impurity is present, or that self-CI is occurring. Self-CI reactions were found to be the most significant cause, in the work for this dissertation, for the inability to perform electron capture. The elution of high concentrations of acids into the ion source, as stated previously, generated mass spectra comparable to that of methane NCI. Instrument Optimization and Background Ions The optimization for negative ion analysis was carried out by examining characteristic PFTBA negative ions at m/z 264 and m/z 414, as well as the m/z 127 ion (I~) found in the background on the TSQ70. The optimization plot is presented in figure A-1. Formation of ions requires indicated pressures greater than 500 mtorr CO2 in the ion source. Examination of the profiles demonstrates the need for precise control over the ion source pressure to maximize sample ion generation efficiency. The optimum indicated pressure of 1200 mtorr is most likely a combined 60 ■*-• c e>o CQ U o u s o CA C« M s _o a> V G "O u en <4-l O CO a 3 60 301 o o u r4 (siunoo 0001 ^) ^isusjiq 302 tradeoff between COj efficiency of electron thermalization and collisional quenching of sample ions by this gas. The negative ion mass spectrum of PFTBA, acquired at an indicated pressure of 1200 mtorr CO2 reagent gas, is shown in figure A-2. All ions found in the mass spectrum, with the exception of m/z 60, are characteristic ions seen with this instrument when methane is employed as the electron moderating gas for NCI. The m/z 60 ion is presumed to be COj"'. This ion has been examined previously due to concerns with modeling the ionized atmosphere above the earth, and for instability problems with CO2 lasers [127]. Based on this literature source, it was proposed that the C03^* ion is formed via collisions of COj with 0~'. Later studies support the attachment of an electron to clusters of (C02)n, whereby stepwise elimination of neutral CO2 or CO occurs from the solvated ion to eventually yield CO3 [128]. Additional negative ion clusters are absent from the background spectra shown here due to the low pressures employed in this study; the optimization for EC reactions occurred at a low pressure, i.e. 1200 mtorr. At pressures below 2000 mtorr, the CO3 and O ions predominate. The appearance of (C02)2 at m/z 88, or higher cluster ions, will not occur until pressures of 2000 mtorr or greater are reached [128]. Ions produced from cluster reactions in the positive ion mode are more abundant in the pressure regime of these studies (figure A-3). Major ions present in the positive ion background are at m/z 32 (02'^'), m/z 44 (C02'^*), and m/z 88, the (C02)2^' ion. Additionally, ions at m/z 45 and m/z 46 are visible in the mass o u o o o Ui u ;-> u § T3 V u e (3 o a. «3 e _o .> so z I < l-l 3 60 304 (%) iCjisnajni aAijBiaH •♦-• a l-r -a 4) u a O o o B o •a u •a o f-i a o § >s u s u a o I en O Oh l-l 00 306 (%) iCjisnajni 9ai;bph 307 spectrum. These are presumed to originate from either wall reactions, or the presence of an impurity such as water. A closer inspection of the low mass range (figure A-4) reveals the presence of HiO"^' and H3O*, at m/z 18 and m/z 19, respectively. The ions at m/z 56, 60, 72, and 76 are generated by CO2 cluster ions of (C02)C*', (CO^)©^*, (C02)C0**, and (C02)02+', respectively [128]. The ions at m/z 30, 34, 45, 46, 47, 63, 89, and 90 are presumed to result from impurities adding hydrogen to the system, either from water, or from surface-bound radicals on the ion source walls [121]. Experiments were conducted involving the addition of water to the system in both positive and negative ion modes. The mass spectra were acquired in the absence of calibration gas. The direct insertion probe was inserted with an empty vial as the blank; the second series contained water in the probe vial. The mass spectrum of the blank in positive ion mode is shown in figure A-5. The relative intensities of the water ions at m/z 18 and m/z 19 should be noted, as well as the ratio of m/z 32 (02~*) to m/z 28 (CO~'). Additionally, the intensity of the m/z 45 ion is approximately 30% of the m/z 44 base peak. The m/z 88 ion is at about 5% RA, with an ion at m/z 89 not observed in this spectrum. This experiment was repeated with water introduced via the direct insertion probe vial. The mass spectrum in figure A-6 clearly shows the presence of water in the system (compared to figure A-5). The intensity of m/z 18 and m/z 19 are increased to almost the intensity of the base peak. There is also an increase in the T3 ■*-> to u •a a O o o o .1^ o o o Oh re ■♦-» a (« E u 309 CB + HI

