i

THE DEVELOPMENT OF AN IN-FIELD RAPID DERIVATISATION TECHNIQUE FOR THE ANALYSIS OF CHEMICAL WARFARE AGENT DEGRADANTS

By

Lucas Dival

A thesis submitted in fulfilment of the requirements for the degree of

Master of Forensic Science (Professional Practice)

in

The School of Veterinary and Life Sciences

Murdoch University

Kate Rowen, John Coumbaros

Semester 1, 2018

ii

Declaration

I declare that this thesis does not contain any material submitted previously for the award

of any other degree or diploma at any university or other tertiary institution. Furthermore,

to the best of my knowledge, it does not contain any material previously published or

written by another individual, except where due reference has been made in the text.

Finally, I declare that all reported experimentations performed in this research were carried

out by myself, except that any contribution by others, with whom I have worked is explicitly

acknowledged.

Signed: Lucas Dival

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Acknowledgements

This work would not have been possible without funding and support from Murdoch

University. To my supervisors Kate Rowen and John Coumbaros, please accept my deepest

thanks and appreciation for your assistance in conducting this research. In particular Kate

Rowen, for proposing the original method to be tested.

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Table of Contents

Title Page ............................................................................................................................... i

Declaration ............................................................................................................................ ii

Acknowledgements .............................................................................................................. iii

Part One Literature Review .................................................................................................... 1-61

Part Two Manuscript ............................................................................................................ 62-88

Blank Page – not numbered

1

Part One

Literature Review

THE DEVELOPMENT OF AN IN-FIELD RAPID DERIVATISATION TECHNIQUE FOR THE ANALYSIS OF CHEMICAL WARFARE AGENT DEGRADANTS – LITERATURE

REVIEW

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Abstract The degradation of various toxic nerve agents in the environment has been documented

throughout literature to result in the formation of methylphosphonic acid. The detection

of this compound is used as an indication of previous use or production of such nerve

agents, however for this detection to be possible methylphosphonic acid must first

undergo derivatisation. This process, at its current stage, can be time consuming. To

validate the need for alternative, faster methods, this literature review shall investigate

the degradation of various nerve agents and current derivatisation processes. It was

evident from the findings attained that a process in which derivatisation of

methylphosphonic acid could be achieved quickly would be of great interest to forensic

specialists.

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Contents

TITLE PAGE 1

ABSTRACT 2

LIST OF FIGURES 5

LIST OF TABLES 5

LIST OF ABBREVIATIONS 6

1. INTRODUCTION 7

2. CHEMICAL WARFARE AGENT OVERVIEW 9

3. DEGRADATION OF ORGANOPHOSPHOROUS NERVE AGENTS 12

3.1 Ethyl N,N-dimethylphosphoroamidocyanidate: GA (Tabun) Degradation 13

3.2 Isopropyl methylphosphonoflouridate: GB (Sarin) Degradation 16

3.3 Pinacolyl methylphosphonofluoridate: GD (Soman) Degradation 19

3.4 O-ethyl S-[2-diisopropylaminoethyl] methylphosphonothioate: VX Degradation 20

4. EXTRACTION TECHNIQUES OF CWA CHEMICAL MARKERS 22

4.1 Soil 22

4.2 Liquids 24

4.3 Solids 26

5. DERIVATISATION TECHNIQUES 26

5.1 Disadvantages of Derivatisation 27

5.2 Factors Influencing the Choice of Derivatisation Method 28

5.3 Current Developments in Derivatisation Techniques 29

5.3.1 Development of Derivatisation Techniques not Requiring Removal of Water 32

5.4 Derivatisation of Organophosphorous Nerve Agents and their Degradants 33

5.4.1 Methyl Ester Derivatives 35

5.4.2 Silyl Derivatives 37

4

5.4.3 Pentafluorobenzyl Derivatives 43

6. CONCLUSION AND RATIONALISATION FOR PROPOSED RESEARCH METHODS 44

7. REFERENCES 47

5

List of Figures Figure Page

1 Chemical structures of various CWAs 12

2 Hydrolysis pathways of GA from 14

3 Hydrolysis of GB 16

4 Hydrolysis of GD in the environment 20

5 Hydrolysis of VX resulting in cleavage of the P-S bond. This

pathway is possible at any pH but is predominant at pH<6 and

pH>10

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6 Hydrolysis of VX resulting in cleavage of the C-O bond,

predominant when the pH is between 6 and 10

21

7 Methylation of pinacolyl methylphosphonic acid (14) by

trimethyloxonium tetrafluoroborate

35

8 The TBDMS derivative of MPA formed by reaction MTBSTFA 39

List of Tables Table Page

1 Historical events regarding the production and use of CWAs 10

6

List of Abbreviations Abbreviation Explanation

CWC Chemical Weapons Convention CWA chemical warfare agent

OPCW Organisation for the Prohibition of Chemical Weapons

MPA methylphosphonic acid EDPA ethyl N,N-dimethylamidophosphoric acid IMPA isopropyl methylphosphonic acid

Pi inorganic phosphate DEMP diethyl methylphosphonate EMMP ethylmethyl methylphosphonate DMMP dimethyl methylphosphonate DIMP diisopropyl methylphosphonate

GC gas chromatography LC liquid chromatography ESI electrospray ionisation

PMPA pinacolyl methylphosphonic acid EMPA ethyl methylphosphonic acid

EA2192 S-[2-diisopropylamino)ethyl] methylphosphonothioic acid

NMR nuclear magnetic resonance GC-MS gas chromatography-mass spectrometry

LC-APCI-MS) liquid chromatography atmospheric pressure chemical ionisation mass

spectrometry NICI-MS negative ion chemical ionisation mass

spectrometry SPE solid-phase extraction

SPME solid-phase micro extraction GC-FPD GC flame photometric detection

TMS trimethylsilyl TBDMS tert-butyldimethylsilyl TMPAH trimethylphenylammonium hydroxide

FPD flame photometric detection NPD nitrogen phosphorus detection AED atomic emission detector

BSTFA O-bis(trimethylsilyl)trifluoroacetamide TMSCl trimethylsilyl chloride

MTBSTFA N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide

SIM selected ion monitoring MRM multiple reaction monitoring PFBBr pentafluorobenzyl bromide

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1. Introduction For the authentication of the adherence to the Chemical Weapons Convention (CWC),

analysis and monitoring for the presence or absence of chemical warfare agents (CWAs),

their precursors and degradation products is integral (1). Implemented in 1997, under the

CWC the production and stockpiling of CWAs is prohibited, with signatory nations required

to dismantle chemical warfare arsenals previously developed. Under the CWC, chemicals

are listed in a series of Schedules based on a range of qualifiers, including their potential

risk to populations. Scheduled chemicals range from precursors and degradants of CWAs

to the CWAs themselves, with the most dangerous chemicals listed under Schedule 1. A

collection of laboratories designated by the CWC’s supervisory body, the Organisation for

the Prohibition of Chemical Weapons (OPCW), undertake analysis of samples from

suspected stockpiles, production facilities and war zones to verify compliance to the CWC.

With the danger of CWAs and the implications of their alleged use or production by states

arises the need for conclusive evidence of the presence of CWAs, their precursors and

degradants in various environmental matrices, with analyses for such often needing

sensitivity to detect target compounds in the parts per billion. Other collections of

laboratories also analyse biological matrices for metabolites and evidence for poisoning by

CWAs (2).

The degradation of CWAs in the environment and in biological systems has been well

documented (this shall be developed later). The degradation products formed, referred to

as degradants from here on, are used in analytical techniques implemented by OPCW

designated laboratories as chemical markers indicating historical exposure of CWAs to a

particular environment or physiology (14).

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Developments made in the analysis for CWAs, degradants and precursors allow for further

improvement in detection and decontamination methods as well as physical protection

from these chemicals. Analysis of trace amounts of CWAs in the environment is also

necessary for remedial action on lands related to the production, storage or use of CWAs

(3).

Of the stockpiled CWAs throughout the world, nerve agents comprise the largest quantity

and are the most potent (4). Nerve agents are organophosphorous electrophiles that, upon

reaction with the nucleophilic serine in the active site of acetylcholinesterase in the

nervous system, inhibit neural activation and thereby shutdown the nervous system (5).

Though they saw their first development around World War I (4, 6), the recent alleged use

of nerve agents by terrorists and nations (7, 8, 9) has drawn considerable interest in the

development of more modern and effective analytical methods.

As shall be described in this review, current techniques used for the analysis of

organophosphorus nerve agent chemical weapons often require sample preparation that

is time consuming and can introduce errors into subsequent detection. A method proposed

here is thought to circumvent this time-consuming step, and so provide a rapid qualitative

answer on whether CWA chemical markers are present in an aqueous sample. The research

conducted will investigate the viability of this method.

This literature review will report on the current methodology utilised for CWA analysis.

Research on the degradation pathways of these agents shall be reported for understanding

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of the target analytes to be used in the proposed method. The extraction methods of such

analytes from various matrices shall be reported for comprehension of preliminary sample

preparation that may be performed prior to the steps in the proposed research. Current

derivatisation techniques shall be investigated to find any gaps in literature, and thus

rationalise the parameters of the proposed method, as well as for an understanding of

possible limitations should the new method be successful.

2. Chemical Warfare Agent Overview The use of chemical weapons by nations or terrorist groups remains a constant threat to

modern society. A class of these chemical weapons, known as nerve or neurotoxic agents,

prove to cause the greatest apprehension due to the toxic physiological action they have

on the body. The continuing possibility of the use of these weapons provides just cause for

alert monitoring for their deployment and for medical treatment for those against whom

they may be used.

The term “chemical warfare” was first coined in 1917 following the use of chlorine gas by

the German Army against the Allied Forces during the first World War, who subsequently

retaliated (10, 11). Chemical warfare has since been defined as “tactical war assets which

use incendiary mixtures, smokes and irritating, vesicant, poisonous or asphyxiating gases”

(11). Earlier occurrence of chemical warfare by this definition has however been recorded,

as documented around 1000BC, in China where arsenic laced smoke was used, and with

poisoned water used in Greece (10). Over time, Chemical CWAs became more complex in

their development and effects upon life (4). While the documented use of CWAs has not

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been in a large a scale as during World War I, several states and terrorist groups have since

been proven to have deployed CWAs as shown in Table 1 (4,6).

