Mass spectrometric investigations of mosquito attraction to human skin emanations


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Mass spectrometric investigations of mosquito attraction to human skin emanations
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ix, 333 leaves : ill. ; 29 cm.
Bernier, Ulrich R., 1966-
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Mass spectrometry   ( lcsh )
Chemoreceptors   ( lcsh )
Mosquitoes   ( lcsh )
bibliography   ( marcgt )
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Thesis (Ph. D.)--University of Florida, 1995.
Includes bibliographical references (leaves 325-332).
Statement of Responsibility:
by Ulrich R. Bernier.
General Note:
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University of Florida
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Full Text







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


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.

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



ACKNOWLEDGMENTS ..................................... ii

A abstract ................................................. viii


1 INTRODUCTION ...................................... 1

Research Objectives ..................................... 1
Entomological Overview .................................. 2
M osquito Physiology ................................ 3
Characteristics of gender and species ............... 3
M osquito sensilla ............................. 4
M osquito vision .............................. 6
M osquito Repellents ................................ 7
M osquito Attractants ................................ 8
Nature of concern for attractant identification ......... 9
V ision .................................... 11
H eat ..................................... 11
M oisture .................................. 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


2 SAMPLING METHODS ................................. 31

Introduction .................................... ........ 31
Sampling Considerations .................. ......... 31
Entomological Sampling ............................ 32
Olfactom eter ............................... 32
Field studies ................................ 36
Mass Spectrometric Methods of Sample Introduction ....... 36
Experim ental .......................................... 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
Experim ental ......................................... 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


Phase differences ........................... 149
Implication to attractant origin .................. 152
Conclusions .......................................... 153
Lactic Acid Reactions ............................. 153
Altering Attraction ............................... 154
Origin of Attraction .............................. 154


Introduction .......................................... 156
Analysis of Multiple Beads Without GC Separation ....... 157
Daughter Library ................................ 157
Compound Class Screening ......................... 158
Experim ental ......................................... 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


Introduction ...................................... ... 205
Sample Introduction and Separation ................... 205
Cryo-focusing GC ........................... 208
Purge and trap GC .......................... 208
M ass Spectrometry ............................... 209
PCI theory ................................ 209
NCI theory ................................ 212
Characteristic ion fragmentations ................ 217
Experim ental ......................................... 220
Identification of Emanations by CI and El 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
Olfactom eter .............................. 227
Results and Discussion ................................. 228
Identification of Emanations by CI and El MS ........... 228


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 W ork .................. ....... ... .............. 288


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

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



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

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.


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


mass spectrometric assays concurrently with samples analyzed for mosquito


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. This 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.


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


females for mating. Additionally, differences exist among species as to preference

of hosts for blood-meals. Culex, spp., generally prefer avian hosts while 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 of Ae. aegypti

and one type of chemosensillum on the palps capitatee 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 ofAe. 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


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].


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

max for absorption is 515 nm. This corresponds to a visual range of 323-631 nm with

a maximum spectral sensitivity at 520 nm obtained from electroretinograms with Ae.

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 1-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 Culiseta


The grooved pegs on Ae. aegypti respond to lactic acid and are inhibited by

the popular repellent diethyl-meta-toluamide deett) [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


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 [4-7,9,11,13,15-24], and chemical (olfactory) attractants [4-6,9,11,13,16,18-24].

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

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]:


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. aegpti 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 1-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 1-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


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.


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].


The thermal neurosensory response can be tonic (continuous) to ambient

temperature or phasic (intermittent) during rapid temperature changes [4].


Temperature changes of 0.05C 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-

56F [14,15].


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 48S of

caged female Anopheles quadrimaculatus 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


[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 wingbeatt 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


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


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.


Emanations from animals including man. The use of 1-octen-3-ol and its

effect on some species of mosquito has been examined [11,20-24]. 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 toAe. 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 forAe. 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


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 Ae. 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,


and various amino acids phenylalaninee, 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-


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


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


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.

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 (El) or chemical ionization (CI).

Electron ionization (El). The first El source is credited to work in 1921 by

Dempster; however, the precursor to the modern El 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 (CI). 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

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 El

operating conditions. Ions generated in the source are extracted into the first

quadrupole mass filter (Q1) 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. The 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.