o "2 ° < G u •-" 3 -i .£: o "3 (U 1> t/1 c/1 re re '> a a c u o '^^ >-. 'tfl •4-t o o Oh VO T3 < 3 -a 3 o ftO 4-» E _C 313 (9 1 I r Si G> O "-o ^ rj (%) /{jisnajui aAijcp^ 314 m/z 19 ion relative to m/z 18 indicating increased Cl-like high-pressure proton transfer reactions. There is a significant increase in the production of m/z 32 (02"^') as well as m/z 45 and m/z 62, the (C02)H"^ and (C02)H20** ions, respectively. The presence of an ion at m/z 89 from (C02)2H^ is also noticeable in this mass spectrum. The mass spectra for equivalent experiments with negative ions are in figures A-7 (normal background) and A-8 (with water added). The addition of water to the system does not seem to generate an appreciable difference, except for the increased abundance of the m/z 16 (0~') and m/z 17 (OH~) ions. Although water may not affect the background ions of CO2 significantly, the 0~* and OH~ ions present may lead to conventional CI proton transfer reactions, generating unexpected fragment ions [129,130]. Selected Examples Impurities such as water, as stated previously, are not the only cause for unexpected results in a negative ion CI mass spectrum. An example of the presence of both wall reactions and self-CI from high ion source pressure can be seen (in figure A-9) for lactic acid. The predominant ions at m/z 179 and m/z 251 are characteristic ions found in mass spectra of lactic acid under saturated ion source conditions (see Chapter 3). These ions are attributable to self-CI reactions. The region of interest pertaining to suspected wall reactions is in the m/z 87 to m/z 90 8 .2 o 3 T3 O a O o l-l a, O U (J -3 o ■4-* 6 o o r-l I o •a § i-i f y 4^ C^ ca tf3 CA CC s a .2 i E ■*-• ■*-• (B (/} 60 ^ Z J2 -^^ 1 o < v • 1— 1 tH h> 3 o i3 318 rt s UJ y T _2_ JL i9 -00 1 1 (Si CO 1 SI 1 1 (SI 1 (S 1 1 0 1 "3) 1 CD (S 1:0 ^o • N a (S (%) iC)isn9;ai aAijBpH e u U o u o s o o 1— t bO B 9i M •a CO "bb 'a a 00 o u u CA l-i B ^ o .H a\ o o o cs 3 BO <£ Ui 60 320 (%) iCjisuajni 3AIJBPH 321 range. Saturated conditions with methane as the reagent gas produces the m/z 89 [M-H]~ ion as the base peak and an intense ion at m/z 87 due to the elimination of H2 from m/z 89, in the gas phase. For conditions below saturation, the m/z 87 ion may be slightly lower in intensity; however, in all cases, the m/z 88 is present but in very low abundance (less than 3% RA). The intensity of the m/z 88 ion, at greater intensity than seen throughout this work, suggests that there is an alternative mechanism of ion formation occurring with CO2 as the reagent gas. This alternative route did not occur to the extent seen here for methane CI studies conducted previously. The best explanation for this is the elimination of Hj prior to electron capture in the gas phase, presumably by a wall reaction, followed by electron attachment to the neutral species forming the m/z 88 [M-H2]— ion. The most common detriment to this work involving CO2 for CE or ECNCI was high ion source pressures resulting from the presence of the sample in the ion source. An example mass spectrum of standard palmitic (hexadecanoic) acid subjected to CE with 1200 mtorr COj is shown in figure A- 10. Although the M""* ion is present at m/z 256, the base peak is the [M+H]"^ ion of hexadecanoic acid at m/z 257. The only route for production of this ion is via self-CI. An additional interesting feature of this mass spectrum is the adduct ions present at m/z 271 and m/z 285. The latter ion at m/z 285 is the typical [M+29]+ ion seen in methane CI mass spectra. The [M+15]^ adduct ion at m/z 271 is fairly uncommon in methane n bO •§ c o u e u u •a u -a o E o E j> 2 3 E § a "^ 2 c " o (U — ■>. -a O .S < «5 U R3 3 M bOO 323 1 1 1 1 I O)