Selective ion-molecule reactions in a quadrupole ion trap mass spectrometer

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Selective ion-molecule reactions in a quadrupole ion trap mass spectrometer
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Coopersmith, Brad Ian, 1968-
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Thesis (Ph. D.)--University of Florida, 1994.
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Includes bibliographical references (leaves 201-209).
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by Brad Ian Coopersmith.
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Full Text









SELECTIVE ION-MOLECULE REACTIONS IN A QUADRUPOLE
ION TRAP MASS SPECTROMETER
















By

BRAD IAN COOPERSMITH


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


1994































To the Big Five: Amanda, Norman, Roslyn, Randall, and Sammantha













ACKNOWLEDGMENTS


I would like to thank Dr. Richard A. Yost for his guidance and friendship

over the past five years. I would also like to thank my committee members, Dr.

James D. Winefordner, Dr. Willard W. Harrison, Dr. Kenneth B. Wagener, and Dr.

Roger L. Bertholf. I want to acknowledge the U.S. Environmental Protection

Agency (Mr. Douglas W. Keuhl, in particular) and the University of Florida Division

of Sponsored Research for providing funding for this research.

I want to thank the Yost group members over the past five years for their

continued insights into human nature. I especially want to thank Dr. Jodie V.

Johnson and Dr. Nathan A. Yates for their help in understanding and the use of the

ion trap. Special gratitude goes out to Dr. Donald M. Eades for his friendship and

support and for showing me that "Let's get a cup of coffee" is not always a bad thing.

I want to thank the current Yost group members Mr. James L. Stephenson, Mr.

Matthew M. Booth, Mr. Timothy P. Griffin, and Mr. Ulrich R. Bernier for many

funny stories and helpful discussions over the past few years. I am deeply indebted

to the fourth floor of CLB for providing me with chemicals throughout this research.

I want to thank my parents, Norman and Roslyn, for their love and support

during my graduate studies. The knowledge that they would always be there if I

needed them, whether it be physically or emotionally, meant a lot to me.








Lastly, I would like to thank my wife, Amanda, to whom I partially dedicate

this thesis. Her love and support during the crazy times here made it all worthwhile.

From getting me Randall to kicking me in the ass when I needed it, her love for me

was shown in her actions as much as in her words. I love her with all my heart and

hope that one day I can show her how much.














TABLE OF CONTENTS


ACKNOWLEDGMENTS ..................................... iii

A BSTRA CT .............................................. viii

CHAPTERS

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

Q ITM S Background ..................................... 2
QITM S Development ............................... 2
QITMS Theory and Operation ......................... 3
Tandem-in-Time Versus Tandem-in-Space ................ 7
Ion-M olecule Reactions ................................... 8
Scope of Dissertation ................................... 11

2 CHARACTERIZATION OF THE QITMS FOR ELECTROPHILE/
NUCLEOPHILE ION-MOLECULE REACTIONS ............. 13

Introduction .......................................... 13
Carcinogen and Mutagen Background ......................... 14
In vivo Carcinogen Detection ......................... 15
In vitro Carcinogen Detection ........................ 16
Mass Spectrometric Approaches ....................... 18
Proposed Methodology .................................. 20
Experim ental ......................................... 29
R results .. .. . . .. . . 31
Ion-Molecule Reaction Spectra Under Static Conditions ..... 31
Reaction Scheme Determination ...................... 35
Kinetics Analysis .................................. 44
Conclusions .......................................... 52

3 STRATEGIES FOR BETTER CONTROL OVER ION-MOLECULE
REACTIONS PERFORMED IN A QITMS .................. 54

Introduction ............................. ............. 54
Experim ental ......................................... 55








Changing the Ion-Neutral Chemistry ........ ........... 62
Pulsed Valve Introduction ........... ..................... 65
Optimization of Pulsed Valve Introduction ....... ....... 66
Spectra Obtained from Pulsed Valve Introduction .......... 72
Sensitivity Comparison Between Pulsed Valve and Constant Pressure
Introduction ............................ ......... 77
M ixture A analysis ....................................... 85
Mixture Analysis with Pyridine ........................ 86
Mixture Analysis with Thiophene .................... 94
C conclusions ......................................... 102

4 INVESTIGATIONS INTO ION/NEUTRAL CHEMISTRY ...... 105

Introduction ......................................... 105
Solution-Phase Carcinogen/DNA Adduct Studies .............. 107
Determination of Site of Reaction .................... 107
Hard/Soft Acid Base (HSAB) Theory ................. 109
Experimental ........................................ 112
Investigations of Multifunctional Nucleophiles ................ 115
Reactions of 2-Thiohydantoin Molecular Ions with Allyl
Brom ide ... .......... ................... 120
Analysis of the Other MFN/Allyl Bromide Adducts ........ 129
Correlation of Nucleophile/Allyl Halide Studies to HSAB
Theory ................................... 136
Reactions of ar,-Unsaturated Carbonyls ......... .......... 137
Reactions Between Pyridine and the a,P-Unsaturated
Carbonyls ................................. 138
Reactions Between Piperidine and the a,P3-Unsaturated
C arbonyls ................................. 141
Correlation of Nucleophile/a4,3-Unsaturated Carbonyl Studies
to HSAB Theory ............................ 143
C conclusions ......................................... 146

5 ISOMER DIFFERENTIATION VIA SELECTIVE
ION-MOLECULE REACTIONS ......................... 147

Introduction ......................................... 147
Methods of Gas-Phase Isomer Differentiation ................. 148
Collision-Induced Dissociation (CID) ......... ....... 148
Energy-Resolved CID ............................. 150
Ion-Molecule Reactions............................ 152
Experim ental ............................... ......... 154
Origins for Isomer Differentiation Investigations ............... 156








Carbocation Differentiation Based on Thermodynamics ..........
Reaction Schem e ................................
Experimental Verification of Thermodynamic Reaction
Schem e ..................................
Identification of the m/z 81 Ion from Allyl Iodide .........
Carbocation Differentiation Based on Steric Hinderance .........
Reaction Schem e ................................
Experimental Verification of Differentiation by Steric
Inhibition .................................
C conclusions .........................................

6 CONCLUSIONS AND FUTURE WORK ...................

C conclusions .........................................
Future W ork ........................................

APPENDIX EQUATIONS USED FOR KINETIC DETERMINATIONS

REFERENCES ...........................................

BIOGRAPHICAL SKETCH ..................................


158
158

163
170
170
170

175
183

184

184
187

192

201

210













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

SELECTIVE ION-MOLECULE REACTIONS IN A QUADRUPOLE
ION TRAP MASS SPECTROMETER

By

Brad I. Coopersmith

August 1994

Chairperson: Dr. Richard A. Yost
Major Department: Chemistry

This dissertation presents two applications for selective ion-molecule reactions

performed in a quadrupole ion trap mass spectrometer (QITMS): (a) the detection

and identification of possible carcinogens and mutagens in environmental samples

and (b) the differentiation of secondary and tertiary carbocation isomers.

Potential carcinogens and mutagens are classified as electrophiles with the

ability to modify nucleophilic sites in the body, including DNA. Initial studies focus

on the characterization of the QITMS to perform the ion-molecule reactions between

a model DNA base nucleophile ion, the pyridine molecular ion, and the allyl halides,

a class of well-characterized mutagens. These studies highlight the problems with

performing these reactions in a tandem-in-time instrument, namely the unwanted

side products due to reaction with the nucleophile neutrals.








Incorporation of pulsed-valve introduction of the nucleophile neutrals is

shown to overcome the lack of spatial separation by affording temporal separation

between the introduction of the neutrals and the reaction period. Initial application

of the gas-phase screening technique is demonstrated for mixtures of carcinogens and

noncarcinogens using the nucleophiles pyridine and thiophene.

Reactions with multifunctional nucleophiles are performed to better model

the DNA/carcinogen reaction in the gas-phase and to better understand the ion/

neutral chemistry. These studies into the ion/neutral chemistry show the ability of

the Hard/Soft Acid/Base theory to predict reactivity and site of reaction. This

correlation will be important for future choices of nucleophile ions.

Differentiation between secondary and tertiary carbocation isomers is

demonstrated based upon thermodynamic considerations. The secondary carbocation

reacts with tert-butanol while the tertiary carbocation is inert. Differentiation

reactions based upon steric hinderance arguments is attempted. While not totally

successful, these steric reactions demonstrate that most solution-phase organic

principles can be extended to gas-phase organic reactions.














CHAPTER 1
INTRODUCTION


This dissertation discusses the design and application of selective ion-molecule

reactions performed in a quadrupole ion trap mass spectrometer (QITMS) to (a)

screen for possible carcinogens and mutagens in environmental samples and (b)

differentiate between isomeric carbocations. In both applications, the gas-phase ion-

molecule reactions attempt to mimic what has been observed in the solution phase.

Electrophile/nucleophile reactions form the basis for the carcinogen and mutagen

screening. Carbocation-based reactions are used to model the isomer differentiation

reactions. The use of selective ion-molecule reactions on the QITMS offers several

advantages over the current methods used for the above applications. Compared to

solution-phase carcinogen and mutagen detection, the QITMS offers improved speed

and the use of on-line chromatographic separation. The selective ion-molecule

reactions permit the formation of distinct product ions for the differentiation of gas-

phase carbocation isomers, whereas other gas-phase techniques lead to the formation

of identical product ions in differing abundances.

This chapter introduces background on both the quadrupole ion trap mass

spectrometer and selective ion-molecule reactions. A brief description of the

operation of the QITMS is presented, including the particular characteristics that

make it desirable for performing the selective ion-molecule reactions. Selective ion-








2
molecule reactions are introduced through a description of general ion-molecule

reactions and a definition of what characteristics make them selective. This chapter

concludes with a description of the dissertation organization.


OITMS Background


QITMS Development


This discussion of the QITMS is not designed to be comprehensive, but

merely to introduce important concepts and principles of the instrument which was

used for this research. Comprehensive reviews of the ion trap have been published

by Todd (1991) and by Cooks et al. (1991).

The concept for the ion trap was first published by Paul and Steinwedel

(1953) in describing the operating principles of the quadrupole mass spectrometer.

These ideas were further developed by Dawson (1976), Dawson and Whetten (1969),

and Todd and coworkers (Lawson et al., 1976; Todd, 1981) by first using the ion trap

as solely an ion source to a quadrupole mass filter and then expanding its capability

to perform mass analysis. The first commercial ion trap, known as an ion trap

detector or ITD, was introduced in 1983 by Finnigan MAT and was based upon the

development of the mass-selective instability scan by Stafford et al. (1984). At that

time, the ITD was simply a low cost gas chromatographic detector. Since then,

QITMS research has been expanded into many varied areas. Among those areas are

the extension of the mass range to over 70,000 Da (Kaiser et al., 1991), the

attainment of mass resolution exceeding one million (Schwartz et al., 1991), the








3

application of tandem mass spectrometry (MSn) to the tenth degree (Nourse et al.,

1992), and the combination of the QITMS with external ion sources such as

electrospray (Van Berkel et al., 1990).


OITMS Theory and Operation


The ion trap consists of three hyperbolic electrodes, two end-cap electrodes

and one ring electrode, that when assembled yield a trapping volume with a

hyperbolic cross section according to ro2 = 2zo2; ro is the center-to-ring distance, and

zo is the center-to-endcap distance. Figure 1-1 shows this cross section for an

assembled ion trap. Radio-frequency (rf) and direct current (dc) voltages are

applied to the ring electrode to create a quadrupolar electric field within the

trapping volume. This field will apply a restoring force (towards the center of the

ion trap) to the ion which is proportional to the ion's displacement from the center

of the trap. The restoring force causes the ions which are trapped to oscillate in a

three dimensional Lissajous orbit (Wuerker et al., 1959). Since this restoring force

is a function of a time-dependent harmonic variable (the rf voltage), it can be

described by the appropriate Mathieu second-order differential equation

(McLachlan, 1947) given in equation (1-1).


S+ (a,- 2qcos2()u = 0 (1-1)
dt2

In the above equation, u can represent either the radial (ring-to-ring), or the axial

(endcap-to-endcap) directions, and is a dimensionless variable which is equal to



































-7


Figure 1-1:


Ion trap cross section showing the center-to-ring distance (ro) and the
center-to-endcap distance (zo).