Date Historical event

1915-1918 Use of CWAs in World War I

1935 Use of mustard gas by Italy in Libya and Ethiopia

1936 First synthesis of the nerve agent tabun by German scientist Gerhard Schrader

1937 Synthesis of sarin by Schrader and associates

1939 Japan uses mustard gas against China

1940-1945 Germany employs Zyklon B, a variant of hydrogen cyanide in gas chambers

1942 Germany begins industrial production of nerve agents

1944 Synthesis of soman by German scientist Richard Kuhn

1950s Synthesis of VX by the British and weaponization by the USA

1984-1986 Use of CWAs by Iraq against Iran confirmed

1988 Use of CWAs by Iraq against Kurdish people confirmed

1994 Sarin attack by Japanese terrorist group Aum Shinrikyo

1994/1995 Use of VX in assassination attempts by Aum Shinrikyo

1995 Aum Shinrikyo uses sarin in Japanese subway Table 1 Historical events regarding the production and use of CWAs (4,6)

CWAs are classified according to their chemical and physiological properties as well as their

nature of use (4). CWAs can also be classified as persistent or non-persistent based on their

volatility (11). More volatile agents such as chlorine gas are less persistent than agents such

as VX with lower volatility, and so are removed faster from the environment by

atmospheric dispersion (11). CWAs are generally classified into the following categories

(10):

i) Vesicants or blistering agents, which cause painful chemical burns and blisters

to mucous membranes such as the lungs or to the skin. Vesicants such as sulfur

mustard can lead to death, however often have an incapacitating effect

requiring long term hospitalisation.

ii) Pulmonary toxicants or choking agents, such as phosgene, which cause choking

by attacking the pulmonary system.

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iii) Cyanogenic or blood agents, which cause respiratory failure by inhibition of the

exchange of oxygen and carbon dioxide between blood cells and tissue cells.

iv) Incapacitating agents, which are often used as riot-control agents such as in the

case of tear gas due to their non-lethal effects (at certain doses).

v) Neurotoxic or nerve agents, which lead to the shutdown of the somatic and

autonomic nervous systems by inhibition of the acetylcholinesterase enzyme

upon which the systems rely.

Nerve agents can also be subdivided into two other classes. The first nerve agents were

classed as G-agents, so named due to their discovery by German scientist Gerhard Schrader

during his research in development of organophosphorous (OP) pesticides (12).

Subsequent development of V-agents (V denoting ‘venomous’) were produced by the

British, and were many times more powerful, stable and persistent than G-agents (13). The

lower volatility of V-agents, as well as their fat solubility allows for their mode of entry into

physiological systems to be through dermal exposure, whereas the introduction of G-

agents is primarily through inhalation (11). Figure 1 (14) shows the chemical structures of

various CWAs; the G-agents tabun, sarin, soman and cyclosarin, V-agents VX and RVX,

nitrogen and sulfur mustards and the vesicant Lewisite 1.

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Figure 1 Chemical structures of various CWAs (14)

Neurotoxic agents are the largest component of modern CWA arsenals, and so are the

focus of this research. The ease of their manufacturer and devastating employment, as well

as the difficulty at which they are able to be detected, makes nerve agents ideal for terrorist

use (15). Although there are confirmed uses of nerve agents in Tokyo, Iran and Syria,

demonstrating the destructiveness of these weapons (7, 8, 9), there are also numerous

unconfirmed reports of their use (11). This shows a strong argument for the development

on the expansion of methods available to detect the use of these nerve agents so that the

victims of these inhumane weapons may have greater hope of justice.

3. Degradation of Organophosphorous Nerve Agents Many CWAs exhibit a reactive electrophilic behaviour, which plays a large part in their

toxicity and degradation mechanisms. In biological and environmental matrices these

electrophilic agents undergo hydrolysis when in contact with water, resulting in polar

products. A majority of these degradants aren’t as toxic as the parent agent from which

they degrade (14), although there are some exceptions such as the agent VX. These

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degradants constitute a large focus for CWA analysis as they can a give a greater indication

for prior use or production of the parent agents due to their longer persistence in the

environment and biological systems. All nerve agents in their raw form are viscous liquids,

though the G-agents tend to present more of a vapor hazard due to their greater volatility

than the V-agents, which by comparison pose more of a surface hazard (16). The higher

solubility of G-agents also makes them more susceptible to hydrolysis (16).

3.1 Ethyl N,N-dimethylphosphoroamidocyanidate: GA (Tabun) Degradation The synthesis of GA (Tabun, ethyl N,N-dimethylphosphoroamidocyanidate) is simple

though often results in many impurities. Steps to purify the mixture are often applied (16).

Depending on the conditions present, a final production sample of GA could also consist of

degradation products as well as impurities should it be in storage for extensive periods of

time. Degradation pathways of GA allow for it to result in the most possible degradants of

the G-agents (3, 17–20). Because of its instability and inefficient production methods, pure

GA is scarce. Analysis conducted on military GA found that impurities consisted of 28% of

the content (19, 21). The degradant diethyl dimethylphosphoramidate was found to

account for the largest of the impurities, with smaller amounts of ethyl

dimethylphosphoramidate, O-ethyl O-isopropyl N-dimethylphosphoramidate, triethyl

phosphate and tetramethylphosphorodiamidic cyanide. Upon reaction with water,

hydrolysis of GA results in the formation of ethylphosoryl cyanidate, dimethylamine, ethyl

N,N-dimethylamidophosphoric acid (EDPA), dimethylamidophosphoramidate, phosphoric

acid and hydrogen cyanide (though this is not unique to the hydrolysis pathways) (16). The

pathways of the formation of these compounds is shown in Figure 2. As will be shown the

case later with other nerve agents, the initial hydrolysis reaction forming the first order

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degradant(s) occurs quickly (in this case the hydrolysis of GA to O-ethyl N,N-dimethylamido

phosphoric acid), with the subsequent hydrolysis reactions occurring at a far slower rate

(22). The favoured pathways under acidic and basic conditions are shown in Figure 2. It is

important to note that in neutral pH conditions, the pathway forming O-ethyl N,N-

dimethylamido phosphoric acid is also favoured (22). Only one literature source has

reported that final hydrolysis to methylphosphonic acid (MPA) is theoretically possible, but

it is likely that such amounts are below limits of detection, though a likely explanation for

this was not provided nor investigated elsewhere (23). Because of this, analysis of GA agent

markers often regards EDPA as the target degradant (24).

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Though these hydrolysis reactions can occur at all pH conditions, the reactions are

accelerated at high or low pH and elevated temperatures (22). Under ambient temperature

and neutral pH GA persists in solution for 14 – 28 hours (25), with a half-life reported to be

about 8 hours under slightly decreased temperature (26). However, other literature offers

half-life values in conflict with what might be expected, with an increase in the half-life of

GA at a lowered pH, with it being 2 and a half hours at pH 5 and 14 hours at pH 3 (27). This

may be explained by the tendency of solutions to approach a pH of 4 – 5 upon hydrolysis

of GA, due to the acidity of the hydrolysis products (22). This may explain that the greater

concentration of OH- in solution drives the hydrolysis reactions forward, albeit low [OH-]

may have a lesser effect in low pH conditions due to its minute amount. This may be

supported by the far shorter half-life of GA in basic conditions, such as a mere 4.5 hours in

alkaline seawater (26).

GA has a volatility of 610 mg/m3 at 25˚C, which is about one twentieth that of water, though

far greater than V-agents (20). These slight volatile properties of GA have an impact on its

environmental persistence. Under average weather conditions (that being no rain, strong

winds or extreme temperatures), GA is reported to last for 1 to 2 days as surface deposition

of it as a liquid (28). It persistence in the environment comes from its absorption into traces

of water. With a Henry’s Law constant of 1.52 x10-7 atm.m3/mol, GA has negligible

volatilisation from its aqueous form in water (22). As expected, at low temperatures the

persistence of GA is greatly increased, extended to 2 weeks when topically deployed on

snow (29, 30).

Figure 2 Hydrolysis pathways of GA from (22)

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3.2 Isopropyl methylphosphonoflouridate: GB (Sarin) Degradation In aqueous solutions, it has been found that hydrolysis of the P-F bond in GB (Sarin,

isopropyl methylphosphonoflouridate) is the favoured position for hydrolysis to occur,

forming isopropyl methylphosphonic acid (IMPA) (31, 32). Current literature has found that

in high pH environments dealkylation of the isopropyl moiety is favoured producing the

compound fluoromethyl phosphonic acid (33); however, subsequent research was unable

to produce the same results (34–50). The hydrolysis product isopropyl methylphosphonic

acid has been shown to undergo further hydrolysis to methylphosphonic acid at a much

slower rate in comparison to GB’s conversion into IMPA (34, 35). At an even slower rate,

this MPA can hydrolyse into inorganic phosphate (Pi) (36). This pathway of hydrolysis is

shown in Figure 3 (37).

The rate of formation of these products is greatly affected by the environmental conditions,

namely temperature and acidity (32, 38, 39). A minimum rate of hydrolysis of GB has been

shown to occur at a pH of 4.5 – 6, with the rate of hydrolysis increasing with greater

concentrations of hydrogen and hydroxide ions (37). Literature reporting the maximum

half-life of aqueous GB has shown large variation, reporting values of 193 hours (40, 41),

238 hours (32) and 312 hours (31), with all methods used therein using aqueous samples

of pH 6 at 25ºC. A large increase of pH has been shown to greatly decrease the half-life,

Figure 3 Hydrolysis of GB (37)

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with it being a mere 3 seconds at pH 12 (42). The hydrolysis of GB is catalysed by both acidic

and basic conditions. Auto-catalysis has been shown to occur at low pH conditions when

the concentration of GB is in the order of 10-4 M, at which point the acidic hydrolysis

products (IMPA and HF) further lower the pH of the solution (40). In basic solutions

hydrolysis occurs at a slower rate with auto-buffering occurring due to the same

mechanism (40). Further catalysis may occur if GB is in the presence of hydroxycations such

as Cu(OH)+ or Ca(OH)+ (43), with half-life reduced to 2 hours in these circumstances. This

explains the faster hydrolysis rate observed for CWA analysis samples of sea water with the

observed half-life reduced to 58.1 minutes (44, 45). A large eighty-fold increase in the

hydrolysis rate of GB has been found to occur by copper (II) chelate-catalysis (39).