N n.
l ^




N *(

eg /
CY ---'



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 1-2(a)). Alternatively, Q1 and Q2 can be held in RF-only

mode while scanning Q3, as shown in figure 1-2(b). Selected ion monitoring (SIM),

shown in figure 1-2(c), is a single-stage mode allowing passage of only selected m/z

values through the mass filter; it can be performed with either Q1 or Q3. The

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 1-3(a)) consists of setting Q1 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 1-3(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 Q1 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 Q1. If Q1 and Q3 are both scanned with


(a) J--

(c) _I

I l


Figure 1-2 Single-stage scan modes with a tandem quadrupole mass
spectrometer set for (a) Q1 full mass spectrum, (b) Q3 full mass spectrum, and
(c) selected ion monitoring using Q1.

(a) -



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.


a fixed mass offset, a neutral loss spectrum can be generated (figure 1-3(c)). The

scan m/z value, for each ion passed through Q1, is offset lower in Q3 by the mass of

the neutral loss sought. The final scan mode, shown in figure 1-3(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. The 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 El 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 El.

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


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


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


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.



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


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.


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.


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














`tf w
=t a
ca o~


consists of one port (port A) for introduction of the sample and the second port

(port B) for the introduction of the control or a second comparative sample. The

sample sizes or media of introduction are limited only by the volume of the ports;

in this figure, petri dishes are shown. In port A, the petri dish has either been

inserted after handling or inserted after deposition of a sample consisting of a sole

compound or mixture of compounds. The dish in port B is a cleaned untreated petri

dish used as a control. A humidity-controlled stream of air, pure gas, or mixture of

gases is passed through the ports, over the samples. Any volatiles desorbed from the

sample will be carried via the gas stream through a trap on each port. The outlet

of the trap then opens into a enclosed chamber where the mosquitoes are held. The

enclosed chamber also contains an exhaust to enhance the airstream effect through

the olfactometer.

Mosquitoes used in these tests are typically female Ae. aegypti, although not

limited to this species or gender. Mosquitoes are usually not fed for a period of time

in order to enhance the response to any attractive compounds. If the volatiles are

odor cues for the mosquitoes, the mosquitoes will be activated to flight and follow

upstream into the appropriate trap. Should mosquitoes not be able to directionally

locate the source of the odor with accuracy but do alight upstream, they will be

collected in the control port trap. After a specified period (typically 3-5 minutes),

the experiment is ended and the mosquitoes trapped in each port counted. This

allows for the percentage of mosquitoes attracted to a specific stimulus to be


Field studies

Studies in the field are less precise in determining the actual percentage

attracted to a sample. Sampling in the field also involves careful consideration in the

design of traps, the release rates of samples, the location of samples, and the

influence of parameters in the outdoors. Some of these parameters are wind speed,

wind direction, humidity, and temperature. Additional concerns are manifested in

the actual population of mosquitoes during the test, i.e. whether or not they are in

close proximity to the test region such that odors could be detected. Trapped

mosquitoes are separated and counted according to species. The information

obtained from these experiments consists of a net capture rate relative to other

species and/or a control. Estimating the total population of unattracted mosquitoes

is not possible via field tests of this nature.

Mass Spectrometric Methods of Sample Introduction

The sampling for this dissertation was conducted numerous ways; a chart

representing this is portrayed in Figure 2-2. The sample in this diagram refers to

emanations or sweat found on human skin. These emanations were either deposited

on glass beads via handling the beads, washing the beads with solvent after handling

the beads, or directly dissolving perspiration into methanol.

Methods of direct analysis of glass beads consisted of either using a single

bead fitted on the end of a solids probe stem or placing multiple beads in an

enclosed glass sample container or "vial". The term "vial" is used to describe any of






L3r E
'S ea

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.


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


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 50C to 3000C over a six minute period.

The mass spectrometer was a Finnigan MAT TSQ70 triple quadrupole mass

spectrometer equipped with a Varian 3400 gas chromatograph. This 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 pA in all cases. The electron energy was 70 eV for El analyses and 100

eV for CI analyses. The manifold temperature was set to 700C while the ion source

temperature was set to 1500C for CI and 170C for EI. The mass ranges scanned

were m/z 10-650 for El 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.

To Mass Spectrometer

Helium In

Toggle for Helium -J




Four Way Cross

Cajon Fitting

-100 mL to 250 mL
Round Bottom Flask

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. The 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


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 10-15C/min ramp up to a final temperature of 190-210C and a final hold of

2.0 to 5.0 min. The transfer line was initially set to 500C and ramped immediately

at 200/min up to 200-210C and held at that temperature for the duration of the


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 pIA with 100 eV electron energy. The ion source temperature was set at

150C and the manifold set at 70C. Prior to analysis, the mass spectrometer was

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 250C 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. Throughout the loading

process the head pressure of helium was set to 0 psig to allow for proper alignment


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 25C to 2500C over 7.5 min and holding at 2500C for

2.5 min. Throughout the cryo-focusing phase, the GC oven was set at 250C and the

transfer line set 400C. 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. The

GC oven ramp consisted of an initial 1.0 min hold at 25C, a 12 min ramp at

15"C/min up to 2100C, and then a hold at 2100C for 5.0 min. The transfer line was

concurrently ramped, after a 1.0 min hold at 400C, up to 2250C at 15OC/min, and held

5.0 min at 2250C. Experiments were performed with either an 18 m x 0.18 mm i.d.