5

Ot/2, where f is the frequency of the applied rf voltage. Operation of the QITMS

with the endcaps held at ground and the rf and dc voltages applied only to the ring

electrode leads to the following equations for au and q,, where u=r for radial and

u=z for axial:

-8eU
a.=-2a,= (1-2)
mrQ2

and

-4eV
-4eV (1-3)
qz=-2qr= (1-3)
mr2o2
where U is the dc voltage applied, V is the zero-to-peak amplitude of the rf voltage

applied, e is the charge on an electron, and m is the mass of the ion.

Depending upon the magnitudes of the rf and dc voltages which are applied,

only ions of certain mass-to-charge (m/z) ratios will be stored. Equation (1-1) can

be solved to find all values of au and qu (and hence all m/z values) which will be

stable, either in the radial or in the axial or in both directions, under a given set of

rf and dc voltages. The set of solutions for stability in both directions, when plotted

in au-qu space, form an envelope called a stability diagram which is shown in Figure

1-2. An ion will be stable if its au and qu values fall within the borders of the

stability diagram.

Typical operation of the QITMS for mass analysis is now described. After the

sample is introduced into the QITMS, electrons are pulsed into the QITMS volume

through one endcap to ionize the sample. A gating pulse, which changes from -180V















0.20-


az= 0, qz= 0.908
0.00



-0.20-



-0.40



-0.60-



-0.80 1.0 10

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40


Stability diagram in az-q, space for all ions.


Figure 1-2:








7
to + 180V, is used to control the introduction of the electrons created by a filament.

During the ionization process, and for most of the mass analysis scan, no dc voltage

is applied so that the QITMS operates along the a,=0 line in Figure 1-2, which

allows for the largest range of stable m/z values possible for a given rf voltage. The

reason for this mode of operation is shown in Figure 1-2 where for a given rf voltage,

all ions are stable along the az=0 line if q, (which is inversely proportional to the

mass) is less than 0.908. After a storage period, the ions are detected by the mass-

selective instability scan (Stafford et al., 1984). This scan consists of ramping the

amplitude of the rf voltage while applying no dc voltage to induce ions of increasing

m/z values to exceed a qz value of 0.908 and thus become unstable in the axial

(endcap) direction. Ions are detected by an electron multiplier which is placed

behind the exit endcap.


Tandem-in-Time Versus Tandem-in-Space


The QITMS offers several advantages which enhance the performance of gas-

phase ion-molecule reactions compared to most other tandem mass spectrometers.

The ion trap, a tandem-in-time instrument, performs each stage of mass spectrometry

in the same volume of space with each analysis step separated in time. This attribute

allows the time of each stage of mass spectrometry to be controlled. As a result, the

QITMS, with reported ion confinement times of 105 seconds (Sugiyama and Yoda,

1990), can act as a gas-phase organic reaction chamber. This control of reaction

time allows the reactions to occur for longer periods than available with most








8
commercial tandem-in-space instruments such as the triple quadrupole mass

spectrometer (TQMS). The TQMS can be modified to trap the ions inside the

second quadrupole (Annacchino, 1993), however, this approach is not a common

practice. The duration for each stage of mass spectrometry, with regard to ion

residence time, on the unmodified TQMS is on the order of microseconds, while on

the QITMS each stage lasts typically for tens to hundreds of milliseconds. This three

order of magnitude difference for reaction times facilitates the detection of

kinetically slow reactions. Also, the QITMS permits the acquisition of time-resolved

data through steady increments of the reaction period. These data can then be used

to yield kinetic information regarding the reaction under investigation. A further

advantage of the QITMS is its ability to perform multiple stages of mass

spectrometry (MS") (Nourse et al., 1992). This MS" capability allows for the

complete identification of mass-selected reaction products, as well as the

determination of reaction mechanisms. One possible drawback to the use of the

QITMS for ion-molecule reactions is the lack of spatial separation between the ions

and neutrals inside the ion trap which can lead to unwanted side reactions due to the

presence of the reagent neutrals and, thus, limit the effectiveness of the selective ion-

molecule reactions.


Ion-Molecule Reactions


Ion-molecule reactions have been investigated for years on instruments such

as the TQMS (Heath et al., 1991; Dolnikowski et al., 1990), the ion cyclotron








9
resonance mass spectrometer (Beauchamp et al., 1974; Tiedemann and Riveros,

1973) and the QITMS (Kinter and Bursey, 1988; Eichmann and Brodbelt, 1993).

These reactions have provided insight into reaction rate constants and kinetics

(Lifshitz et al., 1981; Tiedemann and Riveros, 1974), ion energetic (Orlando et al.,

1989; Orlando et al., 1991), thermodynamics (Jasinski and Brauman, 1980; Meot-Ner

and Smith, 1991), and general gas-phase ion-neutral chemistry (Castle and Gross,

1989).

The ion-molecule reactions investigated in this dissertation are not only

selective, which will be defined later, but they also are mass-selected ion-molecule

reactions (Berberich, 1989). The term mass-selected means that only ions of selected

mass-to-charge (m/z) ratios are permitted to react with the neutrals inside the ion

trap volume. Most applications of ion-molecule reactions are nonmass-selected, in

which both a reagent gas and the analyte are introduced into the ion source of a

mass spectrometer, with the reagent gas at a significantly higher pressure. Due to

its greater pressure, the reagent gas is primarily ionized to yield the reagent ions

which are used for the ion-molecule reactions. One problem with this approach is

that most reagent gases will form more than one reagent ion, and each reagent ion

may react differently. For example, methane, which is used commonly for chemical

ionization (Harrison, 1992), forms predominantly C2H5+ and C3H5, reagent ions

which undergo proton transfer and proton abstraction reactions, respectively, with

most anlaytes. By mass-selecting only one reagent ion, any ambiguity as to which

product ions are due to which reagent ion is significantly reduced. On the QITMS,








10
mass-selected ion-molecule reactions are performed by adding an ion isolation

sequence shortly after the ionization step. A result of the tandem-in-time nature of

the QITMS is that this addition is easily implemented since the change is software

intensive (a change in the computer program controlling the mass analysis scan)

rather than hardware intensive (the addition of an extra mass analyzer).

In designing selective ion-molecule reactions, a reagent ion is chosen such that

reactions with only a single neutral or a single class of neutrals should occur. The

resulting product ion is characteristic of both the reagent ion and neutral. For

example, gas-phase Michael additions will occur if and only if an enolate ion is

allowed to react with a neutral a,/3-unsaturated carbonyl compound (Solomons,

1985). For the ion-molecule reactions studied in this work, the selectivity results in

the formation of a distinct adduct ion; electrophile/nucleophile adduct ion formation

is used to detect the carcinogens and mutagens, and isomer differentiation is based

upon one isomer forming an adduct ion while the other isomer is inert.

One important issue regarding the general applicability of the selective ion-

molecule reactions is ion energetic. All ion-molecule reactions require a certain

amount of activation energy for the reaction to proceed. The selective ion-molecule

reactions possess low energy of activation barriers for the preferred neutral or class

of neutrals and high activation barriers for the unwanted neutrals. For example, the

carcinogen detection reactions are designed so that the nucleophilic reagent ion will

react with the electrophilic carcinogen neutrals based upon the known attraction

between nucleophiles and electrophiles in the solution-phase (Solomons, 1982).








11

However, if the reagent ion were to possess a sufficient amount of excess internal

energy so that it was able to overcome the high activation energy necessary to react

with the undesired neutrals (i.e., nucleophile ions reacting with nonelectrophilic

neutrals), the ion-molecule reaction would lose its selectivity. Therefore, the

energetic related to ion formation and ion identity will be important throughout this

work.


Scope of Dissertation


This dissertation presents two applications of selective ion-molecule reactions

performed in a QITMS: (a) screening for carcinogens and mutagens in environ-

mental samples, and (b) differentiation of carbocation isomers. Chapter 1 presented

an introduction to both the QITMS and selective ion-molecule reactions.

Chapter 2 discusses the characterization of the ion trap to perform the

carcinogen screening reactions. Along with a description of methods currently in use

for carcinogen detection, the proposed gas-phase methodology is presented. The

QITMS is then characterized through the reactions of pyridine ions (model DNA

base ions) with allyl halide neutrals mutagenn neutrals). Experimentally determined

rate constants and extensions of ion-neutral chemistry studies are then used to

explain the observed results.

Chapter 3 introduces two approaches to overcome the problems from the lack

of spatial separation inside the QITMS: the use of alternative nucleophile ions to

alter the ion/neutral chemistry and the use of pulsed-valve introduction of the








12
nucleophile to introduce temporal separation. Altering the ion/neutral chemistry is

evaluated with respect to the degree it effectively removes unwanted nucleophile

ion/electrophile neutral and unwanted nucleophile ion/nucleophile neutral side

reactions. Comparisons of pulsed-valve results (spectral quality and limits of

detection) to those obtained through constant pressure introduction are used to

evaluate pulsed-valve introduction. The analysis of a carcinogen/noncarcinogen

mixture using the proposed gas-phase methodology is presented and used to evaluate

the method's effectiveness at this stage of development.

Chapter 4 investigates the ion-neutral chemistry of other nucleophiles and

electrophiles. Specifically, the reactions of the a,3-unsaturated carbonyls

(electrophiles) and of piperidine (nucleophile) and multifunctional nucleophiles

(containing more than one possible reactive site) are presented. The results from

these investigations are correlated to the Hard/Soft Acid/Base (HSAB) theory to

better predict which model DNA bases will detect which carcinogens and mutagens.

Chapter 5 presents the ability to differentiate between carbocation isomers

through selective ion-molecule reactions. After a brief introduction as to why this

approach is necessary, two approaches are discussed. Chemical differentiation based

upon reactions with tert-butanol and structural differentiation based upon steric

hinderance are presented. Chapter 6, the final chapter, summarizes the overall

conclusions from this dissertation and presents ideas for future work.













CHAPTER 2
CHARACTERIZATION OF THE QITMS FOR ELECTROPHILE/
NUCLEOPHILE ION-MOLECULE REACTIONS



Introduction


The main goal of this research was to use selective ion-molecule reactions as

a gas-phase screening technique for the detection of carcinogens and mutagens. This

chapter will present the first step towards accomplishing that goal, the

characterization of the ion trap to perform the ion-molecule reactions necessary for

carcinogen and mutagen screening. Initially, background information on carcinogen

and mutagen screening will be offered to put this work into perspective with current

techniques. The Ames Test, the most widely used method of carcinogen and

mutagen detection, will be discussed in detail, as it was the impetus for this work.

The specific role of the selective ion-molecule reactions and the use of gas

chromatography/mass spectrometry (GC/MS) will then be given. The ion trap will

be characterized through the reaction of pyridine ions with allyl halide neutrals. This

reaction is examined through the products which are formed and the kinetics of the

system. This chapter ends with conclusions regarding the use of the QITMS for

selective ion-molecule reactions based upon these results.










Carcinogen and Mutagen Background


A carcinogen has been defined as "an agent or process which significantly

increases the yield of malignant neoplasm in a population" (Clayson, 1962). The

exact mechanism by which a carcinogen induces the malignant neoplasm or

cancerous growth is still unknown. However, four broad stages for this mechanism

have been established. These stages are (i) transport from the site of application

and, if needed, metabolic activation of the carcinogen: (ii) interaction of the ultimate

or activated carcinogen with the critical target (most likely DNA); (iii) DNA repair

and replication for fixation of the initial features of the tumor progenitor cell; and

(iv) possible progressive changes in the tumor progenitor cells leading to clinical

cancer (ICPEMC, 1982). In its reactive form, the ultimate carcinogen has been

identified as a reactive electrophile with the capability to modify biological

macromolecules such as DNA (Miller, 1970). Based upon stage (i) and the

definition by Miller, two categories of carcinogens have been defined (ICPEMC,

1982):

(1) Genotoxic carcinogens: agents which significantly increase the occurrence of

tumors in a population and possess the ability to alter genetic information.

(2) Nongenotoxic carcinogens: agents which significantly increase the occurrence

of tumors in a population but need to be activated in order to alter genetic

information.