Most literature has pointed to IMPA and HF being the exclusive hydrolytic products of GB

by cleavage of the P-F bond. However, alcoholic solvolysis has been found to result in ethyl

isopropyl methylphosphonate (46). It is important for research to cover various possible

environments that may be tested for the presence of degradants of CWAs, so that

appropriate subsequent extraction and testing methods may be used with confidence. A

nucleophilic substitution reaction has been found to occur of diethyl methylphosphonate

(DEMP) by methoxide, resulting in the formation of ethylmethyl methylphosphonate

(EMMP) and dimethyl methylphosphonate (DMMP) (47). Such a reaction may not appear

to be related to analysis of GB or its degradants. However, alcohols and hydroxides are

often used in decontamination agents (42). These substitution reactions may occur with

the decontamination agents with the dealkylation of GB occurring after by its degradation.

Therefore, choosing a degradant as an analyte may not be unique evidence of the presence

of the parent agent.

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Conversion of IMPA into MPA has been shown to be possible, but only occurring very slowly

at ambient temperature. Research has been conducted into this dealkylation in acidic

conditions and higher temperatures, with dealkylation occurring at a rate proportional to

the concentration of the protonated phosphonic acid (34). Under conditions attempting to

convert all alkyl methylphosphonic acid into MPA, such results have been repeated at pH 3

and 169˚C for water samples extracted from the environment (26).

Although it has been reported that further degradation of MPA into inorganic phosphate

(Pi) can occur, the research reporting such does not hold firm claim over this (36). Pi has

been shown to result in MPA solutions where added calcium magnesium and iron cations

are present in small amounts (36). Such results may indicate that the C-P bond of MPA is

less stable than what has been established in literature, however Schowanek and

Verstraete’s work requires further development as only inorganic phosphate was

monitored, with the amount detected only twice as much as background concentration of

blanks. The analysis technique used is also not very specific (37).

False identification of the presence of diisopropyl methylphosphonate (DIMP) and MPA has

been shown due to condensation of IMPA on gas chromatography (GC) columns (48).

Because of this, it may be necessary to verify whether the presence of these is due to the

condensation or whether they were present in the initial sample, done so by modifying the

GC technique or using other analyses such as liquid chromatography (LC) combined with

electrospray ionisation (ESI) (49, 50), though to do so may be more experimentally

demanding.

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3.3 Pinacolyl methylphosphonofluoridate: GD (Soman) Degradation Though there is less research into the degradation of GD (Soman, pinacolyl

methylphosphonofluoridate), what has been conducted has pointed to similar end

products as GB. The major pathway of its hydrolysis forms pinacolyl methylphosphonic acid

(PMPA), which can undergo hydrolysis itself to result in the formation of MPA (37). The

half-life of GD in its formation of these products is reported to be 60 hours at pH 6 and 25oC

(27). Also, similarly to GB, the hydrolysis mechanism is catalysed in acidic and basic

conditions (33), and by copper(II) complexes (51).

Research has questioned whether MPA is the final product in the hydrolysis of GD,

suggesting rather that PMPA undergoes no further degradation. It has been shown that for

biological samples of plasma and liver tissue, no further hydrolysis of PMPA occurred (52),

however such was found only in vitro samples. Further research using in vivo testing found

the end product of the hydrolysis to be a complex of MPA bound with the enzyme

acetylcholinesterase (53).

The first order degradant, PMPA, been found to have a very long half-life. Research

indicating that upon storage at pH 6 for 8 weeks, the PMPA:MPA ratio was 250:1 (27).

Extrapolation then shows that the half-life of PMPA is 27 years, which is consistent with

previous research (34). Figure 4 shows the primary hydrolysis pathway of GD in the

environment (3).

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Figure 4 Hydrolysis of GD in the environment (3)

3.4 O-ethyl S-[2-diisopropylaminoethyl] methylphosphonothioate: VX Degradation

The pathways of hydrolysis that VX (O-ethyl S-[2-diisopropylaminoethyl]

methylphosphonothioate) can undergo allows this agent to degrade into far more

degradants than the other nerve agents. Depending of the conditions, hydrolysis can either

occur at the P-O-C or P-S bonds. In acidic conditions (where pH<6), ethyl methylphosphonic

acid (EMPA) can result from the cleavage of the P-S bond, and in basic conditions (pH>10)

cleavage of the same P-S bond can occur, however resulting in the degradant

diethylaminoethylmercaptan (37). EMPA has shown further hydrolysis to form MPA, albeit

at a much slower rate (37). Both reactions occur steadily, however the rate of degradation

is accelerated in basic conditions (37). While the degradation reaction(s) of VX are

prevalent when the pH is less than six or greater than 10, degradation still occurs regardless

of pH. In such cases, tendency towards dealkylation of the ethoxy moiety increases,

resulting in possible formation of S-[2-diisopropylamino)ethyl] methylphosphonothioic

acid (abbreviated to EA2192). The degradant bis(diethylaminoethyl)disulphide can also

result from the dimerization of the mercaptan (37). It is important to note that EA2192 is

reported to possess high levels of toxicity almost reaching that of its parent VX (54). Further

degradation of EA2192 has been reported, resulting in the mercaptan (55), however other

literature shows that this toxic compound has a long half-life, exceeding 1000 hours in

21

aqueous solution (56). The possible pathways for hydrolysis of VX is are shown below in

Figure 5 and Figure 6 (37)

Figure 5 Hydrolysis of VX resulting in cleavage of the P-S bond. This pathway is possible at any pH but is

predominant at pH<6 and pH>10 (37).

Figure 6 Hydrolysis of VX resulting in cleavage of the C-O bond, predominant when the pH is between 6 and 10 (37)

22

The hydrolysis of VX in aqueous solutions has been shown to occur at a far slower rate than

other nerve agents, however it was shown to be more affected by temperature. At low

temperatures, the degradation rate has been shown to have an almost exponential

decrease, with degradation occurring at a tenfold decrease for a decrease of 10˚C (33). High

temperatures also have been shown to affect the favoured pathways of hydrolysis in basic

conditions, with the EA2192:EMPA ratio at approximately 2:1 at 25˚C, lowering to 1:1 at

55.6˚C (38). Additional research into VX hydrolysis occurring at high pH has shown that

other hydrolysis products are possible, with formation of O-ethyl methylphosphonothioc

acid, diisopropylaminoethanol and bis[diisopropylethyleneimmonium] formed from the

cleavage of the S-C bond (55).

4. Extraction Techniques of CWA chemical markers Environmental and biological samples collected in their raw form cannot be run through

analysis procedures. Therefore, techniques are needed to extract analytes from their

matrices so that they can undergo subsequent derivatisation and analysis. Many

techniques are available for the extraction of CWAs and their degradants from various

matrices to achieve this.

4.1 Soil Current recommended extraction techniques of analytes from soil samples often involve

the addition of deionised water or organic solvents, followed by centrifugation, decanting

of the supernatant and filtering through filter paper or syringe filters (14). These steps are

repeated to ensure maximum recovery and purity of the analyte, resulting in the analyte

23

extract within the chosen solvent. Organic solvents used are often ethyl acetate,

dichloromethane or chloroform, with ethyl acetate favoured due to its more inert nature

in soil matrices, particularly in nitrogenous soils. However, ethyl acetate cannot be used in

nuclear magnetic resonance (NMR) analysis, so other solvents are favoured in such a case

(14). In the case of aqueous extraction, water needs to be completely evaporated before

silyl derivatisation and gas chromatography-mass spectrometry (GC-MS) can commence

(14).

Dichloromethane has been shown however, to not be an optimal solvent for organic

extractions. Amines present in the soil or from other sources have been shown to react

with dichloromethane. The background chemical N,N,N',N'-tetramethyl-1,2-

ethanediamine has shown to result in artefacts in analysis due to its reaction with

dichloromethane (57). Its reaction with amines, 3-quinuclidinyl benzilate (BZ) and 3-

quinuclidinol has also been shown to occur (58). Subsequent analysis of the same samples

using ethyl acetate as a solvent demonstrated its favoured inert nature over

dichloromethane in these circumstances, with no reactions or artefacts observed with this

solvent.

As shall be reported later, metal cations in the analyte solution can have adverse effects on

subsequent derivatisation efficiency, as well as chromatographic techniques. A strong

cation exchange resin can be employed before subsequent derivatisation to remove these

ions from the extract.

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While there is little literature available relating to the extraction of analytes from soil in real

cases of illegal deployment of CWAs, it has been reported that extraction techniques have

been successful in recovery of analytes from soil samples several years old. This is shown

in the case of organic extraction of soil samples in Iraq using chloroform as a solvent (59).

Sulfur mustard and the degradant thiodiglycol, as well as IMPA and MPA were identified

from soil samples collected from a Kurdish village using deionised water extraction with

subsequent silylation and GC-MS analysis (60). Several years later, analysis by liquid

chromatography atmospheric pressure chemical ionisation mass spectrometry (LC-APCI-

MS) verified identification of thiodiglycol and MPA (61).

Efficiency of extraction from soil has a heavy dependency on the soil type and chemical

composition, with poor results exhibit with increased polarity of analytes. After polar nerve

agent degradants were extracted from various soil samples, poor recoveries were shown

after GC-MS analysis of their silyl derivatives (62). The carbon alkali earth metal content of

the soil was shown to have a great impact on the efficiency of the techniques used.