DB-5 column (df=0.25 lpm) or a 20 m x 0.25 mm i.d. Carbowax column (df=0.25


The mass spectrometer was operated in NCI mode, positive and negative ion

mode (PPINICI), or El mode. For NCI and PPINICI experiments the reagent gas

was methane at 1650 mtorr and 1660 mtorr (indicated) pressures, respectively. The

ion source and manifold temperatures were 1500C and 70C, respectively, for CI and

1700C and 70C, respectively, for El. 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 pA.

The conversion dynode was set at +5 kV for positive ion CI and El, and -5 kV for

negative ion CI. The 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. The

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



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 -1600C for three minutes and heats up to -16C 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 -20C and then heated

to 1560C for desorption of trapped volatiles onto the focusing trap. The cryo-

focusing trap was set a -1600C for concentration and was heated ballistically

(approximately 10 seconds) to 1500C to purge volatiles onto the head of the GC


The GC employed was an HP5890 series I with a 30 m x 0.25 mm i.d. DB-1

column (df= 1 pm). For analyses involving glass beads, the column was initially cryo-

cooled at -350C and held for 3.0 min. Subsequently, the column was ramped at

12C/min up to 1800C, then ramped at 250C/min from 180C to 2250C and held for

5.0 min at 2250C. For analyses from the Tedlar bag, the column was held 6.0 min

at -35C then ramped at 60C/min to 1800C and 12C/min from 180C to 225C, and

held for 10.0 min at 225C.

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 tiA with an electron energy of 100 eV. The electron

multiplier was set to -1200 V. The ion source temperature was set at 1800C; there

is no heater for the manifold. Prior to analysis, the instrument was tuned with


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

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molecular ion (M') of cholesterol and m/z 387, the carbon-13 isotope of the

molecular ion. Positive identification of cholesterol during this initial phase was

difficult to obtain due to the abundance of fragment ions from other species found

in the mass spectrum over the selected peak; background subtraction was not able

to fully remove the interferant masses due to the width of the desorption profiles.

Trace components of lower m/z become increasingly difficult to identify due

to overlapping profiles of fragment ions when examining the mass chromatograms

for particular m/z values (see discussion of figure 2-8). Additionally, re-examination

of each mass over the entire scan range, or sections of the RIC devoid of discernible

peaks, makes identification a tedious process. The positive identification of

cholesterol was achieved in the work of Chapter 5.

During this first series of experiments with a single bead, different subjects

were involved in handling the beads. Figure 2-6 is the RIC from the mass

spectrometric analysis of a single bead rubbed by Mr. Dan Smith; this individual has

been consistently found to be the most attractive person to mosquitoes in USDA

tests conducted with the olfactometer. Comparing figure 2-6 to figure 2-4 (the RIC

of a bead rubbed by the author of this dissertation) it is evident that the traces are

very similar. This comparison also demonstrates that examination of this nature is

insufficient to ascertain differences among people in terms of relating what

components and what abundance of components is present on the skin causing the




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Figure 2-7 is the RIC of a second bead rubbed by Dan Smith of the USDA.

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+H]* 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



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ions (M+') of aliphatic fatty acids, some of these ions may be due to fragment ions

from higher molecular weight compounds (e.g. longer chain fatty acids in the same


Thermal Desorption from Multiple Beads

The first approach to improving identification of components was to develop

methods of increased sample size to obtain greater ion signal for the identification

of trace components. The apparatus initially constructed for this purpose was

described in the experimental section and depicted in figure 2-3. Figure 2-10 is an

illustration of the NCI results of thermal desorption from multiple beads in a round

bottom flask. In this case, the 100 handled beads were spiked with additional lactic

acid prior to heating and analysis of the beads. There is an erratic response of the

RIC past scan number 1100. This problem was characteristic for the analysis of a

large number of beads. This may arise from saturation of the electron-capture/NCI

process in the ion source due to the increased abundance of sample components

from the large number of beads. Alternatively, this problem could arise from the

desorption of volatiles from the surface of the beads, where non-uniform heating

produces warm spots and "burping" occurs from volatiles escaping around the beads.