In vivo Carcinogen Detection


Carcinogen testing can be accomplished either in vivo (inside a living

organism) or in vitro (outside the living organism). In vivo testing has focused

primarily upon long-term animal bioassays. Since human testing would be the most

effective but is not supported by the medical community, most animal testing is

performed upon mice and rats due to their similar genetic make-up to humans. The

objective of these long-term tests is to observe the animal under study for the

development of neoplastic lesions due to exposure to various doses of a test

substance by an appropriate route (i.e., inhalation, ingestion, etc.) (Hamm, 1985).

By performing these assays on living animals, the overall body chemistry is not

altered as it would be if the assay were performed in a single organ which was

removed from the animal. This advantage allows both carcinogen initiators and

promoters to be tested (Periano et al., 1975; Solt et al., 1983). While this approach

would seem to be the most effective method for determining carcinogenicity, it

suffers severe drawbacks. First, any animal test will cost between 0.5 and 1.5 million

dollars and will take a minimum of three to four years to complete (Hamm, 1985).

Second, there is no standard methodology for performing the animal tests (OECD,

1983). Other problems with animal testing arise from the number of variables which

must be taken into account. Among the many factors are the type of animal, the

number of animals, their diet, their drinking water, their caging, and the room

temperature (Hamm, 1985).








16
Other in vivo methods consist of detecting the formation of covalent binding

between chemicals and DNA. Among these methods are the use of radioactively

labelled chemicals (Warren, 1984), the use of specific anti-adduct antibodies

(Shamsuddin et al., 1985; Nehls et al., 1984), and the use of 32P postlabelling

(Randerath et al., 1984). These methods show some promise; however, they are

limited due to the very small quantity of DNA adducts which are formed in the

tissue. This limitation allows detection solely by pure chemical means and is only

applicable in certain cases (i.e., when the chemical marker is highly fluorescent or

radioactive).


In vitro Carcinogen Detection


As an alternative to the long-term in vivo methods, short-term in vitro

methods have been developed. These tests rely upon mutagenicity testing to reveal

the presence of carcinogens due to the beliefs that all carcinogens are mutagens and

that tumor formation involves genetic alteration (ICPEMC, 1982). Also of primary

importance to the in vitro tests is that they mimic the in vivo test conditions.

Genotoxic carcinogens will be detected without the need for metabolic activation;

however, nongenotoxic carcinogens need metabolic activation to become active

carcinogens. Therefore, the more closely the in vivo test conditions are mimicked,

the greater the chance of detecting the nongenotoxic carcinogens.

The most widely used in vitro test is the Ames Test (Ames et al., 1975; Ames,

1984). The Ames Test is performed by first mutating the bacteria Salmonella








17
typhimurium to prevent normal histidine production. The test sample is then

incubated with the bacteria colonies for two days at 37C, and afterwards, the

number of bacteria colonies which have reverted to allowing histidine production are

counted (Ames, 1984). The observation of a significantnumber of revertant colonies

above background indicates the presence of a mutagen in the test sample. In most

applications of the Ames Test, a liver homogenate known as S-9 is added to the petri

dish to mimic the in vivo conditions by acting as a metabolic activator (Miller and

Miller, 1977). In contrast to the in vivo tests, the in vitro Ames Test is much cheaper

and faster. One chemical can be adequately tested with regard to dosage in

approximately two weeks for $1000 to $1500, depending upon the protocol used

(Zeiger, 1985).

The in vitro tests may be cheaper and faster, but they do not possess the

overall reliability found using in vivo tests. The major reason for this occurrence is

that while all carcinogens are believed to be mutagens (Zeiger, 1985), all mutagens

are not carcinogens. As an example, 2-aminopurine has been found to mutate DNA

in bacteriological assays, but has not been found to be carcinogenic in either humans

or animals (Barrett, 1987). Similarly, tetrachlorodibenzo-p-dioxins, TCDDs, are a

known class of carcinogens which yield a negative Ames Test (ICPEMC, 1982). The

Ames Test has been found to have a success rate between 50 and 95% for the

identification of carcinogens from positive mutagenicity results (ICPEMC, 1982).

This success rate is to be class dependent for the chemicals under investigation

(ICPEMC, 1982).








18
The Ames Test has been successfully applied to the identification of

carcinogens in complex mixtures: the pyrolized amino acids formed during heating

(Bjeldanes et al., 1982) and the nitropyrenes formed from diesel exhaust (Sugimura

and Takayama, 1983). This success is tempered by the inability to combine the

Ames Test with on-line chromatography and spectroscopic identification. Having a

response time of two days and detection limits on the order of 100 nanograms, the

Ames Test cannot be combined with on-line chromatography, which requires a

response time of milliseconds and detection limits less than one nanogram.

Therefore, analysis of complex mixtures by the Ames Test requires repetitive

incubation and fractionation until the fraction containing the mutagen is found.

Once found, the mutagen must then be identified by a separate off-line spectroscopic

analysis (e.g., IR, NMR, MS).


Mass Spectrometric Approaches


Mass spectrometry has found use in nucleic acid analysis due to its ability to

yield structural information on a small quantity of sample. This ability combined

with "soft ionization" techniques (methods which allow for the formation of the intact

large biomolecule ion) has permitted the structural characterization of both modified

(i.e., carcinogen adducts) and unmodified nucleotides (Burlingame et al., 1983). Of

the various "soft ionization" techniques, fast atom bombardment (FAB) and matrix-

assisted laser desorption ionization (MALDI) have been the most extensively used.








19
The combination of FAB with tandem mass spectrometry has been applied

to the structural differentiation of isomeric nucleotides and dinucleotides as well as

being used for mixture separation (Crow et al., 1984; Cerny et al., 1986). However,

extensions to small oligonucleotides (3 to 6 nucleotides) resulted in complex spectra

which prevented proper sequencing of the oligomers (Cerny et al., 1987).

Performance of MALDI inside a Fourier transform mass spectrometer

(FTMS) has been shown to be capable of differentiating between methyl guanosine

isomers formed in the solution-phase and has been used to sequence oligonucleotides

consisting of four nucleotides (Hetitch, 1989; Hetitch and Buchanan, 1991). The

FTMS has the ability to fragment the oligonucleotide ions one nucleotide at a time,

permitting the detection of DNA modifications through DNA sequencing. However,

these sequencing methods may not be able to detect actual DNA modifications in

human tissue where there is normally one modification for every 108_1010 normal

nucleotides (Wolf et al., 1992). Studies are currently underway in which constant

neutral loss (CNL) scans on a TQMS are used to simplify the spectra to aid in the

detection of DNA modifications (Wolf et al., 1992; Bryant et al., 1992).

Derivitization of the nucleotide with trimethylsilane prior to analysis by CNL scans

has been reported to lower the detection limit of nucleoside-carcinogen adducts to

1 ng (Bryant et al., 1992). This detection limit corresponds to the detection of one

nucleoside-carcinogen adduct per 106 normal nucleosides.










Proposed Methodology


The mass spectrometric approaches discussed above focus on detecting

carcinogens by identifying nucleoside-carcinogen adducts which are formed in the

solution phase. Previous work in our laboratory (Freeman, 1991; Freeman et al.,

1990; Freeman et al., 1994; Anacchino, 1993) was the first demonstration of a strictly

gas-phase method, namely the use of selective ion-molecule reactions via mass

spectrometry for carcinogen screening. In that work, the ion-molecule reactions were

performed on a triple quadrupole mass spectrometer (TQMS). Various reports have

concluded that at some point during carcinogenesis, there is an

electrophile/nucleophile reaction between the ultimate carcinogen and the DNA

nucleoside (Miller, 1970; ICPEMC, 1982). Based upon those reports, the gas-phase

ion-molecule reactions were designed to be electrophile/nucleophile ion-molecule

reactions.

Ionized nucleophiles (model DNA bases, DNA bases, or nucleotides) were

formed in the TQMS ion source and were mass-selected by the first quadrupole.

These ions were passed into the second quadrupole (collision cell) where possible

carcinogens and mutagens were simultaneously introduced via gas chromatography

(GC). As the ionized nucleophiles traversed the second quadrupole, they reacted

with the electrophilic neutral carcinogens to form the nucleophile/electrophile adduct

ions. These product ions were then mass-analyzed by the third quadrupole and were

subsequently detected.








21
In that work (Freeman, 1991; Freeman et al., 1991), alternating scans

(selective ion-molecule reaction with a model DNA base ion and charge exchange

with methane molecular ions) were employed to obtain two complementary

chromatograms for the test sample. This scanning procedure is shown conceptually

in Figure 2-1. While only one GC column was employed, the alternating scan modes

gave the effect of performing two separate GC/MS analyses for each injection. First,

methane molecular ions were mass-selected and introduced into the second

quadrupole where they reacted with all compounds via charge exchange. After

detection of the charge exchange product ions, the nucleophilic model DNA ions

were mass-selected and introduced into the second quadrupole to undergo selective

ion-molecule reactions with only the electrophilic carcinogens. As this process was

repeated, two complementary chromatograms were produced. Reactions with the

methane molecular ions yielded a chromatogram which indicated the number of

components in the sample. This value was the upper limit for the number of

possible carcinogens in the sample. The selective ion-molecule reactions lowered

that number by producing a chromatogram which only had peaks if the compound

eluting from the GC column reacted with the model DNA ions, thus indicating which

peaks in the methane chromatogram represented possible carcinogens or mutagens.

The degree of fragmentation for each compound during the methane charge

exchange reactions was not given (Freeman, 1991). Therefore, the identification of

each was assumed to be a prior. Electron ionization would have provided complete

structural information but could not be performed because the carcinogens were not










Environmental Screening


IAAJX


MS


GC


IKT-[


Carcinogen
Detector






Figure 2-1: Depiction of proposed gas-phase ion-molecule screening to yield two
complementary chromatograms.








23
introduced into the ion source. Picogram limits of detection were reported for the

allyl halides, a class of well characterized mutagens, and the analysis speed was

improved significantly over that of the Ames Test (Freeman, 1991).

The TQMS method suffered several drawbacks. The TQMS is capable of

performing up to two stages of mass spectrometry: the reagent ion isolation and the

scanning of the reaction products. Therefore, when unknown product ions (i.e. ions

other than the desired adduct ion) were formed, they could not be fragmented to

indicate their structure. Another problem with the TQMS is the short reaction time

it permits for the reaction between the nucleophile ions and the carcinogen neutrals.

The ions can only react with the neutrals for as long as both reside inside the second

quadrupole. Unless the TQMS has been modified to permit ion trapping in the

collision cell (Anacchino, 1993), the residence time is on the order of microseconds.

This short reaction time biases the detection towards fast reaction kinetics and

permits slow reacting carcinogens, such as acrolein, to pass through undetected

(Freeman, 1991).

To overcome these shortcomings, a method using the QITMS is proposed.

This method for detecting carcinogens and mutagens on the QITMS consists of

performing gas chromatography/mass spectrometry (GC/MS) with alternating

electron ionization (El) and selective ion-molecule reaction scans. This proposed

method should overcome the problems of the TQMS method to yield better

detection and identification. Also the MS" capabilities of the QITMS allows the

identification of unknown product ions.








24
A sample containing possible carcinogens would be injected onto the gas

chromatograph which would separate the sample into its individual components. The

other end of the GC column is interfaced to the QITMS so that as the individual

components eluted off the column, they would enter directly into the ion trap

analyzer region. While the components eluted, they would undergo alternating El

and selective ion-molecule reaction scans. Similar to the TQMS method, the

selective ion-molecule reaction scan will indicate potential carcinogenicity if the

compound is reactive with a model DNA base ion. However, since the QITMS is

a tandem-in-time instrument (see Chapter 1) and the reaction time can be extended

into the millisecond time scale, slow reacting carcinogens should have ample time to

react which should remove any detection bias. Another characteristic of tandem-in-

time instruments is that the ion source and analyzer region are the same. This

characteristic gives the QITMS the ability to perform an El scan on the compounds

as they elute off the column, which will produce a mixture of molecular and fragment

ions, thus allowing structural information to be obtained so that the compound may

be identified.