4.2 Liquids To extract analytes from liquid environmental samples, an extraction is performed by first

returning the sample to a neutral pH, then using an organic solvent such as

dichloromethane, ethyl acetate or chloroform to perform a liquid-liquid extraction (14),

which separates any non- or semi-polar analytes to be collected in the organic extract, with

polar analytes such as alkylphosphonic acids, in the aqueous extract. The use of a strong

cation exchange is favoured for the removal of interferent metal cations from this extract

(14).

25

In the same sense as presented in the literature regarding soil analysis, there is little

reporting extraction methods of CWA analytes from liquids. However, what is reported has

shown success, with applications shown to munition deployment (63), as well as

production sites (64) in which case liquid-liquid extraction was applied to soil samples

which were shown to have degradants of GB present. While the techniques used in

extraction from aqueous matrices has been shown successful, the majority of errors has

shown to arise from the need for complete evaporation of water from aqueous extracts

(65). Derivatisation methods described later herein may minimise or circumvent some of

these problems.

Where LC-MS analysis is used for alkyl phosphonic acid degradants, hydrogen fluoride can

be used as an extraction solvent for the polar analytes, with an additional 10% NaCl shown

to be a sensitive technique for liquid-liquid extractions (66). This method has been shown

to have greater effectiveness where environmental liquid extracts may have a high nitrogen

content; however, this method has the disadvantage of HF being highly toxic and

hazardous.

As with soil matrix extractions aforementioned, choice of organic solvent is important, with

ethyl acetate and dichloromethane exhibiting the same disadvantages and artefacts (57,

58).

26

4.3 Solids The extraction of analytes from solid materials such as concrete, wood, metal or cloth is a

simple process in which the solid is placed in the presence of an excess (such that it is

completely immersed) of an organic or aqueous solvent, followed by agitation or sonication

in its closed container (14). Cutting, grinding or crushing materials to increase surface area

may enhance the extraction yield. Organic solvents that may be used include acetone, ethyl

acetate or dichloromethane. Deionised water, methanol or acetonitrile solvents are

suggested for aqueous extraction of the polar degradants of nerve agents, with subsequent

concentration to dryness necessary for derivatisation (14). In implementing these

extraction methods, sarin and its degradants were able to be detected on metal fragments

four years after the initial exposure to the chemical agent (60).

Organic solvents for extractions from solids exhibit the same disadvantages as described

previously with soil and liquid extraction methods (57, 58).

5. Derivatisation Techniques The volatility of the degradants of CWAs necessitates derivatisation. Many of the CWA

degradants have inadequate volatility required for GC analysis, or other such properties

such as thermal instability that inhibit the ability to detect these compounds (67).

Derivatisation refers to the conversion of an analyte into another chemical species

preceding its detection in chromatographic or other detection techniques. Derivatisation

lowers the reactivity, instability or volatility of CWAs or their degradants. Volatility of

analytes is often lowered by the replacement of a labile hydrogen attached to a heteroatom

with a less polar and non-labile group (13), which in doing so removes the compound’s

27

ability to form hydrogen bonds. The reactive electrophile properties exhibited by many

such compounds means they cannot be run through chromatography due to their

detrimental interactions with the column or other nucleophiles used in the analysis. For

example, phosgene, a toxic chemical listed on the Annex on Chemicals of the CWC (68) is a

reactive electrophile. Derivatisation of this compound reduces its reactivity and volatility

such that the derivatives formed can be run through chromatography. Upon derivatisation,

the sensitivity of some analytical methods is increased, or otherwise allows for a more

sensitive method to be used. For example, perfluorinated derivatives are used when target

compounds are at a concentration generally not above 1 part per million, often in the low

parts per billion range such as often seen in the analysis of biological samples like blood or

urine. In the case of thiodiglycol, pentafluorobenzoyl and heptafluorobutyryl derivatives

are used (69–71), or pentafluorobenzyl ester derivatives formed from alkyl alkylphosphonic

acids (72–76) and alkylphosphonic acids (14), with the former case allowing analysis by the

very sensitive technique of negative ion chemical ionisation mass spectrometry (NICI-MS).

5.1 Disadvantages of Derivatisation The derivatisation process can introduce errors in quantitative analysis by chromatography.

Issues often arise from foreign material introduced to the reaction from the extraction of

the target CWA or degradant compound from the environmental (or otherwise) matrix.

These undesired contaminants, including water, can inhibit the derivatisation reaction, or

can react with the derivatising agent itself, which may not only inhibit the process of

derivatising the target compound but also introduce background products in the analysis

(67). For this reason, many derivatisation processes require the removal of all water from

the extract to complete dryness (67). This necessity may be a major time-consuming step

28

in the analytical process, traces of water not fully removed from the analyte may react with

both the targeted degradant or the derivative formed (67). Furthermore, the volatility of

some degradants of many CWAs raises the issue of loss of analyte during the evaporation

process (77). Preparation of analytes has been examined by Kuitenen (78). It is proposed

that identification of derivatives does not pose as strong an evidence of CWA presence as

by identification of the intact agent or degradant itself. This may remain true for when

derivatisation by methylation is used, or some derivatives of phosgene, however most

cases show that when a suitable derivative of a compound is chosen it is sufficient proof of

the presence of that compound in the original extract (67).

5.2 Factors Influencing the Choice of Derivatisation Method Summaries of derivatives to be chosen for chromatographic analysis have been given by

Blau and Halket (79) and Taguchi (80). As with all reactions, derivatisation should aim to be

rapid and selective to the target compound with minimum energy required. While rapidity

is often achieved, sufficient selectivity has only been observed for a select few analytes.

Common derivatisation reactions involve a nucleophilic moiety with a reactive electrophilic

derivatising agent (67). However, selectivity for derivatisation reactions involving CWAs

and their degradants is difficult to achieve as these compounds are electrophilic

themselves, and many contaminants found in environmental and biological samples are

nucleophilic. Derivatisation reagents chosen therefore need to react with electrophiles, as

in the case of the target CWA compounds, and with fewer electrophiles found in

environmental extracts (67), greater selectivity may result.

29

Derivatives formed also need to have properties such that when processed through a

chromatographic analysis, their retention time within columns allow for easy separation

from undesired compounds such as environmental contaminants or interferents.

Derivatives also need to have a high level of uniqueness from the sample origins, in that

the derivative must be so rare in the environment from which samples are taken such that

their presence can only be evidence of a CWA. It is required that derivatives possess

features that allow for detection by the analysis used, such as chromophores for

spectrometry or heteroelements for detection by atomic emission detection (AED). It is

also important to consider the safety of the target compound derivatised, the derivative

and the derivatising agent itself, as in the case of diazomethane (14) that is unstable to

detonation. The compounds must be thermally stable and not be reactive with traces of

moisture in the laboratory. Such is not the case seen in the commonly used silylating agents

and derivatives, which are sensitive to moisture (67).

5.3 Current Developments in Derivatisation Techniques A review of new developments in derivatisation methods has been given by Wells (81),

including acknowledgement of the silylation method. It has been described that with the

increase in accessibility of GC-NICI single stage and tandem MS, fluorinated derivatives

required for these analyses have seen a more widespread implementation. This increased

use of fluorinated derivatives has also led to the development of other reagents to be used

in complement. The complementary use of derivatising agents allows for the monitoring of

more than one ion to confirm identification of the presence of a derivative. The lowest

detection limits are reached when derivatisation results in a single unique ion in its mass

30

spectra, however doing so decreases specificity due to it being difficult to analyse the

effects of possible interferents or contaminants. In cases where interferants may be

observed with the use of pentafluorobenzyl derivatives of alkyl alkylphosphonic acids, 4-

(trifluoromethyl)-2,3,5,6-tetrafluorobenzyl derivatives may be implemented by reaction of

the analyte with the corresponding bromide of this compound (TTBB). This derivatising

agent can, in its complementary or substitutionary use with the agent pentafluorobenzyl

bromide also be used as a confirmation in analyses of environmental matrices (82).

However, this research investigating TTBB, and current available literature has not tested

for optimised conditions for this TTBB derivatising agent, and so maximum yield (and

therefore the future potential use of the methods presented therein) have yet to be

understood. Derivatisation of fatty alcohols forming 4-carboethoxyhexafluorobutyryl

derivatives by microwave heating with the corresponding chloride of the derivative allow

for less volatile products instead of conversion into heptafluorobutyryl esters previously

used (83). Far greater retention times were also achieved with these derivatives over

heptafluorobutyryl derivatives, however such methods have yet to be directed towards

application to CWAs and their degradants, particularly to those relating to

organophosphorous nerve agents. Until then, 4-carboethoxyhexafluorobutyryl derivatives

remain only applicable to biological analysis and fatty alcohols. New methods now

sometimes involve reagents which show that it is possible for polar analytes to be

derivatised directly in an aqueous solution.

A widespread issue with derivatisation methods is the requirement to use the derivatising

agent in a large excess to drive the reaction to completion. However, doing so can introduce

error in the form of introduced chemical background, or in cases where a column is used,

31

reduce the product lifetime of the column (67). A review posed by Rosenfeld (84) of

analytical techniques involving new technology demonstrated the ability for derivatisation

to occur with analytes, reagents or catalysts held on a solid support. With methods

involving solid-phase extraction (SPE) or solid-phase micro extraction (SPME), the solid

support in those cases may be the solid phase itself. This allows for derivatisation to occur

in situ, therefore occurring alongside the extraction. This method allows for the removal of

excess derivatising agent by washing, which removes the possibility of the associated

problems aforementioned. Such methods are also an avenue to develop automated

detection methods. In methods where analytes are supported on the solid support,

mechanical action can remove the excess. Currently, most research investigating SPE/SPME

methods involve the derivatisation of carboxylic acids (85), phenols (86) and carbonyls (87)

often utilising the same derivatising agents applicable to CWAs and their degradants,

however future research may widen the applicability of these methods. This technology

has seen application in pentafluorobenzyl derivatives of acidic analytes deposited onto an

ion-exchange resin (88), however the high temperatures optimal for the methods

presented therein may be inappropriate to adapt to analysis of volatile compounds, yet this

can be investigated in future research regardless. Pentafluorobenzyl derivatives of

organophosphorous acids implementing a polymer supported phase transfer catalyst is

also reported possible with this method (89), as well as a polymeric

pentafluorobenzoylating derivatising agent (90). The research presented by Jedrzejczak

and Gaind (90) presented a method which was quite sensitive, with testing done on

butylamine as a model amine, and future directions of this method may investigate its

possible use on other analytes. A longer time of reaction may also further optimise this

method, which was only carried out for 10 minutes at 60˚C. The results reported by Miki

32

et. al (89) show that the methods can give a high yield of up to 96% given high enough

temperature and long enough time, however doing so may result in loss of volatile analytes.