The problems associated with the analysis of a large number of beads were

rectified by reducing the number of beads and size of the sample container used to

hold the sample in the GC oven. The RIC in figure 2-11, obtained by using 5

rubbed beads in a small tube, demonstrates that the erratic fluctuations in the RIC

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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 H2 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.


.fl "

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Thermal Desorption from Multiple Beads/Crvo-focused GC Separation

Cryo-focusing GC/MS has been previously used in the detection of volatile

emanations from humans [68]. The collection and desorption of human emanations

was achieved by the use of charcoal, and re-focusing accomplished via the use of

three successively colder traps. Most of the experiments reported in this dissertation

employed a single stage of cold-trapping. The use of on-column cryo-focusing solved

the problem of wide desorption profiles due to inefficient compound removal from

the headspace above the beads in the previous series of experiments. Due to the

similarity in size of the 3" x 1/4" glass tube (use for multiple bead studies above) and

the glass injector insert in the GC injection port, experiments were conducted using

the insert as the sample holder. By reversing the injection insert, beads could be

placed into the insert. Using an insert with a glass frit allowed the beads to be

supported above the column entrance and more importantly, allowed free passage

of volatiles past the beads. Additionally the design of the injection port allows for

a directional sweep of helium over the beads and out of the injector through the

column entrance; the enclosed vials for multiple beads have less directional flow.

The additional advantage of using the injection port to initially desorb volatiles is

that the column could then be held at ambient room temperature with a section of

it cryo-focused while the beads were heated external to the GC oven.

The injection insert, when inserted reversed, held up to 12 beads. Figure 2-13

is the RIC for the first such experiment involving the maximum number of beads

(12). Two points are evident when examining this figure. First, this was by far the

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best separation achieved at the time for the analysis of beads, greatly improved over

direct thermal desorption. The second point is that the peak shapes demonstrate

that the column is overloaded and that a reduction in the number of beads analyzed

is necessary for better chromatographic peak shapes. The components found in

lesser abundance in the sample show less fronting, i.e. evidence of more abundant

components overloading the column sample capacity.

Figure 2-14 demonstrates this point by the examination of characteristic

cholesterol ions. These ions are the [M-H]- ion at m/z 385, the M-' ion at m/z 386

and the carbon-13 isotope of the M-' ion at m/z 387. The peak shape near scan

number 800 is a significant improvement over previous experiments where

cholesterol was detected (including the thermal desorption profiles shown in figure


Reducing the number of beads to from 12 to 5 and employing a slightly longer

polar (Carbowax) column further improved peak shape, as can be seen in figure 2-15.

A Carbowax column was effectively used previously for thermal desorption and

sniffing mass spectrometric fruit odor analysis [69]. This figure demonstrates an

analysis employing PPINICI, to allow for detection of both positive and negative ions

via alternating scans between positive and negative ions [70]. The complementary

El analysis of 8 rubbed beads is found in figure 2-16. The data in this figure was

acquired with a slightly slower temperature ramp of the GC oven. Comparison

between the positive and negative ion CI RICs in figure 2-15 and the RIC for the

El analysis in figure 2-16 shows that peaks can be clearly correlated between the two

.- I0




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experiments. Additionally, the top trace on figure 2-16 is that of the molecular ion

of cholesterol at m/z 386. The major peaks found in the RIC of these figures are

aliphatic fatty acids; the examination of the cholesterol molecular ion demonstrates

the ability to detect trace components. The quality of figures 2-15 and 2-16 are

similar (although slightly lower) to that of the subsequent cryo-focused analyses

found for studies in Chapter 5.

Purge and Trap/GC Separation

Thermal desorption/purge and trap has been used previously for studies such

as identification of fruit fragrances and analysis of volatiles from smoke [69,71].

However, purge and trap was initially avoided in this study due to the possibility of

sample discrimination by the trap, i.e. being unable to desorb some volatiles back off

of the trap for analysis. However, upon analysis via a three-stage microscale purge

and trap GC/MS system, it was found that a great number of trace components could

be concentrated and identified, as readily seen in the RIC traces for figures 2-17 and

2-18. Data for figure 2-17 were acquired from rubbed beads and data for figure 2-18

(using a heating rate approximately half of that for figure 2-17) were acquired from

direct introduction of volatiles from the skin by sampling a Tedlar bag with the hand

contained therein.

The microscale purge and trap system does not allow for detection of the

acids and other polar compounds; they are lost somewhere in the system, possibly

in the nickel-plated lines or perhaps in the first drying trap. The removal of the

ll C)



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a.) E -~
bo 4.) %4-

.0 0


4-4 0.