As in the TQMS procedure, the alternating scans will produce two

complementary chromatograms for the sample. The El chromatogram would

indicate the number of components in the sample and the identity of each. The

selective ion-molecule reaction chromatogram would indicate which components are

possible carcinogens. This procedure would permit carcinogenic detection (selective

ion-molecule reaction chromatogram) along with the on-line chromatographic








25
separation (GC introduction of the sample) and spectroscopic identification (El

chromatogram) which the Ames Test lacks.

The basis for this gas-phase screening is the similarity between solution-phase

and gas-phase reactions. There have been several publications in the literature

(Pellerite and Brauman, 1980; Angelini and Speranza, 1981) which indicate the

strong similarity of reactions in both media. Figure 2-2 demonstrates the electro-

phile/nucleophile reactions in both the solution and gas-phases (Freeman, 1991). In

the body, where the solution-phase reactions occur, the ultimate carcinogen acts as

a reactive electrophile and reacts with the DNA base acting as a nucleophile; the

result is a DNA/carcinogen adduct. In the gas-phase reaction, only the detection of

genotoxic carcinogens is possible. The lack of metabolic activation in the gas-phase

prevents nongenotoxic carcinogens from being activated to the ultimate carcinogen.

The genotoxic carcinogens act as reactive electrophiles and react with nucleophilic

model DNA base ions to form a model DNA base/carcinogen adduct ion.

Model DNA base ions are used since it is easier to get them into the gas-

phase than if they were actual DNA bases. The model DNA base is chosen so that

it is structurally similar to the actual DNA bases, thus giving similar reactivity.

Figure 2-3 shows the structures of the five DNA bases and pyridine. For this initial

work, pyridine was chosen as the model DNA base ion due to is similar structure to

the DNA bases. The allyl halides were chosen as the carcinogens because they

represent a class of well-characterized mutagens. Studies have shown that

nucleophile ions (N', m/z N) react with the neutral allyl halides (EX) under a
















Modeling DNA/Carcinogen Reactions




Reaction in the Body


Carcinogen
(electrophile)


+ DNA Base
(nucleophile)


-- DNA adduct


Reaction in the Gas-Phase


Carcinogen
(electrophile)


+ Model DNA base
(neutral or ion)


- Product
(ion)


Figure 2-2: Comparison of reactions in the body with the proposed gas-phase screening
reactions.









NH

\
N

AO
H

Cytosine


0
CH
3CH NH

N-kO


Thymine


NH
2

KI
>aV


0

NH

NAO
H


Adenine




o
N NH

N NA NH
2


Uracil


N


Guanine


Figure 2-3: Structures of the five DNA bases and of pyridine.


Pyridine










N'-


CH2=CH-CH2X


Ionized
Nucleophile


Allyl Halide
Neutral


m/zN


X .


N-C-C CH=CH2

Nucleophile/Allyl
Adduct Ion


Halide
Radical


m/z [N+41]


Figure 2-4: Reaction for nucleophile ions with allyl halide neutrals.








29
variety of mechanisms (i.e. SN1, S~2) to produce a nucleophile/allyl adduct (NE, m/z

N+41) and the halogen radical (X) (Freeman et al., 1990; Eder et al., 1982a). This

reaction sequence is shown in Figure 2-4. The formation of the pyridine/allyl adduct

ion will be monitored for the characterization of the ion trap to perform these gas-

phase screening reactions.


Experimental


All pyridine/allyl halide ion-molecule reactions were performed on a Finnigan

MAT Ion Trap Mass Spectrometer (ITMS'"). The allyl halides (Sigma Chemical

Company, St. Louis, MO and Aldrich, Milwaukee, WI) and the pyridine (Fisher

Scientific, Orlando, FL) were obtained from the manufacturer and used without

further purification. Samples for the constant pressure studies were introduced

through Granville-Phillips (Boulder, CO) Series 203 variable leak valves. The valves

were heated to a constant temperature of 700C by wrapping them in heating tape

controlled with a variac. All pressures reported were measured by a Bayard-Alpert

ionization gauge mounted on the vacuum chamber and are uncorrected. Sample

pressures ranged from 1 x 107 torr to 3 x 106 torr.

The scan function used for performing the ion-molecule reactions is presented

in Figure 2-5. Ionization (step A) at q(N' of pyridine)=0.23 was followed by two-

step rf/dc isolation (step C) (Gronowska et al., 1990; Yates et al., 1991) of the

pyridine molecular ion [N ]. After isolation, the pyridine molecular ions were

allowed to react with the both the pyridine and allyl halide neutrals present inside

















C 0


- - ~
*- C




N u



*C3



o
Cl








Cu
Acc










S"





S/2















o Z
3 C



























-- u
(0
0r
.1-

5Q











C tu
--:

'3U
- S ^
| C 0 r
.2 -
CO
^ ^

*^. O
Cg CS
L S .2
o ca*.
g
P5 ro"
.i (0 m






m vs S **; (/
a j- 2 *













E~u


U



CC








31
the ion trap for up to 500 ms (step D) at a q(N')=0.3. Mass spectra were acquired

with the axial modulation (530 kHz and 6Vp.) mass-selective instability scan (step

E) (Stafford et al., 1984). The product spectra shown were obtained after the 500

ms reaction period.

Resonant excitation collision-induced dissociation (CID) was performed on

all product ions. Unless indicated otherwise, spectra obtained by CID utilized the

following procedure: following two-step rf/dc isolation (Gronowska et al., 1990;

Yates et al., 1991), the selected product ion was cooled for 10 ms at a ring RF

voltage level corresponding to q(product ion)= 0.3; the selected product ion was then

resonantly excited for 10 ms at the same RF level. Relative rate constants were

obtained by solving the pseudo-first order integrated rate equations for the product

ions. The equations and their derivations are presented in the Appendix.


Results


Ion-Molecule Reaction Spectra Under Static Conditions


Initial investigations focused upon the characterization of the ion trap to

perform the electrophile/nucleophile ion-molecule screening reactions. These

reactions were performed under static conditions, where each reactant is present

inside the QITMS at a constant pressure. Each allyl halide neutral was present at

a pressure of 2.5 x 107 torr. The pyridine pressure was 1.3 x 107 torr for the allyl

chloride reaction and was 2.4 x 107 torr for the allyl iodide reaction. The product

spectra for the reactions of pyridine molecular ions with allyl chloride and allyl









a) 79
N+.
1.00 80
NH*
.^
120
NE +
0.50-




0.00



b)
80
1.00 Nr

206
r NX+

0.50- 285
120 N 2X-
NE+ N



0.00

0 50 100 150 200 250 300
m/z




Figure 2-6: Product ion spectra for the reaction of pyridine molecular ions with (a) allyl
chloride and (b) allyl iodide neutrals.








33
iodide neutrals for 500 ms are shown in Figures 2-6a and 2-6b, respectively. In both

product spectra, the desired product ion (NE', the pyridine/allyl adduct ion) at m/z

120 was less abundant than the protonated nucleophile (NH) at m/z 80. For the

allyl chloride reaction, the m/z 120 ion was 60% of the relative abundance of the m/z

80 ion, while for the allyl iodide reaction its relative abundance was about 33%.

There was a significant difference in the amount of pyridine molecular ion remaining

for each reaction. This discrepancy is most likely due to the different pyridine

pressures used in each reaction, where at the higher pyridine pressure, reactions with

the pyridine neutrals depleted the number of pyridine molecular ions faster than at

the lower pyridine pressure. Also, while reaction with the allyl chloride did not

produce any pyridine/chlorine adduct ions, reaction with the allyl iodide did lead to

the formation of pyridine/iodine adduct ions (the pyridine/iodine adduct ion (NX)

at m/z 206 and the dipyridinium iodide ion (N2X') at m/z 285).

From the spectra in Figures 2-6a and 2-6b, a few characteristics of ion-

molecule reactions in the QITMS are apparent. Reactions with the neutral

electrophile as well as reactions with the neutral nucleophile can occur. The latter

reactions accounted for the most abundant product ion in both spectra, the

protonated nucleophile (NH') at m/z 80. Second, solution-phase results may not

always be accurate in predicting gas-phase results. In the solution-phase

mechanisms, the ionized nucleophile reacts at either C1 or at C3 of the allyl group.

There is no mention of ionized nucleophile reacting with the halide atom. In the

gas-phase, reaction at the halide atom competes with reaction at the allyl group








34
because the ions are unsolvated and their intrinsic reactivity controls which products

are formed.

These two characteristics and the spectra shown in Figures 2-6a and 2-6b

highlight the problems that unwanted ion-molecule reactions will cause for the gas-

phase screening of carcinogens and mutagens with regard to both sensitivity and

selectivity. As was seen for the relative abundances for the m/z 80 and m/z 120

product ions, competing reactions (such as those with the nucleophile neutrals)

deplete the ionized nucleophile population and reduce the extent of the desired

reaction with the electrophile neutrals. Therefore, the response per unit of analyte

(i.e. the sensitivity) is reduced.

Ideally, the ion-molecule product ion spectra will have only one product ion

present, an adduct ion between the model DNA base and the carcinogen or

mutagen. This ion's formation is based solely on the reaction proceeding because

the neutral is a carcinogen or mutagen. The formation of multiple product ions from

the reactions with both electrophile and nucleophile neutrals reduces the selectivity

of this detection procedure. The multiple product ions indicates that the overall

reaction is not specific for the detection of carcinogens and mutagens. While one

product ion may be due to the compound being a carcinogen or mutagen, the other

product ions may be due to other characteristics of that compound. Therefore, for

the reactions between the pyridine ions and the electrophile allyl halide neutrals, it

is critical to understand the reaction pathways for the formation of each product ion

in order to design the screening reactions so that the nucleophile/allyl adduct ion










(NE') will be the most abundant product ion formed.


Reaction Scheme Determination


To better understand the reactions between the pyridine molecular ions and

the allyl iodide and pyridine neutrals, the reaction scheme was elucidated through

a combination of MS" and time-resolved data acquisition. Figure 2-7 shows the MS4

sequence from a separate experiment used to identify the m/z 285 product ion of

Figure 2-6b. Figure 2-7a, obtained with the first stage of mass spectrometry, is the

electron ionization (EI) spectrum of the neutrals initially present in the QITMS.

Following isolation of the pyridine molecular ion (N') at m/z 79 and its subsequent

reaction for 500 ms with the neutrals present in the QITMS, the spectrum in Figure

2-7b was obtained. As mentioned earlier, unwanted product ions, such as the m/z

285 product ion, were produced in addition to the desired pyridine/allyl adduct ion

(NE') at m/z 120. Isolation and collision-induced dissociation (CID) of the m/z 285

product ion was performed and the resulting spectrum is presented in Figure 2-7c.

The only fragment ion occurred at m/z 206, due to a loss of 79 mass units. Note that

the m/z 286 ion, presumably the 3C isotope of the m/z 285 product ion, was not

affected by resonantly exciting the m/z 285 ion and therefore remained in the CID

spectrum. The m/z 206 fragment ion was further fragmented by CID without prior

mass isolation. The CID spectrum for the m/z 206 fragment ion, shown in Figure

2-7d, yielded a single fragment ion at m/z 79, due a loss of 127 mass units. Again

the m/z 286 ion remained in the fragmentation spectrum. While its intensity relative











1.00





0.50





0.00


1.00


0.50


0.00



Figure 2-7:


168
EX+.


80
NH+


285
N2X+


120
NE+


206
NX+


50 100 150 200 250 300
m/z
MS4 sequence for the reaction of ionized pyridine with neutral allyl
iodide and neutral pyridine.
(a) MS': Electron ionization of pyridine and allyl iodide neutrals.
(b) MS2: Product ion spectrum following the isolation of ionized
pyridine; (c) MS3: Isolation and CID of the m/z 285 product ion; (d)
MS4: CID of the m/z 206 fragment ion.












1.00-





0.50 -





0.00



d)

1.00-


0.50


0.00


I. a


' I


' I


Figure 2-7--continued.


206
NXw


' I


' I


' I


206
NXw


50 100 150 200 250
m/z


300








38
to the fragment ions has increased due to ion ejection during CID, the absolute

intensity of the m/z 286 ion has remained constant.