5.3.1 Development of Derivatisation Techniques not Requiring Removal of Water

With the polarity of many analytes used in CWA analysis, an issue arises in the difficulty

with which it must be separated from an aqueous matrix before derivatisation (67). Liquid-

liquid extraction and SPE may be ineffective or inappropriate for the conditions present,

and the complete removal of water from aqueous extracts may be time consuming and

introduce error (67). Because of this, many efforts have been directed to developing

methods that allow derivatisation to occur directly in the aqueous matrix, with derivatives

formed to be subsequently extracted. One such possibility for this process is the use of

chloroformate derivatising agents, examined by Hǔsek (91), with which a method was

developed that does not require the complete expulsion of water from analytical samples.

Hexyl chloroformate has been shown to be very effective in this method when applied to

aqueous polyhydroxy and polycarboxy analytes (92) with such methodology only requiring

2 to 3 minutes, and has shown use with ethylene glycol (93); however, this technique is

limited in its use in that oxalic and formic acids cannot be analysed as their derivatives are

the same as the by-products resulting from the hydrolysis of the derivatising agent. Amino,

hydroxyl and carboxyl moieties have been able to be derivatised in aqueous solutions using

pentafluorobenzyl chloroformate (94) and 2,2,3,3,4,4,5,5-octafluoropentyl chloroformate

(95), and so show possibility of developing further in relation to CWA analysis. It is

suggested that other halogenised derivatising reagents derived from perfluorinated

alcohols may pose as possible research subjects in their synthetic chemistry.

33

A rapid screening technique for alkyl phosphonic acids is proposed by Subramaniam et al.

(96) utilising similar techniques proposed by this research. Direct derivatisation was

achieved using a highly fluorinated phenyldiazomethane reagent, 1-(diazomethyl)-3,5-

bis(trifluoromethyl)benzene, reacting with aqueous samples of various alkyl phosphonic

acids with acetonitrile, assisted with ultrasonication at room temperature. The resulting

fluorinated derivatives could then be screened rapidly using NICI-MS, taking little more

than 5 minutes of sample preparation, achieving ppb sensitivity from an original 25μL

aqueous sample without the removal of the water. Data analysis of this research for one

analyte tested, EMPA, suggested a strong interaction between the derivatising agent and

the amount of water introduced into the reaction from the original aqueous sample (97).

It was therefore indicated that the reaction yield could be increased by including a

particular amount of water in the original sample, which would reduce the strength of the

interactions between polar chemicals and the vial surface. The methods of Subramaniam

et al.’s research did not test for various concentrations of the original analyte, suggesting

that the 25μL was sufficient for the sensitivity achieved. While different amounts of water

affecting the reaction yield is only posed on the basis of data analysis, it could pose for

future research into the optimisation of Subramaniam et al.’s methods. Future research

may also indicate whether the same may hold true when using non-silylated vials, as such

were used in Subramaniam et al.’s methods.

5.4 Derivatisation of Organophosphorous Nerve Agents and their Degradants Nerve agents generally have adequate volatility and stability to be used in GC analysis

without prior conversion into a derivative (67). An exception to this however, is the agent

34

VX, which has shown that where it is run through GC analysis at low concentrations, its

interaction with adsorptive sites within the column can cause poor peak shapes and a large

amount of background noise (67). This is often seen when analyses are performed on air

samples, where its low concentration may prove problematic when testing for recent use

of this agent. This issue may be overcome by converting the phosphonothilate of VX to a

phosphonofluoridate. Such a reaction saw its use as the basis for detectors of nerve agents

(67). Another method presented by Fowler and Smith (98) overcomes this issue by allowing

air samples believed to contain VX to pass through a filter imbued with silver fluoride, which

forms ethyl methylphosphonofluoridate upon reaction with VX present in the air. The ethyl

methylphosphonofluoridate is then adsorbed onto Chromosorb 106 to then undergo

analysis via GC flame photometric detection (GC-FPD). This method was found to have

moderate sensitivity, however its effectiveness and ease of interpretation of results had a

high dependence on any background compounds in the air samples, which could be

expected to be high in some real-life applications. The recovery of VX using this method

was also dependant on the concentration of the agent in the air samples. Because of this

the methods presented may likely find their best use in areas with recent exposure to VX.

A derivatisation method analogous to that presented by Fowler and Smith utilised silver

fluoride in a column, with low concentrations of VX in benzene solution (99). This method

could also be applied to the compound O,O-diisopropyl S-benzyl phosphorothiolate.

The high polarity and low volatility of phosphonic acids, major degradants of nerve agents,

means that they are not ideal for GC analysis (67). Because of this, they are converted into

methyl, pentafluorobenzyl, trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS)

derivatives, which then allows them to be analysed by GC (14).

35

5.4.1 Methyl Ester Derivatives Upon reaction with methylating derivatising agents, acidic degradants of nerve agents are

readily converted into their methyl or dimethyl ester derivatives, as shown in Figure 7(100).

A commonly used methylating derivatising agent is diazomethane. The selective reactivity

of diazomethane towards acidic analyte give this derivatising agent an advantage over

other reagents available (67). When performed along other derivatisation methods, doing

so can provide complementary evidence for the identification of CWAs, which can be useful

for laboratories unable to perform LC-MS as a complementary analysis. It has been shown

that in an organic solvent, alkyl methylphosphonic acids can be converted into their

conjugate methyl ester derivatives rapidly with diazomethane. This has been achieved

within 15 minutes where the derivatisation agent was used in excess to the analyte,

yielding a derivative at greater than 99% (101). As mentioned previously, the excess of the

derivatising agent may cause issues, and so this may be solved in this case by separation of

the alkyl methylphosphonic acid reagents in a dry organic solution prior to derivatisation.

The high volatility of diazomethane also allows for the easy removal of excess reagent.

Figure 7 Methylation of pinacolyl methylphosphonic acid (14) by the derivatising agent trimethyloxonium tetrafluoroborate from (100)

Derivatisation by diazomethane results in products that are more stable in aqueous

conditions than silyl derivatives, and their mass spectra are easier to interpret (14). While

36

diazomethane can be used with acidic analytes such as is often the case with nerve agent

degradants, it cannot be used on non-acidic analytes such as thiodiglycol, a precursor and

hydrolysis product of mustard gas (14). Other disadvantages posed by the use of

diazomethane are its toxicity and potential to detonate unexpectedly, which is especially

susceptible to doing so when on rough surfaces, in contact with some metal ions or during

crystallisation (14). Because of this, when using this reagent all glassware must be clean

and free of any scratches, and the agent must not come into contact with metals. It has

been shown that with derivatisation of MPA extracted from polluted water, reaction with

diazomethane formed the derivative dimethyl methylphosphonate, resulting in poor peak

shapes. Further, with the short retention of this derivative within the column, and

interference with background compounds, interpretation was even more difficult (101). In

addition to these properties, chromatographic analysis results may not provide solid

identification as the methyl ester cannot be differentiated in the results as either its ester

form or acid form (67).

Despite the advantages of methyl ester derivatives, many laboratories opt for the use of

the more versatile silyl derivatives for acid degradants. However, due to some of the

aforementioned disadvantages of methyl ester derivatives and the diazomethane agent,

research developments have been targeting the possibility of methylating derivatising

agents that are easier to store and handle over diazomethane. Trimethylsilyldiazomethane

has been shown applicable to derivatise the CWA degradants EA2192 and phosphonic acids

(102), with this agent more stable compared to diazomethane. Degradants IMPA and TMPA

have also been identified at concentrations of 100ng in 10mL aqueous solutions with their

methyl ester derivatives formed upon reaction with trimethylphenylammonium hydroxide

37

(TMPAH) (103). While this method was not as sensitive as others available, it avoided the

need for complex sample preparation. GB and GD degradants could be detected in this

method in up to 2 weeks exposure in water, soil, sand and grass environments. The results

undertaken in the paper by Tørnes and Johnsen however show an interesting increase in

detection from the first week passing to the second week, exhibited in the sand, soil and

grass environments with no correlation between the analytes. This may give cause to

repeat the experiment under different conditions or with testing occurring at times closer

together. Investigating this method further, Sega et al. (104) utilized similar techniques on

MPA, EMPA and IMPA degradants recovered from groundwater, successfully identifying

the analytes at concentrations 2–9ng/mL. A review of alternative agents to TMPAH with

similar methods has been given by Amijee et al. (105). While TMPAH readily derivatises the

analytes, its high pH (~13) can cause deterioration of the column and has low selectivity.

Phenyltrimethylammonium fluoride is proposed by Amijee et al. for better life of the

column, or phenyltrimethylammonium acetate which has been shown to have better

selectivity (105).

5.4.2 Silyl Derivatives Methods involving the derivatisation of polar CWA degradants or precursors into their

trimethylsilyl (TMS) or tert-butyldimethylsilyl (TBDMS) derivatives are some of the most

broadly applicable. OPCW laboratories utilize TMS derivatives for onsite analysis, and

TBDMS derivatives for offsite analysis due to their greater resilience to reaction with

moisture (14). TMS or TBDMS derivatives, sometimes abbreviated to just silyl derivatives,

can be analysed by a variety of methods including flame photometric detection (FPD),

nitrogen phosphorus detection (NPD), atomic emission detector (AED) and MS (67). The

38

formation of a silyl derivative is shown in Figure 8 (24). As with methyl ester derivatisation,

formation of silyl derivatives may suffer from excess use of the derivatising agent, albeit

only exhibiting negative effects when using FPD or NPD, which may be coated in silica

deposits from the excess reagent (67).