.0 c

~ 0

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acids was a benefit rather than a hinderance in this case. Based on the thermal

desorption/cryo-focusing GC results, the aliphatic fatty acids are the most abundant

components in emanations; their removal permits the analysis of trace level

components constituting other compound classes.


All sampling methods in this section satisfy the initial criterion of sampling

volatile emanations in a manner similar to that which mosquitoes encounter.

Specifically, this refers to sampling a chemically unmodified sample volatilized into

the gas-phase. Direct analysis of a single bead with thermal desorption via the use

of a direct insertion probe was found to be inadequate in terms of temporal

resolution and the ability to detect trace level components. The use of multiple

beads in an enclosed glass sample container increased the sample size for the

purpose of detecting trace level components; however, the temporal resolution was

even worse than the single bead method due to the headspace volume of the sample

container which had to be heated and swept by helium to transfer volatilized

compounds onto the column. The problem with poor temporal resolution was

resolved via the use of on-column cryo-focusing. This method is used for much of

the identification of compounds in Chapter 5. Purge and trap introduction was

employed as an alternative method to detect volatile components; furthermore, it

discriminates against fatty acids, which are predominant in cryo-focused analyses.

This allows for detection of some trace components (low to nonpolar) not readily


detected in the cryo-focused analyses. Additionally, purge and trap analyses

involving the sampling of the hand enclosed in a Tedlar bag provided the most

accurate sampling introduction method with respect to analyzing airborne volatiles

emanated from the skin.



Lactic Acid as a Model Compound

At the inception of the work for this dissertation, lactic acid was the only

widely recognized mosquito attractant [5]. This compound only attracts the Ae.

aegypti species of mosquito. Due to this aforementioned attribute and due to the

abundance of lactic acid on the skin, it is the most studied compound in this

dissertation. The studies reported in this chapter are arranged in three sections, as

described below.

Reaction Studies

The identification of lactic acid can be achieved through knowledge of its

characteristic fragmentations and associative reactions in the mass spectrometer;

therefore, these reactions will be examined. The extent of these reactions and the

abundance of ions is dependent upon operating parameters and sample conditions

[72,73]. Additionally, mass spectrometer ion sources have slightly different designs;

therefore, some differences in relative abundances are inherent to the mass

spectrometer. These studies are beneficial to studies involving identification of lactic



acid in complex samples due to the lack of a molecular ion (M'") formed by El of

lactic acid and due to the presence of additional ions, characteristic of instrument

parameters, which contribute to the difficulty in identifying this compound by library

searching of El spectra.

Altering Attraction

Previous work with altering attraction via acidification consisted of the

addition of sulfuric acid to solutions of carboxylic acid salts in acetone. The purpose

of the sulfuric acid addition was to increase the concentration of the free acid versus

the sodium salt rather than to examine possible enhancement of attraction [19].

Mosquitoes responded to these acidified solutions by exhibiting a greater activation

to flight; however, there was little increase in directional attraction.

The increase in attraction effected by the acidification of lactic acid solutions

had not been examined. Logically, addition of acid should increase the proportion

of free acid (volatile) relative to the dissociated form (involatile). This should

increase attraction; addition of base should produce the opposite effect. The utility

of mass spectrometry in this case was in its application to the examination of possible

solution-phase reactions involving lactic acid and the methanol solvent. Specifically,

this is an examination for products of acid-catalyzed or base-catalyzed esterification.

Analysis of Methanolic Perspiration Solution

The final study in this chapter involves the direct dissolution of components

in perspiration into methanol. This work is aimed at determining the residence of

lactic acid in the matrix on the skin. By examination of each phase for lactic acid,

insight was gained into the abundance and distribution of lactic acid in perspiration.


Reactions of Lactic Acid

Most of the lactic acid mass spectra found in this section were acquired from

direct thermal desorption of handled glass beads. The sample was desorbed from

5 beads which had been rubbed in the palms of the hands for 5 min by the author

of this dissertation. The beads were placed in a 1/4" o.d. glass tube (maximum 25

beads) which was inserted into the apparatus described in chapter two (figure 2-3).

Helium, at 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 temperature was

ramped via initially holding the column at 280C for 1.0 min followed by a 12 min

ramp at 15C/min to 2070C, with a 5 min hold at that temperature. The transfer line

was concurrently ramped from 50C to 2100C at 200C/min once the run was started,

and held at 2100C for the final 10 min of the analysis. Ionization was effected by

isobutane CI at an indicated source pressure of 1647 mtorr. The ion source

temperature was set at 1500C and the manifold temperature at 70C. The electron