Based upon the information from the CID spectra in Figures 2-7c and 2-7d,

the m/z 285 product ion was identified as the dipyridinium iodine adduct ion (N2X).

The MS4 fragment ion at m/z 79 (Figure 2-7d) corresponded to the pyridine

molecular ion (N'). Addition of an iodine atom (127 mass units) to the pyridine

molecular ion resulted in the formation of the pyridine/iodine adduct ion (NX') at

m/z 206 (Figure 2-7c). Addition of a second pyridine molecule (79 mass units) to

the pyridine/iodine adduct ion (NX') yielded the dipyridinium iodine adduct ion

(N2X) at m/z 285.

The remaining product ions were also subjected to MS" analyses to determine

their structures. Table 2-1 lists the product ions along with the fragment ions which

were formed from each upon MS". The pyridine/iodine adduct ion (NX') at m/z 206

fragmented through homolytic cleavage at the pyridine-iodine bond to yield the

starting pyridine molecular ion (N') at m/z 79. The pyridine/allyl adduct ion (NE')

at m/z 120 fragmented via a 1,3-hydrogen shift to form the protonated pyridine

(NHI) at m/z 80. Both of these fragmentations are shown in Figure 2-8. The

protonated pyridine (NH') was resistant to fragmentation under the conditions

employed. The inability to obtain any fragment information on this ion was due to

the high energy necessary to fragment even electron ions with aromatic resonance

stabilization (Lossing and Holmes, 1984). Under the resonant excitation conditions

used, these ions were ejected from the ion trap before they could acquire sufficient






















Table 2-1

Fragment Ions Obtained from Collision Induced Dissociation of the
Product Ions from the Reaction of Pyridine Ions with Allyl Iodide Neutrals


Product Ion m/z


Product Ion Symbol


Fragment Ions (Stage)'


206(2); 79(3)


79(2)

80(2)

None


a Stage refers to the stage of mass spectrometry required to observe that particular
fragment ion. For example (2) refers to MS/MS, (3) to MS/MS/MS, etc.


285

206


N2X+

NX

NE+

NH+















m/z 206









CJN-CH2-CH=CH2


('N.


+ I.


m/z 79








-- C=CH,


m/z 120


+ CH2=C=CH,


mNz -H
m/z 80


Figure 2-8:


Fragmentation mechanisms for the m/z 206 and m/z 120 product ions
from the reaction of pyridine molecular ions with allyl iodide.









kinetic energy to induce fragmentation upon collision with helium.

The reaction pathways were determined by acquiring signal intensities as a

function of time as demonstrated in Figure 2-9. Visual inspection of this plot gives

insight into the reaction mechanism. In the early stages of the reaction (<50 ms),

the four product ions at m/z 80 (protonated pyridine, NH+), at m/z 120 (pyridine/allyl

adduct ion, NE'), at m/z 206 (pyridine/iodine adduct ion, NX'), and at m/z 285

(dipyridinium iodine ion, N2X+) initially increased while the m/z 79 pyridinee

molecular ion, N+) ion decreased. After 50-60 ms, the production of the m/z 206

product ion began to decay, while the rate of production of the m/z 285 product ion

(dipyridinium iodine adduct ion, N2X ) increased. This sequence is characteristic of

A-B--C consecutive reactions, where A forms B and then B forms C (Laidler,

1987). For the pyridine ion/allyl iodide neutral reaction, A corresponds to the

pyridine molecular ion (N'), B corresponds to the pyridine/iodine adduct ion (NX+),

and C corresponds to the dipyridinium iodine adduct ion (N2X+). This sequence

agrees with the MS4 spectra presented in Figure 2-7 in that both Figures 2-7 and 2-9

indicated that the m/z 285 product ion was formed from the reaction of the pyridine/

iodine adduct ion (NX') at m/z 206 ion with neutral pyridine. Further support for

this mechanism was found when the reaction of the isolated pyridine/iodine adduct

ions (NX') at m/z 206 with neutral pyridine and neutral allyl iodide resulted in the

sole production of the dipyridinium iodide ion (N2X') at m/z 285.

The time-resolved and MS" data were combined to obtain the reaction scheme

for pyridine ions reacting with pyridine neutrals and ally iodide neutrals which is

























3000-




a 2000-


m/z 120
1000-

m/z 2

m/z 79
0

0 100 200 300 400 500
Reaction Time (ms)




Figure 2-9: Signal intensity versus time for the reaction of pyridine molecular ions with
allyl iodide.











+


II
u
1 +

O


u
u
I




C2
E


+ 0
Z ~N
2


+
X
z~


------>*
CO
en
19


oz


*

0O








44
shown in Figure 2-10. This reaction scheme suggests that when the pyridine

molecular ion (N+) reacts with the allyl iodide neutral, there will always be

competition between the formation of the pyridine/allyl adduct ion (NE*) via loss of

the halogen radical and the formation of the pyridine/iodine adduct ion (NX+) via

loss of the allyl radical. These formation of the two products competes with the

desired reaction between the nucleophile ions and the allyl halide neutrals, shown

in Figure 2-4. Also, the presence of the pyridine neutrals introduced two more

unexpected product ions, the protonated pyridine (NH) at m/z 80 and the

dipyridinium iodine adduct ion (N2,X) at m/z 285.


Kinetics Analysis


When the screening reactions are performed on the QITMS, the competing

side reactions due to the inherent reactivity of the ions towards interfering neutrals

need to be minimized. Therefore, the rate constants for the formation of the four

product ions from the reaction of pyridine ions with the pyridine and the allyl iodide

neutrals were determined to define strategies which might minimize these side

reactions.

The reaction scheme in Figure 2-10 shows that each product ion results from

the reaction of one ion with one neutral, thus indicating bimolecular rate constants.

Typically, however, the number of each neutral species present was several orders

of magnitude greater than the number of each ionic species; therefore, the neutral

concentration can be considered constant which leads to pseudo-first order kinetics








45
for each product ion formation. Differential equations describing each product ion

formation (Laidler, 1987) were integrated with respect to time to yield the integrated

rate equations for each product ion formation as functions of reaction time and

[N']j. The integration used for each product ion are presented in the Appendix

and the integrated rate equations are listed in Table 2-2. The integrated rate

equations were plotted accordingly and the resulting slope was used to obtain the

desired rate constant. The plots used to determine the slopes are listed in Table 2-3

and the calculations necessary to evaluate the rate constants from the integrated rate

equations are given in the Appendix. For example, kT was found from the slope of

a plot of ln[N ] versus time, as shown in Figure 2-11. To demonstrate that the

inherent rate constants are pressure independent, as one would expect, a

combination of neutral allyl iodide and neutral pyridine pressures were used to

determine the product ion formation rate constants which are presented in Table 2-4.

The spectra resulting from pyridine to allyl iodide pressure ratios of 4:1 and of 1:10

are presented in Figures 2-12a and 2-12b, respectively.

This kinetic/pressure analysis of the reaction pathways revealed several aspects

regarding the inherent reactivity of the pyridine molecular ions towards both the allyl

iodide and pyridine neutrals. First, reaction with the pyridine neutrals is preferred

over reaction with the allyl iodide neutrals. The rate of formation of the protonated

pyridine, k4 (3.8x101- cc/molec s), is larger than the rates of formation of the

pyridine/allyl adduct, k, (7.2x10H- cc/molec s), and of the pyridine/iodine adduct, k2

(2.5x10'1 cc/molec s), combined. Second, if the pyridine ions were to react only with






























(U











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C,
0











c.
W o:
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8.00






7.00






6.00






5.00


0.00 0.10 0.20
Time (s)


Figure 2-11: Plot of In[N+] versus time to obtain the value of kT.


Best Fit Line:
y = -10.226x + 7.727
2-= .998


0.30






































































efmc r- 0
c~d- ( ~


6\O O\ 00


Cu
Cu





Ud










u
c


B
U



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-Cu




o
(4~ 0

f =



CuC

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cu


cu
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(c




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m

u

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00
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o~.

3 u
ac



U,
U)

p, M

[" c=

'4-


aCu:











80
NWr


285
N2Xf


120
NE+


206
NX


206
NX+


1.00-


80
NH


120
NE+


285
N2X+


AI ________ -. U~ .


' I
100


' I
150


m/z


200
200


250
250


300
300


Figure 2-12:


Product ion spectra for the reaction of pyridine molecular ions with
pyridine and allyl iodide neutrals. (a) Pyridine pressure is four times
that of allyl iodide. (b) Allyl iodide pressure is ten times that of
pyridine.


1.00


0.50


0.00


0.50 -





0.00-








51
the ally iodide neutrals, the pyridine/iodine adduct ion (NX+) at m/z 206 will always

be more abundant than the pyridine/allyl adduct ion (NE+) at m/z 120 because the

rate of formation of the m/z 206 ion is larger than the rate of formation of the m/z

120 ion. Both of these aspects are demonstrated in Figure 2-6b. In this spectrum,

the protonated pyridine (NH') at m/z 80 was the most abundant product ion, as

predicted by the kinetics. Also, the sum of the signal intensities for the

pyridine/iodine adduct ion (NX+) at m/z 206 and the dipyridinium iodide ion (N2X+)

at m/z 285 is larger than the signal intensity for the pyridine/allyl adduct ion (NE')

at m/z 120. Remembering that this sum represents rate constant k, (refer to the

Appendix and Table 2-3), this observation supports the kinetic determinations that

k2 is greater than k1.

In changing the relative pressures of each reactant neutral, the kinetics of the

reaction system was changed slightly. The entire kinetics discussion thus far has

focused upon the system exhibiting pseudo-first order kinetics because the number

of neutrals was several orders of magnitude greater than the number of ions. The

inherent rate constants, those due solely to the thermodynamics of the system, were

shown in Table 2-4 and are pressure insensitive. The observed rate constants are the

product of the inherent rate constant times the pressure of the neutral reactant. By

changing the relative pressures of the pyridine and allyl iodide neutrals, the observed

rate constants are changed. This change manifests itself in the relative abundances

of the product ions generated as shown in Figures 2-12a and 2-12b. In Figure 2-12a,

the pyridine pressure is four times greater than the allyl iodide pressure. This








52
pressure ratio favors the formation of product ions due to reaction with pyridine

neutrals, thus the relatively high intensity for the protonated pyridine (NH) at m/z

80 and for the dipyridinium iodide ion (N2X') at m/z 285. In contrast, the allyl

iodide pressure is ten times greater than the pyridine pressure for the spectrum

shown in Figure 2-12b. This pressure ratio increases the rate of formation of the

pyridine/allyl adduct ion (NE') at m/z 120 and the pyridine/iodine adduct ion (NX)

at m/z 206 relative to the reaction conditions which yielded the spectrum shown in

Figure 2-12a.


Conclusions


The ability to screen for carcinogens and mutagens inside an ion trap via

selective ion-molecule reactions is improved when only one product ion is formed

with a significant extent of reaction. This chapter has demonstrated two factors

which must be taken into account when trying to perform these selective ion-

molecule reactions. The thermodynamics of the reactions between the reagent ions

and all neutrals which are present inside the QITMS is the first factor since it will

indicate which product ions are formed. The thermodynamics for formation, as

shown through the inherent rate constants, of the pyridine/iodine adduct ion (NX')

were more favorable than those for the formation of the pyridine/allyl adduct ion

(NE') when pyridine ions reacted with allyl iodide neutrals. Therefore any reaction

between pyridine ions and allyl iodide neutrals will result in two product ions with

the desired product ion being less abundant. The second major factor is the pressure








53
of each neutral which is present. The pressure determines the observed pseudo-first

order rate constants and thus controls the extent of production of each product ion.

There are two general approaches to remedy the problems mentioned above.

Changing the ion/neutral chemistry through a change of the ionized nucleophile is

one method. This change will alter the overall thermodynamics of the system, and

if done correctly, will reduce the production of side products upon reaction with the

electrophilic carcinogen. In other words, reaction between an alternate ionized

nucleophile and allyl iodide might produce only one product ion, the desired

nucleophile/allyl adduct ion (NE'). A second approach to reducing the production

of side product ions is to reduce the pressure of the nucleophile neutrals during the

reaction period. As seen with the pyridine/allyl iodide system, reactions due to the

pyridine neutrals accounted for as many product ions as the reactions due to the allyl

iodide neutrals. Reduction of the nucleophile pressure during the reaction period

will minimize the reactions with the neutral nucleophile.