Derivatisation of polar compounds into their silyl esters by reaction with the derivatising

agent, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), or with BSTFA with an additional 1%

catalyst of trimethylsilyl chloride (TMSCl) occurs efficiently. Recommended operating

procedures state that optimal conditions for the reaction to occur are at 60°C for 30

minutes in an organic solvent (14). Using the 1% TMSCl catalyst with BSTFA, it has been

reported that the reaction can occur at 80-100% efficiency when only running at 60°C for

15 minutes in hexane (106). It is also important to note that this research conducted by

Creasy et. al (106) showed that there was no difference found in derivatisation efficiencies

between hexane and acetonitrile solvents, especially when current recommended

operating procedures suggest that acetonitrile be used for silyl derivatisation reactions

(14).

It could be suggested that current recommended operating procedures consider hexane as

an alternative, as it has been reported that acetonitrile as a solvent for the reaction has

resulted in broad and poorly defined peaks in GC analysis (107). Silyl derivative formation

from phosphonic acids has been investigated using BSTFA in acetonitrile and toluene

solvents (107). The effect of including a 1% catalyst of TBDMSCl, as well as an alternative

derivatising agent of imidazole with TBDMSCl were tested. The derivatisation occurred

efficiently regardless of the inclusion of a catalyst with N-tert-Butyldimethylsilyl-N-

39

methyltrifluoroacetamide (MTBSTFA), which was chosen as an ideal agent over the

imidazole/TBDMSCl, which gave a lower yield with numerous by-products of the reaction.

It was suggested that attempts to remove these by-products may overcomplicate the

process and may reduce the recovery yield. Although efforts to do so were untested, with

the more efficient methods proposed in the same paper, future research into such may not

be worthwhile. Derivatisation by MTBSTFA was successful in ambient temperature,

reaching a completion after 30 minutes, however was found to be optimised at 60°C for 1

hour. An alternative to MTBSTFA is be tert-butyldimethylsilyl cyanide, which reportedly

derivatises acid degradants efficiently at ambient temperature (67), however the research

investigating this has yet to be published.

Figure 8 The TBDMS derivative of MPA formed by reaction MTBSTFA (24)

While no research has yet undertaken comparison of properties between TMS and TBDMS

derivatives, it is accepted that TBDMS derivatives are often favoured for their resistance to

water (79), and so exhibit greater stability. The greater resilience to water exhibited by

TBDMS derivatives are reported (67), however it is only stated that such characteristics are

assumed in cases where they have chosen as the favoured derivatisation pathway. No such

research has been published testing the truth of this assumption. It has been shown that

derivatives of IPMPA and MPA stored in isolation at ambient temperature did not undergo

any significant chemical changes over 6 days (107) and are suggested to be able to be

40

stored frozen for a month further, if not longer. It has been suggested that TMS derivatives

of phosphonic acids have greater long-term stability than is believed, with detectable

presence of the derivatives after 7 months of isolated storage (106), however this lifetime

was achieved when the derivatives were stored with the derivatising agent BSTFA, so it

could be possible that an incomplete derivatisation of the analyte could have occurred,

with reactions ongoing for the lifetime of the storage. In order to determine the true

lifetime of TMS derivatives it may be more worthwhile for them to be stored in an organic

solution (such as acetonitrile) without any derivatisation reagent.

The negative effects calcium and magnesium ions have upon the derivatisation reactions

of phosphonic acids poses an issue for analysis particularly to those involving extracts from

soil samples, and has affected analyses so far as to give a false negative result for the

presence of MPA (67). These effects have been quantitated for the degradants MPA, EMPA,

IPMPA and PMPA, with derivatisation of MPA exhibiting the greatest adverse effects from

the presence of metals (108). Current recommended operating procedures in OPCW

laboratories utilise a cation exchange resin which removes metal ions from the aqueous

solution (109), which was utilised by Kataoka et al. (108), confirming that the cation

exchange resin solves this issue. The yield of MPA derivatives was low however, which was

attributed to the likelihood of poor recovery of MPA from the soil matrices used in that

research. This research by Kataoka et al. also spiked derivative solutions with metal ions,

with interference with the GC-MS occurring after this step. With moderate yields obtained

of phosphonic acids from soil matrices without metal ion spiking, it may be concluded that

extraction processes may have removed metal ions that may have been present in the soils

which could have otherwise been introduced to the derivatisation reaction. However, with

41

the soil matrices used lacking a stated content of metal ions, it may be possible that there

were simply no metals that could have been introduced in the first place. In any case, the

increased yields of derivatives shown in this paper after use of cation exchange resin give

good reason for its implementation in cases where it is likely to encounter contamination

by metal ions. Even with its implementation, the strong cation exchange allows for neutral

and anionic compounds to pass through, which may interfere with the conversion of the

phosphonic acids into their derivatives. Another solution has been proposed by Kataoka

with other authors who separated anionic phosphonic acids on an anion exchange resin,

which could then be eluted with hydrochloric acid (110). This research encountered

different measures of success based on the soil matrices from which the phosphonic acids

were extracted. Highly saline soil showed low success likely due to the chloride ions coating

the exchange resin and hence inhibit binding of the targeted derivative analytes. While this

research showed its effectiveness on phosphonic acids and removal of interfering metal

ions, future research repeating the methods used therein upon different types of soils may

allow for understanding of its limitations to real life situations.

Methods which can combine extraction with derivatisation pose an avenue for valuable

future research, as simple methods may be particularly useful for in-field testing of samples

where a quick result may be desired. A possible method for use on soil samples involves

the soil to reside with BSTFA and pyridine with dichloromethane (111), though such

methods were conducted on mustard class CWAs (HT and HQ agents) and their degradants.

Though success was achieved with the silyl derivatives of these compounds, amendments

to the method presented therein may be required for the same success to be achieved with

organophosphorous nerve agents. A similar method has also been successful with wet soil,

42

though it has been reported that different soil types have a great effect upon the success

of the reaction (112). This method also had success with the CWA degradants thiodiglycol,

ethyldiethanolamine and benzilic acid.

The degradation of CWAs to phosphonic acids is such that it allows for the implementation

of silyl derivatisation for a long time after the initial suspected use of nerve agents. The

degradant IMPA has been successfully identified after its conversion into its silyl derivative

4 years after the use of a chemical weapon (60), with detection possible in soil and painted

metals. The detection of sarin in the metal samples was, however, attributed to the

likelihood that sarin’s absorbability into paints protected it from hydrolysis in the

environment (60). Thus, such a long lifetime of CWAs may be less for unpainted metals.

The complementary use of selected ion monitoring (SIM) or multiple reaction monitoring

(MRM) allows for the detection of fragmentary ions formed by TBDMS derivatives, which

gives a high level of confidence in the final result of testing (67).

AED detection has been used to give quantitative analysis for phosphonic acid degradants.

Testing for MPA, alkyl MPAs, EMPA and IMPA, an anion exchange cartridge was used for

solid phase extraction, followed by silyl derivatisation and AED detection, though only

moderate sensitivity was achieved to a low ppm range (106). An alternative method used

GC-MS-MS analysis of TMS derivatives of phosphonic degradants, which achieved

quantitation to 200–500pg (113). The sensitivity of this method was greater than those

expected for TBDMS derivatives (67). A method investigated by Rohrbaugh involved silyl

derivatisation of various VX degradants, with undegraded VX in the samples also analysed

(28). Many degradants were able to be detected by this method of analysis by electron

43

impact (EI) and chemical ionisation (CI) MS by methane, however the degradant EA 2192

was unable to undergo derivatisation due to its zwitterionic property. Therefore, this

method is presented ideally as a rapid and selective screening test for alkyl

methylphosphonic acids.

After the use of GB by terrorists in Tokyo, biological analysis used derivatisation by BSTFA

with a 10% TMSCl catalyst, which reportedly increased the reaction efficiency (114). It may

be possible for future research to investigate an optimisation for a reagent:catalyst ratio.

5.4.3 Pentafluorobenzyl Derivatives Pentafluorobenzyl ester derivatives most commonly see their use with analysis of trace

amounts of phosphonic acid degradants, which is often the case in biological samples (67).

As mentioned previously, analysis of silyl derivatives was used in the Tokyo case (114),

however the sampling that was used occurred only shortly after the deployment of the GB,

meaning that the chemical weapon had not been degraded much in the biological matrices.

Pentafluorobenzyl esterification of degradants allows for the analysis to be undertaken

weeks after the exposure of a CWA often with limits of detection below 1ng/mL (67).

While conversion of analytes into their silyl or methyl derivatives is often a fast and efficient

reaction, pentafluorobenzyl ester derivatives, formed by reaction of phosphonic acids with

pentafluorobenzyl bromide (PFBBr), are formed slowly and require more complex

conditions. The initial success of pentafluorobenzyl ester derivatives required reaction of

various phosphonic acids in acetonitrile with the agent PFBBr for 200 to 400 minutes (101);

far longer than the half an hour to an hour required for silyl derivatisation. With issues of

44

decomposition of the analytes in this study, a subsequent study showed that alkylation of

the analyte could be achieved more efficiently with the addition of sodium or potassium

salts in tetrahydrofuran with sodium hydride (115). These conditions raised the pH which

would prevent the hydrolysis of the alkoxy moiety.

Numerous solvents have been investigated for their efficiency for pentafluorobenzyl ester

derivatisation for biological matrix analysis, with dichloromethane, ethyl acetate and

acetonitrile all giving similar yields for the reaction, however dichloromethane was found

to perform easier concentration of the analyte (72). The direct comparison of the solvents

showed that derivatisation could be optimised at 50°C for 1 hour.

An advantage of pentafluorobenzyl ester derivatives is their sharp and well-shaped peaks

when analysed by GC, with longer retention in comparison to methyl esters (67). With

pentafluorobenzyl derivatives often directed to use in sensitive NICI-MS, the loss of the

C6F5CH2 functional group forms a base peak at [M–181]− relating to the anion (67). Selected

ion monitoring (SIM) can then be utilised to give high sensitivity due to the large proportion

of ion current in this ion (67).