CHAPTER 3
STRATEGIES FOR BETTER CONTROL OVER ION-MOLECULE
REACTIONS PERFORMED IN A QITMS


Introduction


The previous chapter identified the two major factors which must be

considered when performing ion-molecule reactions in a QITMS. The

thermodynamics (i.e. heat of reaction, activation energy, etc.) between the reactant

ions and all neutrals which are present inside the QITMS during the reaction period

determines which product ions can be formed. The pressures of each neutral govern

the extent to which each reacts with the reactant ions. Based upon these

characteristics, the lack of spatial separation inherent to the tandem-in-time QITMS

was shown to be problematic for the gas-phase reactions between pyridine and the

allyl halides. The ionized nucleophile pyridinee M' ions) could not only react with

the nucleophile neutrals, but preferred to react with them as opposed to reacting

with the electrophilic allyl halide neutrals.

This chapter will present two approaches which were investigated to overcome

the problems resulting from the lack of spatial separation. The first method was to

change the ion-neutral chemistry by using an alternate ionized nucleophile. In this

case, piperidine ions were reacted with the allyl halides in order to prevent the

production of the nucleophile/halide adduct ions. The main focus of this chapter will








55
be on the second approach, namely the use of pulsed valve introduction of the

nucleophile to introduce temporal separation between the ionized nucleophile and

the nucleophile neutrals. Once characterized, the pulsed valve is evaluated with

respect to spectral quality and compared to that obtained by the static pressure

approach presented in Chapter 2. Both methods of introduction are then used to

detect and quantify allyl halide mixtures. Finally, the pulsed valve introduction and

alternate nucleophile approaches are combined and applied to the analysis of a

mixture of carcinogens and noncarcinogens.


Experimental


The allyl halides (Sigma Chemical Company, St.Louis, MO and Aldrich,

Milwaukee, WI), pyridine (Fisher Scientific, Orlando, FL), and piperidine (Fisher

Scientific) were obtained from the manufacturer and used without further

purification. Samples for the constant pressure studies were introduced through

Granville-Phillips (Boulder, CO) Series 203 variable leak valves. The valves were

wrapped with heating tape and were heated to a constant temperature of 70C. All

pressures reported were measured by a Bayard-Alpert ionization gauge mounted on

the vacuum chamber and are uncorrected. Sample pressures ranged from 1 x 10'

torr to 3 x 106 torr.

All experiments were performed on a Finnigan MAT Ion Trap Mass

Spectrometer (ITMST"). A Varian 3300 gas chromatograph was used for gas

chromatography (GC) introduction of the mixtures. The GC transfer line for








56
introduction into the ITMS, designed by Hail et al. (1989), consists of a capillary

column passed through a 1 m long stainless steel (s.s.) tubing (1/16" o.d.) which is

resistively heated by applying an AC voltage provided by a variable transformer

(Variac) and step-down transformer across the s.s. tubing. The s.s. tubing is passed

into a modified solids probe shaft to allow for easy insertion of the transfer line into

the ITMS chamber through the solids probe lock.

Pulsed valve introduction of the nucleophiles was through a Series 9 pulsed

valve (General Valve Corp., Fairfield, NJ). An expanded view of the pulsed valve

experimental setup is shown in Figure 3-1. The hardware consists of a pulsed valve

mounted on 1/4" stainless steel tubing, which was inserted into the ITMS chamber

through a 1/4" bored-through Cajon adapter welded to the flange. A teflon sleeve,

containing two sets of relief holes, was used to act as a splitter for the pulsed sample.

The teflon sleeve was held in place by a stainless steel brace (not shown) mounted

to the chamber flange. The exit orifice of the pulsed valve was 0.006 in. in diameter.

Ion Catcher Software, ICMS* (developed by Nathan A. Yates at the

University of Florida), was used to allow software control of the TTL signal on the

ITMS Scan acquisition processor (SAP) Adapter board. This TTL signal triggered

a pulsed valve controller built at the University of Florida. The duration of the

control signal emitted from the pulsed valve controller was measured by a LeCroy

(Chestnut Ridge, NY) 9400 dual 125 MHz digital oscilloscope.

The scan function used for performing the pulsed valve ion-molecule reactions

is presented in Figure 3-2. A TIL trigger pulse (step A) was generated prior to









57







I-









E I -









E u
r
-o
U,>




SI I














21-
0
L -
tF ii l z









I-
rV a.'
< l
I C/3

I~ -^ ^-
^1~ O
































































0 0

- c o


-=


.r- -












o .

0- 0












0
Og -
0 0
0> O

























O a-





a-o
a a
o v 2
9? 5 6
vi -

0 .S (
'^3 *- -
<- H
3 U -L
*aeo^

O L"S;
laS|
-^ _, B










3 -<3
c 5 .
EO a;
*<3 t3
a .2


=,


U Ir

- -Cllj -


U

EP








59
ionization. This pulse was used to open and close the pulsed valve so that the

nucleophile sample may be introduced into the ITMS chamber. After an appropriate

delay (step B), ionization (step C) at q(nucleophile)=0.23 was followed by reagent

ion formation (step D) and two-step rf/dc isolation (step E) (Gronowska et al., 1990;

Yates et al., 1991) of the nucleophile molecular ion [N'-]. After isolation, the

nucleophile molecular ions were allowed to react with the both the nucleophile and

electrophile neutrals present inside the ion trap for up to 500 ms at a q(N+)=0.3

(step F). Mass spectra were acquired with the axial modulation (530 kHz and 6Vp.p)

mass-selective instability scan (step G) (Stafford et al., 1984). The product spectra

shown were obtained after the 500 ms reaction period.

For the calibration studies, gas chromatography was carried out on a J & W

Scientific (Folsom, CA) DB-5 (27.5 m long, 0.250 mm i.d., 0.25 pm film thickness)

capillary column in the split mode (split ratio(SR)=52:1) with helium carrier gas at

an inlet pressure of 8 psig. The GC oven was temperature programmed from 35C

to 120C at 200C/min after an initial hold time of 3 minutes. One microliter

injections were made in triplicate at an injection port temperature of 2000C and a

transfer line temperature of 200C. Allyl halide (chloride, bromide, and iodide)

standards were prepared in octane in the following manner: the 50, 25, 10, and 5

nmol/pL solutions were prepared from a 500nmol/pL stock solution; the 1000, 500,

250, 100, and 50 pmol/pL solutions from the 10 nmol/pL solution; the 25, 10, 5, 1,

and 0.5 pmol/pL solutions from the 100 pmol/ipL solution. Quantitation was

performed on the selected ion-molecule product ions (m/z 120 for allyl chloride and








60
both m/z 120 and m/z 206 for allyl iodide) through the Quantitation Program of the

ICMS software. Calibration curves obtained for pulsed valve introduction used a

control pulse of 2.23 ms. The pulsed valve was located one-half inch away from the

QITMS and the teflon sleeve was not used. Constant pressure calibration curves

used a pyridine pressure of 3 x 10-7 torr (uncorrected).

For the gas-phase reactivity studies, gas chromatography was carried out on

a J & W Scientific (Folsom, CA) DB-5 (27.5 m long, 0.25 mm i.d., 0.25 pm film

thickness) capillary column in the split mode (SR=52:1) with helium carrier gas at

an inlet pressure of 8 psig. The GC oven was temperature programmed from 30C

to 1500C at 10C/min after an initial hold time of 3 minutes. One microliter

injections were made at an injection port temperature of 2000C and a transfer line

temperature of 2000C. Two carcinogen/noncarcinogen mixtures were prepared with

the components listed in Table 3-1. Each mixture was equimolar (59.6 nmol/pL);

mixture 1 was prepared in octane and mixture 2 was prepared in pentane. Each

mixture was reacted with pyridine and thiophene molecular ions. Data for these

studies were acquired by alternating an electron ionization (El) scan with a selective

ion-molecule scan while the mixtures were eluting off the GC column.

Chromatograms for the El and selective ion-molecule scans were obtained as

follows. For the El chromatogram, the reconstructed ion current (RIC) for each

scan was plotted as a function of time. For the selective ion-molecule

chromatogram, the intensities for both the nucleophile molecular ion and the

protonated nucleophile ion were subtracted from each scan. The resulting RIC's













Table 3-1

Properties of the Nucleophiles and the Analytes Comprising the Two
Equimolar GC Mixtures, Listed in Their Order of Elution


MW IE(eV) Supplier


MIXTURE #1:

Acrolein
Allyl Chloride
Propyl chloride
2-Bro mopropane
Allyl Bromide
Benzene
Cyclohexane
Allyl Iodide
Propyl Iodide
Allyl Isothiocynate
m-Xylene
Decane

MIXTURE #2:

2,3-Dichloropropene
Epichlorohydrin
Chlorobenzene
Ethylbenzene
Styrene
Bromobenzene
m-Dichlorobenzene

NUCLEOPHILES:


56
76
78
122
120
78
84
168
170
99
106
142


10.10b
9.90
10.82
10.07
10.16
9.25
9.86
9.30
9.27
NRc
8.56
9.65


NR
NR
9.06
8.77
8.43
8.98
9.11


Pyridine
Thiophene


9.25
8.87


Fluka (Buchs, Switzerland)
Aldrich (Milwalkee, WI)
Eastman Kodak (Rochester, NY)
Eastman Kodak


Aldrich
Fisher Scientific
Fisher Scientific
Aldrich
Eastman Kodak


(Orlando, FL)


Aldrich
Chem Service (West Chester, PA)
Alfa Products (Danvers, MA)


Aldrich
Eastman Kodak
Chem Service
Fisher Scientific
Fisher Scientific
J.T. Baker (Phillipsburg, NJ)
Aldrich


Aldrich
Aldrich


MW listed is for most abundant isotope
Ionization energies listed in Lias et al., 1988.
No ionization energy recorded in Lias et al., 1988.


Electrophile










were plotted as a function of time.


Changing the Ion-Neutral Chemistry


Aromatic radical cations have been reported to form stable adducts with 2-

alkyl iodides, but not with either 2-alkyl bromides or 2-alkyl chlorides (Gross et al.,

1977; Miller and Gross, 1983; Holman and Gross, 1989). The aromatic/2-alkyl iodide

adduct ions then decompose to Wheland-type structures with the expulsion of neutral

iodine. This selected reactivity was attributed to the high polarizability of the iodine

atom (Miller and Gross, 1983; Holman and Gross, 1989). The iodine atom was

better able to accommodate a positive charge than either the chloride or bromide

atoms and thus the aromatic radical cations bonded at the iodine atom of the alkyl

iodides more easily than at either the chlorine or bromine atoms.

These characteristics can be observed in the spectra shown in Figures 2-6a and

2-6b. Reaction of pyridine molecular ions with allyl chloride produced no chlorine

adducts (Figure 2-6a); however, reaction with allyl iodide produced two iodine

adduct ions (Figure 2-6b). The formation of the pyridine/iodine adduct ions was

likely enhanced by a combination of the aromaticity of pyridine, which allows it to

resonantly stabilize the positive charge of the adduct ion, and the ability of the allyl

group to exist as a radical (i.e. is a good leaving group). In the studies above, no

aromatic cation/iodine adducts were formed through cleavage of the alkyl group

because the alkyl groups were not good leaving groups. Since the allyl group is a

good leaving group, the use of a non-aromatic ionized nucleophile should reduce the










a)
80
1.00 N+

206
NX +

S 0.50- 285
120 N+
NE+



0.00


b)
86
1.00 N





S 0.50-
126
85 NE+
N+-

0.00

0 50 100 150 200 250 300
m/z




Figure 3-3: Product ion spectra for the reactions of allyl iodide with the molecular
ions of (a) pyridine and (b) piperidine.










formation of iodine adduct ions upon reaction with allyl iodide.