6. Conclusion and Rationalisation for Proposed Research Methods

The method to be investigated proposes that an extract of aqueous MPA undergoes

derivatisation by MTBSTFA with an added organic layer of hexane. The derivatisation of

MPA at the phase interface between the aqueous and organic layers allows for the

derivatives formed to migrate to the organic layer. Subsequent analysis of this organic layer

45

by GC-MS therefore eliminates the need for a complete evaporation of the water from the

aqueous sample. This process, if successful, will allow for a fast, qualitative answer on the

presence of MPA in the initial aqueous solution.

In order for the proposed method be applicable to the largest amount possible of

circumstances involving CWA use and manufacture, the target analyte should have

parentage through degradation pathways of as many CWAs as possible. As has been shown

by previous research, MPA is the result of many organophosphorous nerve agent

degradation (with the exception of tabun). Should the method proposed be successful and

demonstrate application in real life contexts, it could therefore prove that, by identification

of MPA in the sample, that the parent organophosphorous was likely present previously.

Regardless of the success of the proposed method for analysis of MPA, future research

could target other first order degradants, such as IMPA or EMPA, which could thus provide

a selective answer on which nerve agent may have been present (should the method prove

successful). It may be necessary to expand the method thusly to application to other

degradants in future research, as the persistence of nerve agents before their degradation

into first order degradants occurs in the order of days, further degradation takes places in

the order of years to form MPA (depending on conditions). Expansion of the research would

therefore also allow its possible application to suspected sites of CWA use or production

where recent exposure is concerned where very little degradation may have taken place.

An issue in choosing MPA, or other degradants for that matter, is the uniqueness of the

chemical species to that environment. It has been reported that MPA is a degradation

product of some fire-retardant chemicals (67), however the only found source reporting

such did not have the statement of such proved by the research conducted therein, nor

46

provide a source for the statement. Other research however, has shown that melamine

salts of methylphosphonic acid can have possible application as a flame retardant in

polymers (116). However, the uptake in use of such compounds in commercial polymer

products subsequent to those research findings could not be found, and the fate of those

compounds over time has not been reported. It remains thus, that should it be possible

that the identification of compounds in CWA analysis could be indicative of another parent

compound or origin other than that of CWA use or production, it is imperative that it be

reported in findings.

The methods presented use an aqueous solution of MPA as representative of an

environmental extract. As shown, this could be from soil, liquid or solid samples collected.

An exception where the method may not show application towards is air samples, for which

current methods use collection by Tenax® tubes, followed by thermal desorption of the

captured sample (14). Organic solvents such as dichloromethane have shown success in

extraction of the sample, however to do so is not recommended due to the degradation

caused on the Tenax® tube (14). Regardless, Tenax® tubes are not applicable to the polar

degradants of nerve agents due to their low volatility.

Derivatisation by MTBSTFA has been chosen to form TBDMS derivatives of MPA, due to

their greater stability in moisture over TMS derivatives as previously discussed. The

derivatising agent is also safe to use. While the use of a catalyst has shown increased

efficiency, as none were available to use, MTBSTFA without the catalyst was found

satisfactory for budget saving measures. Future research aiming to improve the proposed

method could investigate optimisation by catalyst use.

47

Hexane has been chosen as an organic solvent as it has been shown to have greater shaped

peaks in GC-MS analysis of silyl derivatives as aforementioned. The derivatisation shall run

with sonication and heating at 60˚C for 15 minutes, which has shown high efficiency in

derivatisation in previous research.

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Part Two

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THE DEVELOPMENT OF AN IN-FIELD RAPID DERIVATISATION TECHNIQUE FOR THE ANALYSIS OF CHEMICAL WARFARE AGENT DEGRADANTS

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Abstract The recent uses of banned chemical warfare agents (1) indicate the need for the

development of methods that can verify the use of chemical warfare agents. Current

techniques use the presence of the agents’ degradation products within the environment

as an indication of their use (2); however, these are often time consuming in their method

of derivatisation of the degradants for further analysis. A faster and simpler process in

pursuit of a qualitative answer on the presence of these degradants will be valuable to

monitor adherence to the Chemical Weapons Convention in field and military operations.

Methylphosphonic acid (MPA) is considered to be a common degradation product

through hydrolysis of various V and G nerve agents in the environment (2). In this study

MPA was derivatised using N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide

(MTBSTFA) with an added organic layer of hexane. The addition of the aqueous MPA,

derivatising agent and the organic layer, with stirring and heating, resulted in migration of

the GC-amenable derivatives into the organic layer, with subsequent removal of the

organic layer, drying and analysis of the organic fraction by GC-MS. This process

eliminated the need for a complete removal of water from the original aqueous MPA

sample in a process that could take little more than 30 minutes. A series of aliquots of

MPA with decreasing concentrations were tested using this method of derivatisation.

Subsequent GC-MS analysis showed the presence of the derivatives, with sensitivity to

1000 ppm of MPA in the original aqueous sample.

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This study demonstrated a possible method for the in-field rapid derivatisation technique,

which, coupled with field-deployable GC-MS equipment, would allow for rapid verifiable

analysis for degradants of chemical warfare agents in the environment.

Keywords: Forensic science, chemical warfare agents, methylphosphonic acid, MTBSTFA,

derivatisation, gas chromatography mass spectrometry, silylation

Introduction Since the implementation of the Chemical Weapons Convention (CWC) in 1997, increased

efforts have been directed to the development of analytical techniques pertaining to the

detection of chemical warfare agents (CWAs), their precursors and degradants in various

environmental and biological matrices (3,4). Of the stockpiled CWAs throughout the

world, the largest quantities stored are of nerve agents (5). Because of this and the

devastation they cause, great focus is directed to the detection of the compounds of this

CWA class.

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The reactive electrophilic behaviour of nerve agents is responsible for the similar

degradation mechanisms among several agents, with many resulting in a final hydrolysis

product of methylphosphonic acid (MPA) (6), as shown below in Figure 1 (7). The

presence of MPA is used as a chemical marker as an indication of likely nerve agent

production, use, or improper storage at a previous point in time (8). Analytical techniques

resulting in the rapid detection of MPA in environmental extract samples would allow for

a quick answer to the direction of further forensic efforts, as well as any threat to life.

Utilisation of liquid chromatography-mass spectrometry (LC-MS) has been used to screen

for other nerve agent degradants (9,10,11) with minimal sample preparation and time

consumption. However, with gas chromatography-mass spectrometry (GC-MS)

equipment more common in laboratories, particularly mobile or in-field laboratories (12),

rapid analytical techniques screening for MPA utilising GC-MS will prove valuable.

Unfortunately, many nerve agent degradants (including MPA) have inadequate volatility

required for GC analysis, or other properties such as thermal instability that inhibit the

ability to detect these compounds by GC-MS (13). To overcome this, the chemical

markers are derivatised, in most cases by either methylation (14), silylation (15), or

Figure 2 Degradation pathways of four G-type and two V-type nerve agents, resulting in MPA (7).

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pentafluorobenzylation (13,16). The derivatives formed by these processes lowers the

reactivity, instability or volatility of CWA degradants, thus allowing the compounds to be

analysed by through gas chromatography.

Silylation of polar CWA degradants or precursors into their trimethylsilyl (TMS) or tert-

butyldimethylsilyl (TBDMS) derivatives shows widespread applicability. Laboratories

performing analysis for the Organisation for the Prevention of Chemical Weapons (OPCW)

utilize TMS derivatives for onsite analysis, and TBDMS derivatives for offsite analysis due

to their greater resilience to reaction with moisture (8). In order for efficient

derivatisation and GC-MS analysis to occur, many analytes must be concentrated to

dryness prior to the derivatisation (13). Given many extraction methods of CWA

degradants from the environment result in an aqueous sample of the target analyte (8),

the complete removal of water from the extract can prove to be time consuming, and

even introduce error (13). Therefore, methods which will allow for derivatisation to occur

without the complete removal of water from environmental extracts could show

applicability to a more rapid and less laborious screening technique for GC-MS analysis of

CWA degradants.

At present, there are no literature reports of successful silyl derivatisation of CWA related

analytes without prior removal of the water component from aqueous samples. The

method presented here was proposed to allow for derivatisation of an aqueous MPA

sample without the need for complete evaporation of the sample to dryness. By addition

of a silylating derivatising agent and an organic layer, and continued agitation, it was

proposed that derivatisation may occur between aqueous and organic components.

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Subsequent GC-MS analysis by selected ion monitoring (SIM) and total ion chromatogram

(TIC) was utilised to identify the presence of MPA derivatives in the organic layer.

Aqueous solutions with concentrations of 1000 to 0.01 ppm were used in the two phase

derivatisation process.

Experimental Reagents

Solid methylphosphonic acid (98%, 5g) was obtained from Sigma-Aldrich (St. Louis, MO,

USA) as a target chemical marker for nerve agent degradation (14 from lit). N-(tert-

butyldimethylsilyl)-N-methyltrifluroacetamide (TBDMSTFA) was utilised as the

derivatising agent (>97%) and was also obtained from Sigma-Aldrich, stored at 2-8˚C as

recommended by the supplier. Analytical grade reagents (AR) of acetonitrile and hexane

were supplied from Mallinckrodt (St. Louis, MO, USA) and Sigma-Aldrich respectively, and

were previously stored at Murdoch University (Perth, Western Australia).

Helium gas was used as the carrier gas for GC-MS analysis (BOC, ultra-high purity).

MPA Samples

Solid MPA (0.2550g (±0.1mg)) was dissolved in deionised water made to a volume of

250mL (±0.23ml). 1mL of this solution was diluted to a volume of 10mL (±0.040mL). Serial

dilutions were made in this way such that 5 solutions resulted, approximating 1000mg/L,

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100mg/L, 10mg/L, 1mg/L and 0.1mg/L. A final blank solution constituted of only

deionised water.