This hypothesis was tested by performing the mass-selected reaction of the

piperidine molecular ions, the saturated analog of pyridine, with allyl iodide. The

product ion spectra of this reaction is compared to that of the pyridine molecular ion

/allyl iodide reaction in Figure 3-3. The inability of the piperidine to resonantly

stabilize the positive charge prevented the formation of piperidine/iodine adducts, as

evidenced by the lack of product ion formation at m/z 212 (piperidine/iodine adduct

ion) and at m/z 297 (dipiperidinium iodine ion). However, elimination of iodine

adduct ion formation did not prevent the production of the piperidine/allyl adduct

ion, NE*. Similar to the pyridine ions, with both reagents present at equal pressures,

the piperidine ions were more reactive with the piperidine neutrals than with the allyl

iodide neutrals because the self-protonation product ion (NH') at m/z 86 was more

than twice as abundant as the piperidine/allyl adduct ion (NE') at m/z 126.

In Chapter 2, the effects of competing ion-molecule reactions on both

selectivity and sensitivity were discussed. By changing the ion/neutral chemistry, one

primarily affects the selectivity of the ion-molecule reaction. For the test system, the

allyl halides, the selectivity obtained using piperidine molecular ions is much greater

than that obtained using pyridine molecular ions. For the pyridine/allyl halide

reactions, pyridine/iodine and pyridine/bromine adduct ions were observed to form

upon reaction with allyl iodide and allyl bromide, respectively. For piperidine, there

is a higher degree of selectivity due to the reduction of most, if not all, side

reactions. Reaction of the piperidine molecular ions with the allyl halide neutrals








65
produced only one product ion, the piperidine/allyl adduct ion at m/z 126

(Remember, the protonated piperidine ion results from reaction of the piperidine

molecular ions with neutral piperidine).


Pulsed Valve Introduction


The preceding section demonstrates that changing the ion/neutral chemistry

can alter the selectivity of the ion-molecule reaction. However, the lack of spatial

separation still permitted reaction between the nucleophile ions and the nucleophile

neutrals even when the more selective reagent ion (piperidine) was used. As

discussed in Chapter 2, the competition between the nucleophile neutrals and the

electrophile neutrals to react with the nucleophile ions will have a significant effect

on the sensitivity of the ion-molecule reaction. The results obtained thus far

indicated the need for separation between the nucleophile ions and nucleophile

neutrals during the reaction period. The inherent reactivity between the ions and the

neutrals dictate which product ions were formed; the pressures of the neutral

reactants control the extent of product ion formation. Therefore, by reducing the

number of nucleophile neutrals present inside the reaction volume during the

reaction period, the nucleophile ions will react preferentially with the electrophile

neutrals.

Temporal separation between the nucleophile ions and nucleophile neutrals

through pulsed valve introduction was chosen for its reported success for use with

ion-molecule reactions (Einhorn et al., 1991; Emary et al., 1990). In those previous








66
studies, the pulsed valve was mounted external to the ITMS chamber. The gas pulse

width (FWHM), determined by following the variation of the N2' signal intensity as

a function of time from the opening of the pulsed valve to introduce N2, was found

by Emary et al. (1990) to decrease as the connecting tubing was shortened; a pulse

width (FWHM) of approximately 50 ms was reported for 2.5 cm of connecting

tubing. The ultimate goal for this work, as stated previously, is the real-time

screening of environmental samples for carcinogens and mutagens. To accomplish

this goal, pulsed valve introduction of the nucleophile must permit quick removal of

the nucleophile neutrals in order to prevent their interference during the ion-

molecule reaction period and to allow a fast sampling speed which will adequately

sample over a gas chromatographic peak. The pulse width resulting from the

external configuration was too long to permit the quick removal of the nucleophile

neutrals. To minimize the diffusional broadening of the gas pulse as it traverses the

tubing and thereby reduce the duration of both the pulse width and the scan function

(i.e. allow for quick removal of the neutrals), the pulsed valve was mounted inside

the vacuum chamber (Figure 3-1). This setup allowed the distance between the

pulsed valve and the ion trap to be easily adjusted, without breaking vacuum, by

sliding the stainless steel tubing through the Cajon adapter.


Optimization of Pulsed Valve Introduction


The concept behind achieving temporal separation between the nucleophile

ions and nucleophile neutrals through the use of pulsed valve introduction of the








67
nucleophile is presented in Figure 3-4. After the valve is pulsed open and closed,

a pulse of nucleophile neutrals is introduced into the ITMS chamber. This gas pulse

will diffuse into the ion trap analyzer volume, and as it does so, the nucleophile ion

intensity due to the pulse increases. If ionization is performed at the apex of the gas

pulse, then the maximum number of nucleophile ions will be produced, with the

pressure of the nucleophile neutrals decreasing immediately afterward. If the time

required for isolation of the nucleophile molecular ions is adjusted so that by the end

of that period the nucleophile neutral pressure has returned to within 20% of the

base pressure, the electrophile pressure will be significantly higher than the

nucleophile neutral pressure and only reaction with the electrophile neutrals should

occur to any extent.

Three major factors influenced the effectiveness of the pulsed valve when

mounted inside the ITMS chamber: (1) the distance between the valve and the ion

trap; (2) the length of the delay, ionization, and isolation times; and (3) the duration

of the pulsed valve open period. The distance between the valve and the ion trap

was found to control the amount of the pulsed sample which was available for a

given reaction, the time necessary to reach the apex of the gas pulse, and the sample

pulse width. Figure 3-5 presents the total ion intensity (measured as the RIC) for

pyridine as a function of both the distance between the valve and the ion trap and

the time between the closing of the pulsed valve and ionization (referred to as the

delay time) for an open-time of 1.93 ms. Figure 3-1 shows that the teflon sleeve into

which the valve was positioned has two sets of relief holes. These relief holes act as
























































(sg.mn a Ba !qia) ajnssajdj I~qed


68




0

o
0



0






1 1
...




. O
~,d
0i



0







0


o
*a











E
a
*





*M
o1

&4


E
O
I

B -r
o
~II
I

















50000



40000



8 30000



20000



10000



0






Figure 3-5:


Delay Time (ms)


Effects of distance on observed pulse width. Distance between pulsed
valve and QITMS is (A) 0"; (B) 0.5"; and (C) 0.75". Control pulse
duration is 1.93 ms.








70
a splitter for the pulsed sample. When the pulsed valve was positioned at the front

of the sleeve, the valve orifice was located next to the ion trap. Therefore, when the

valve was opened, nearly all of the sample which was introduced went directly into

the ion trap. Placing the pulsed valve at this location resulted in the largest neutral

population inside the QITMS as shown by curve A in Figure 3-5. When the pulsed

valve was backed away from the ion trap, but still positioned inside the teflon sleeve,

enough space was created between the pulsed valve and the relief holes that some

of the sample was split away. Part of the sample entered into the ion trap, with the

rest of the sample passing through the relief holes into the vacuum manifold. This

splitting of the sample pulse lowered the ion intensities as shown by curve B in

Figure 3-5. Curve C demonstrates that when the valve was located beyond the end

of the teflon sleeve, the greatest degree of splitting occurred.

Figure 3-5 also demonstrates that after closing the pulsed valve, both the

length of time needed to reach the apex of the sample pulse and the sample pulse

width (FWHM) decreased as the distance between the valve and the ion trap

increased. This tendency can be understood in terms of conductance, or the rate at

which the neutrals are introduced and removed from a given volume of space. The

overall rate of change of the number of neutrals is a function of both their rate of

introduction and their rate of removal. This can be described as:


d[Neutral]
d[Neur = rate of introduction rate of removal (3-1)
dt

Integration of equation (3-1) with the appropriate functions for the rates of








71
introduction and removal should produce a curve that increases logarithmically for

a certain length of time and then decreases exponentially. The length of time that

the curve increases is dependent upon (a) the difference in the rates of introduction

and removal and (b) the length of time the valve is opened. For a given valve open-

time, the amount of sample introduced is constant due to the constant conductance

of the pulsed valve. The rate of neutral removal, however, changes depending upon

the position of the pulsed valve. When the pulsed valve is located at the front of the

teflon holder, the entire sample is introduced into the ion trap; therefore, the rate

of removal is only due to the conductance of the ion trap. As the pulsed valve is

backed away from the ion trap but still inside the teflon holder, the rate of removal

becomes a function of the conductance of both the ion trap and the relief holes of

the teflon holder. The conductance of the ion trap remains constant, but the

conductance of the teflon holder increases as the pulsed valve is displaced farther

from the ion trap due to the incorporation of the relief holes.

In terms of equation (3-1), when the pulsed valve is opened for a constant

time, the rate of introduction is a constant. As the rate of removal increases, two

things will happen to the curve from equation (3-1). First, the time needed to reach

the apex will shorten. Second, once the pulsed valve is closed, only the rate of

removal remains active. Therefore, with increasing conductance, the exponential

decay will increase which will result in a steeper slope on the backside of the curve

and will shorten the pulse width. These trends are evident from both the positions

of the sample pulse apices and the corresponding sample pulse widths for curves A,










B, and C in Figure 3-5.

While the experiments were not performed, it seems reasonable that the

open-time for the pulsed valve determines the total amount of sample introduced

into the vacuum chamber. From the preceding discussion, the QITMS system has

a certain conductance based upon the distance between the pulsed valve and the ion

trap. Based upon this conductance, there would be a maximum open-time for the

pulsed valve where the nucleophile neutrals could be removed efficiently. If the

pulsed valve was open for longer, more sample would be introduced than could be

removed prior to the reaction period, resulting in a significant amount of neutral

nucleophile present which would interfere, as shown in Figures 3-3a and 3-3b.


Spectra Obtained from Pulsed Valve Introduction


While Figure 3-4 presented an ideal representation of using a pulsed valve

and the preceding discussion outlined the influence of various factors when the

electrophile neutrals were not present, one other factor needs to be taken into

account when the electrophile neutrals are present. As was mentioned previously,

once the sample was introduced, the delay, ionization, and mass isolation times

needed to be long enough to allow the neutral nucleophile to be reduced and thus

prevented from competing with the desired reactions. However, as shown in Figure

2-8, once N is formed (during the ionization period), it will begin to undergo ion-

molecule reactions with all of the neutrals present inside the ion trap. Those

product ions formed during the ionization and post-ionization periods will reduce the








73
effective N' population available for ion-molecule reactions following the mass

isolation of the N+ ions. As such, the effective N- ion population will be dependent

largely upon the production of N` ions during ionization (which is dependent upon

the delay and ionization times) and the loss of N'- ions due to ion-molecule reactions

prior to the mass isolation (a function of ionization time, post-ionization time, and

delay time). Each of these periods was optimized.

The pulsed valve open-time used in this section was 1.65 ms. This pulse

duration resulted in the observed gas pulse for pyridine shown in Figure 3-6, with a

pulse width (FWHM) of 10 ms. This narrower pulse width for the internal

configuration compared to that for the external configuration (Emary et al., 1990)

will allow for more frequent sampling across a gas chromatographic peak; the scan

function time from the 10 ms gas pulse width with a 300 ms reaction period was 380

ms. Based upon the observed pyridine gas pulse width of 10 ms (FWHM), the

pyridine/allyl iodide reaction was performed using a 2 ms delay after pulsing followed

by a 2 ms ionization time (resulting in ionization at the apex of the pyridine pulse),

a cool time of 10 ms, and a 3 ms two-step rf/dc isolation of the N- (m/z 79) ion of

pyridine.

The product ion spectrum for pyridine introduced via pulsed valve reacting

with allyl iodide for 500 ms is shown in Figure 3-7a. In contrast to the constant

pressure spectrum shown in Figure 3-3a in which NH' is the major product ion,

pulsed valve introduction resulted primarily in the formation of the pyridine/iodine

adduct ion at m/z 206 and the pyridine/allyl adduct ion at m/z 120. This product ion
























1.00.

0.80.

0.60.


40 50 60


Delay Time (ms)


Figure 3-6: Observed gas pulse width for a 1.65ms control pulse and the pulsed
valve located next to the QITMS. Ion intensity is the RIC from the
El scans of pyridine.





















79 120
N80 N


1.00-





0.50-





0.00


126
NE+


86
NIW


*


I I I I I I I I I 1
50 100


Figure 3-7: Product ion spectra for the
pyridine and (b) piperidine
introduction of the nucleophil


150
m/z


200


I250
250


I30
300


reaction of the molecular ion of (a)
with allyl iodide using pulsed valve


206


1.00





0.50





0.00


285
N2X+








76
spectrum was due to the ionized nucleophile reacting almost entirely with the

electrophilic allyl iodide neutrals. Also, the product ion distribution appeared as

predicted by the rate constants given in Table 2-4. The ratio of m/z 206 (NX') ion

intensity to the m/z 120 (NE) ion intensity was 3:1, the same ratio as that of rate

constant k2 (the formation of m/z 206) to rate constant ki (the formation of m/z 120).

As expected, the use of pulsed valve introduction limited the extent of reaction with

the neutral nucleophile by significantly reducing its amount present during the

reaction period. However, the undesired side reactions were not totally eliminated

as evidenced by the slight formation of the protonated pyridine (NH') at m/z 80 and

the dipyridinium iodine adduct ion (N2X') at m/z 285. Since the reaction period was

initiated when 80% of the neutral pyridine had been removed, there were still a

small number of pyridine neutrals present to react. By rearranging the integrated

rate equations (Table 2-2) for the m/z 80 and m/z 206 product ions to solve for the

neutral pyridine pressure, [N], and using the determined rate constants, the

uncorrected pressure of neutral pyridine present during the reaction period from

pulsed valve introduction was estimated to be 4 x 108 torr.

Similarly, piperidine was introduced through the pulsed valve for reaction with

allyl iodide. The product ion spectrum presented in Figure 3-7b was obtained using

similar values for the delay, ionization and isolation times to those used for pyridine.

For this case, the only major side reaction (see Figure 3-3b), the formation of the

protonated nucleophile (NH+) at m/z 86, was significantly inhibited.








77
Sensitivity Comparison Between Pulsed Valve and Constant Pressure Introduction


In Chapter 2, the lack of spatial separation was hypothesized to reduce the

sensitivity of the selective ion-molecule reactions. Competing reactions with the

nucleophile neutrals would reduce the number of ionized nucleophiles which are able

to react with the electrophile neutrals. This reduction is expected to impact most at

low concentrations of electrophile neutrals, since given the small number of

electrophile neutrals, any reduction in product ion formation may prevent their

detection.

Figure 3-8 presents the log-log calibration curves obtained for the reaction of

pyridine ions with allyl iodide (Figure 3-8a) and allyl chloride (Figure 3-8b) using

pulsed valve introduction of the pyridine neutrals. Both curves demonstrate a linear

dynamic range of two orders of magnitude and limits of detection (LOD) around 10

pg on-column. The slopes of 0.94 and 0.91 for the allyl iodide and allyl chloride

curves, respectively, are close to the expected slope of unity. The observed LOD's

agree with those reported by Freeman (1991) for calibration curves acquired on the

TQMS. These comparable LODs indicate that the temporal separation obtained

from pulsed valve introduction is just as effective as the degree of spatial separation

inherent to the TQMS.

A linear dymanic range of three to four orders of magnitude was observed in

the TQMS studies (Freeman, 1991). The narrower LDR with the QITMS is a result

of the lack of spatial separation inherent to the QITMS. Pulsed valve introduction

permits temporal separation between the nucleophile neutrals and the nucleophile
















Calibration Curve for Pyridine/Allyl Iodide
Pulsed-Valve Introduction


slope = 0.94


I I 1 111111


I I 1111111


I I 111111


100 1000 10000
Amount on-column (pg)


I 1111111
100000


Figure 3-8:


(a) Calibration curve for the reaction of pyridine molecular ions with
allyl iodide, using pulsed valve introduction.


-Y
0
Q


3


10000




1000




100


-I ---- '"`- ---- ----- ---


I I 1111111
10


I














Calibration Curve for Pyridine/Allyl Chloride
Pulsed-Valve Introduction


slope = 0.91


Ii


S I 11111111 11111111 1 i I 1 11 1 I i 11111i 1 i 111i11
0.1 1.0 10.0 100.0 1000.0 10000.0
Amount on-column (pg)


Figure 3-8 (continued): (b) Calibration curve for the reaction of pyridine molecular
ions with allyl chloride, using pulsed valve introduction.


100000


10000



1000



100








80
ions during the reaction period. However, pulsed valve introduction does not enable

any temporal separation to be attained between the nucleophile neutrals and the

electrophile neutrals during the ionization and reagent ion formation periods, thus

limiting the linear dynamic range.

Previously, the problems during the reagent ion formation period were

identified, namely that the ions and neutrals react immediately, reducing the effective

ionized nucleophile population (i.e. the number of ionized nucleophiles left at the

start of the reaction period). When the nucleophile neutral pressure is significantly

greater than the electrophile neutral pressure during reagent ion formation, the ion

losses due to reactions with the electrophile neutrals are less than those due to

reactions with the nucleophile neutrals. As the electrophile pressure increases with

increasing sample concentration, the ion losses during the reagent formation period

increase due to the greater number of electrophile neutrals available for reaction.

Eventually, the ion losses increase to the point where the effective nucleophile ion

population is significantly lower than it was for lesser electrophile concentrations.

The result is a leveling off in the calibration curve.

While not complete, there is a large degree of spatial separation during both

the ionization and reagent ion formation periods when performing these reactions

on the TQMS. On the TQMS, the electrophile neutrals are introduced into the

collision cell, not the ion source, and thus do not compete during those periods. As

a result, the effective ionized nucleophile population reacting in the collision cell

remains fairly constant with increasing electrophile concentrations. Therefore, the








81
log-log calibration curves performed on the TQMS should have wider linear dynamic

ranges, as observed by Freeman (1991). The calibration curves on the TQMS

eventually become limited when the entire nucleophile ion population is exhausted

upon reaction in the second quadrupole with the electrophile neutrals.

The log-log calibration curves for pyridine ions with allyl iodide and allyl

chloride obtained with constant pressure introduction of pyridine are shown in

Figures 3-9a and 3-9b, respectively. Similar to the pulsed valve calibration curves,

the constant pressure calibration curves have slopes near unity, 1.01 for allyl chloride

and 0.89 for allyl iodide; however, the constant pressure curves show narrower linear

dynamic ranges and higher LOD's. Allyl iodide has an LOD of 15 pg and a linear

dynamic range of 1.5 orders of magnitude. Allyl chloride has an LOD of 70 pg and

a linear dynamic range of only one order of magnitude.

The smaller linear dymanic ranges and higher LOD's are due to competitions

with the nucleophile neutrals during the reaction period and with the electrophile

neutrals during the reagent ion formation periods. The latter competition was just

discussed as the reason for limiting the high end of the linear dynamic range.

Competition with the nucleophile neutrals during the reaction period will reduce the

effective nucleophile ion population which can react with the electrophile neutrals.

As mentioned previously, this reduction should result in higher LODs.

A mathematical approach can be used to demonstrate that pulsed valve

introduction results in greater sensitivity than constant pressure introduction. A

linear calibration curve will have the following form:




















100000


10000 --




1000-




100


-I ---- ---


Calibration Curve for Pyridine/Allyl Iodide
Constant Pressure Introduction







I


slope = 0.89


I I I 111111


I I I 1111
) 100


Amount on-column (pg)


Figure 3-9:


(a) Calibration curve for the reaction of pyridine molecular ions with
allyl iodide, using constant pressure introduction.


I1 11 11111 IIII 1I 11 I1
1000 10000 100000


*
















Calibration Curve for Pyridine/Allyl Chloride
Constant Pressure Introduction


slope = 1.01


II


I I 1111111 I I 111111


1 11111111 i1 111111 111111


1.0 10.0 100.0 1000.0
Amount on-column (pg)


10000.0


Figure 3-9 (continued): (b) Calibration curve for the reaction of pyridine molecular
ions with allyl chloride, using constant pressure introduction.


10000


1000




100




10


-I_ ___ ~~_






















Table 3-2

Sensitivity Factors for Pyridine/Allyl Halide Calibration Curves


Method of Introduction

Pulsed-valve

Pulsed-valve

Constant Pressure

Constant Pressure


Electrophile

Allyl Iodide

Allyl Chloride

Allyl Iodide

Allyl Chloride


a) Determined from the y-intercept of the extrapolated linear portion if the log-
log calibration curve (see text for further explanation).


Log [m]a

3.27

3.23

2.75

1.74








85

y = mx + b (3-2)

where y is the peak area, x is the amount on-column, m is the slope or sensitivity,

and b is the blank response. If b=0 and the base ten logarithm of both sides is

taken, one gets:


log[y] = log[x] + log[m] (3-3)

Therefore, the y-intercept, determined by extrapolation of the linear portion of the

log-log calibration curve to where log[x]=0, is of the sensitivity. Table 3-2 lists the

sensitivities obtained from extrapolation of the linear portions of the log-log

calibration curves. The sensitivities for the pulsed valve calibration curves are both

about 3.25. For the constant pressure curves, the sensitivities are 1.74 for allyl

chloride and 2.75 for allyl iodide. These diminished sensitivities for the constant

pressures calibration curves support the argument of added competition from the

pyridine neutrals. The more reactive allyl iodide can compete with the pyridine

neutrals better than the allyl chloride, thus the sensitivity for the allyl iodide curve

is reduced less than the sensitivity for the allyl chloride curve.


Mixture Analysis


From the preceding discussions, two concepts have become apparent. First,

pulsed valve introduction of the nucleophile allows for maximum sensitivity during

the reaction period by minimizing reactions with the neutral nucleophile. Second,

changing the ion/neutral chemistry alters the product ion distribution and the








86
selectivity of the reaction. With those two concepts in mind, the gas-phase screening

method was attempted on two carcinogen/noncarcinogen mixtures with the ionized

nucleophiles pyridine and thiophene. The compositions of the two mixtures is

presented in Table 3-1.

One point which needs to be made is that while these gas-phase ion-molecule

reactions are used to screen for carcinogens and mutagens, they are actually designed

to identify electrophiles. Remember, the ultimate carcinogen has been classified as

a reactive electrophile (Miller, 1970), and the selective ion-molecule reactions have

been based upon nucleophile/electrophile adduct formation. Therefore, these gas-

phase screening reactions should detect direct acting carcinogens through adduct

formation, but they should not be able to detect carcinogens which must be

metabolically activated because in their inactivated form, these latter carcinogens are

not reactive electrophiles.


Mixture Analysis with Pvridine


Despite its lack of selectivity with the allyl halides (i.e. it forms more than one

product ion), ionized pyridine was used as a starting point to evaluate its usefulness

for identifying direct acting carcinogens. Figure 3-10 presents the two

complementary chromatograms obtained from the alternating El and selective ion-

molecule scans for mixture #1. Table 3-3 lists the components of the mixture, their

corresponding peak number, their Ames test results, and their gas-phase results. The

first observation which can be made from these chromatograms is that the early

















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89
chromatographic peaks were undersampled. Not only is 2-bromopropane absent

from the El chromatogram, but the first five peaks are each only two or three data

points wide. At least five data points are needed across a GC peak for proper

sampling. This undersampling was due to the narrow GC peaks and the length of

the scans. The high flowrate used with the GC column (1.44 mL/min) enabled the

early components to elute off the column as peaks which were less than one second

wide. As a result of the combined length of the scans, only one or two scans of each

(El and selective ion-molecule) type of scan could be performed. If the compound

eluted primarily during the selective ion-molecule scan, as is probably the case for

2-bromopropane, then that compound would be absent from the El scan. Since 2-

bromopropane is a noncarcinogen (see Table 3-3), no reaction during the selective

ion-molecule scan is expected. The result is that 2-bromopropane is absent from

both chromatograms.

To determine that the length of the scans was mainly responsible for the

undersampling, the El and ion-molecule chromatograms acquired for a similar

mixture using a different column (J&W DB-5, 20m, 0.18mm i.d., 0.4 pm film

thickness), but the same El and selective ion-molecule scans. The chromatograms

are displayed in Figure 3-11. The components of mixture #3 are listed in Table 3-4

along with their gas-phase and Ames test results. The slower flowrate used with this

column (0.674 mL/min) permits the necessary number of each scan to be performed.

Now the early portion of the chromatogram has very good separation of the mixture,

but wider peaks to prevent undersampling. The only drawback to this column was



























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