Derivatisation

TBDMS derivatives of MPA were formed by addition of 10µL MTBSTFA along with an

added organic component of AR hexane (1mL) to 1mL of each of the aqueous MPA

samples, as well as the blank. With optimal conditions for MPA derivatisation by

MTBSTFA reported to be 60˚C for 30 minutes (8), the reaction vials were placed under

such conditions with vigorous stirring using an oil bath on Yellow Line MST Digital

magnetic stirrer and heater. The reaction vials were then left for 15 minutes to allow for

cooling and to allow for the separation of the organic and aqueous components into

layers. The superior organic layer was then pipetted off and dried by passing through a

magnesium sulfate packed drying pipette, to then undergo analysis by GC-MS. This

process was repeated for 4 successive batches, with adjustment(s) as follows:

- In the third and fourth batches a greater volume of 2mL of hexane and aqueous

MPA were added to the reaction vials, along with a greater excess of 200µL of

MTBSTFA. Due to poor recoveries after passing through a drying pipette, organic

layers were transferred directly to GC vials for analysis.

A second derivatisation of MPA was done to confirm GC-MS parameters of the derivatives

formed. This was done according to recommended protocol (8), with 0.0267g (±0.1mg) of

solid MPA dissolved in AR acetonitrile to a volume of 25mL. 2mL of this solution was

derivatised with 200µL of MTBSTFA for 30 minutes at 60˚C with vigorous magnetic

stirring, then pipetted off for GC analysis.

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Instrumentation

A Shimadzu GCMS-QP2010S (Shimadzu Australasia, Rydalmere, NSW, Australia) with

ultra-high purity helium carrier gas (BOC, Sydney, NSW, Australia). A BPX-5 (5% phenyl

polysilphenylene-siloxane) capillary column (30m, 0.25mm i.d., 0.25µm film thickness)

was used for all samples. A summary of all the GC-MS parameters can be found in Table

1.

Table 2 GC-MS parameters used

GC parameters

Instrument Shimadzu GCMS-QP2010S Carrier gas Ultra-high purity helium (99.999%,

constant pressure 16.2 psi) Injection mode Split Column oven temperature 200˚C Injection temperature 250˚C Total flow 14.0mL/min Column flow 1.00mL/min Linear velocity 38.7cm/sec Purge flow 3.0mL/min Split ratio 10.0 Oven program 80˚C (1min) 20˚C/min → 280˚C Hold

(6min) Column BPX-5 (5% phenyl polysilphenylene-

siloxane)

MS parameters

Ion source temperature 200˚C Interface temperature 200˚C Solvent cut time 3min Micro scan width 0 Detector voltage 0kV (relative to tuning result) Threshold 1000 Start time 3.25min End time 17.00min Scan speed 1250 Start m/z 45.00 End m/z 600.00 GC program time 17.00min

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For Batch 3 and 4 SIM was performed, with SIM occurring alongside TIC for Batch 3, which

was done by making the GC-MS program to collect the TIC and SIM data from two

different runs associate them with the single sample from which that data was collected.

SIM and TIC analysis was done separately for Batch 4. The selected ion had a m/z ratio of

267, the base peak belonging to the MPA bis[(dimethyl)(tert-butyl)silyl] ester (17).

Results and Discussion Initial samples run through GC-MS analysis in Batches 1 and 2 exhibited complex

chromatograms initially attributed to be a result of a large amount of column bleed.

Chromatogram peaks that formed resulted in a similarity search of associated NIST mass

spectra database relating to organic compounds, commonly with siloxane moeities. None

of the peaks had any relation to the target MPA derivative, nor the alternative mono-

ester (singular TBDMS chain) derivative that may form.

It is reported that such chromatogram characteristics may not be a result of column

bleed, as such usually manifests as an increase in the baseline of the chromatogram at

the more elevated oven temperatures (18). The chromatograms of the samples in

Batches 1 and 2 however exhibited repeated peaks, commonly with mass spectra

displaying signals for ions of m/z 73, 147, 221 and 281; 73 consistently being the most

abundant with slight changes of the abundancy of the other ion signals. No such increase

of the baseline was observed as described in the chromatography troubleshooting (18).

According to the troubleshooting guide, column bleed chromatogram peaks usually show

associated signals for ions at m/z 73, 207 and 281 with 207 being the most abundant in

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relation to the other peaks for 5% phenyl-95% methyl substituted silicone (18). This is

contradictory to the spectra exhibited for samples of batches 1 and 2, where mass spectra

peaks at 73 were most abundant. As such the repeated peaks may not be a result of

column bleed. The mass spectra also share the same values, their relative abundance

changes between their associated chromatogram peaks. These differences of abundances

are not as drastic as the examples provided by troubleshooting (18), so it is difficult to see

whether these peaks are indeed a result of column bleed or contamination from

homologous series. Very low abundances of other ions such as m/z 267 and 295 of the

chromatogram peaks with retention times of 10.833 and 11.242 minutes could also

confirm the likelihood of such peaks arising from contamination from homologous series

rather than column bleed. These repeated mass spectra can be seen in Figure 2, with the

slight changes in ion abundances shown.

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Figure 3 Mass spectra of the peaks shown in the above chromatogram given at elution times of 9.658 (A), 10.342 (B), 10.833 (C) and 11.242 (D). Spectra A and B seem to share common ions, as well as spectra C and D.

Silicon based homologous series can result from silicon based lubricants, GC septa, and

liners or septa of vials or bottle caps (18). As such, anything coming in contact with the

sample or introduced into the gas flow of the GC can introduce these anomalies. Due to

the small volume of recovery from the samples of Batches 1 and 2, vial inserts necessary

to ensure sample intake by the GC-MS were used. These have shown to possibly

exaggerate such bleed in the chromatograms (19), and even degrade some analytes (20),

though such degradation of MPA derivatives in literature has not yet been shown to occur

in other literature. Similarity searches for the mass spectra of the peak series result in

different organo-silicon compounds, which can confirm the likelihood of contaminants.

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No ions of m/z 43, 57, 71 and 85 were found, which denote hydrocarbon contamination

often originating from lubricants, pump oils, hand lotions or gas traps/regulators (18).

Neither were any peaks found containing a strongly abundant peak at m/z 149, relating to

phthalate from plastics (18). Future efforts testing the method presented herein are

advised to use PFTE lined vials (or vial inserts) in the case small volumes are recovered, as

doing so has been shown to eliminate possible bleed sourced from the vial or septa (21).

After the inconclusive results of Batches 1 and 2, the BPX-5 column was replaced with a

new column of the same type. A new glass liner and septum was also installed in the

injection port. Volumes of all reactants were also increased for subsequent batches 3 and

4. Doing so circumvented the need to use inserts in the GC vials which ensured the

volume of sample within was sufficient to be injected in to the GC machine. As mentioned

previously, it has been found that vial inserts septum and column bleed can be

exaggerated by these vial inserts (21). As such, subsequent testing was hoped to not

exhibit the issues aforementioned. Drying by passing samples through magnesium

sulphate packed pipettes was also avoided in subsequent batches to maximise recovery

and avoid possible contamination that may have been introduced.

As a result, samples from batches 3 and 4 showed clean chromatograms resulting from

the organic layer of the samples. As shown below in Figure 3, peaks in the

chromatograms were distinct with no bleed interfering with the results for Batch 3.

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Figure 4 Chromatogram of the 1000ppm sample from Batch 3, along with the mass spectrum associated with the eluted compound at 8.223 minutes.

As shown above in Figure 3, most notable is the chromatogram peak at 8.223 minutes

belonging to the 1000ppm sample, which gives a similarity of 94% to the NIST database

mass spectrum of the desired derivative MPA bis[(dimethyl)(tert-butyl)silyl] ester on the

Shimadzu database. The method presented herein therefore shows success at this

concentration of 1000ppm. Its absence in the blank sample is also confirmatory of this

success. Very minor peaks were visible in this 1000ppm samples relating to alkanes such

as decane at 7.143 minutes and octane at 7.810 minutes. Such peaks were not visible at

all in the other samples of lower concentration, with these chromatograms appearing

completely smooth. This may show that miniscule amounts of contamination occurred

only in this 1000ppm sample, or possibly that the derivative formed has reacted with the

column. Though no literature could be found that supports this, it is important to note

that current recommended operating procedures do not support the use of the 5%

Phenyl Polysilphenylene-siloxane column, rather reporting a SE-54: (5%-phenyl)(1%-

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vinyl)-methylpolysiloxane fused silica column is ideal (8). A 5%-phenyl-methyl-

polysiloxane (HP DB-5) column has also reported success for detection of MPA derivatives

as well as other alkyl phosphonic acids relating to CWA degradants (22). However, due to

time and budget limitations with this research, the BPX-5 column was deemed sufficient.

The large peak at 5.103 minutes shown in the 1000ppm sample gives a 97% similarity

(NIST database) of 1,3-ditert-butyl-1,1,3,3-tetramethyldisiloxane. This peak is also present

in the 100ppm sample with the same mass spectra and same elution time of 5.103

minutes, though at a lower intensity. This peak relates to hydrolysis of the MTBSTFA

derivatising agent (23). Its presence in the blank sample confirms this (albeit in a very

small amount). The size of this peak appeared to decrease with concentration of MPA in

the original samples. While this is interesting to note, the presence of it shown in the

chromatogram of the blank sample does not give any indication of the presence of MPA

in the original aqueous samples.

The peak present in all samples (with the exception of 1000ppm) at 3.917 for 100ppm,

3.930 for 10ppm and 1ppm, 3.942 for 0.1ppm, and 3.943 for the blank sample give a

similarity of 79% for N-methyl-N-(trimethylsilyl)trifluoroacetamide (Shimadzu database).

The presence of this peak may be related to another reaction undergone by the

derivatising agent. Its structure is similar to N-methyl-2,2,2-trifluoroacetamide reported

as a hydrolysis product of MTBSTFA (23), though different with an additional methyl

group on the nitrogen as well as a trimethylsilyl group. The presence of this compound

could arise from the possibility that it may actually be excess derivatising agent within the

samples, though the Shimadzu database is matching it to the TMS version of the

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compound instead of the TBDMS version. Alternatively, it could have arisen from

degradation of the derivatising agent to form the TMS equivalent during the injection or

chromatography processes before being processed through the mass spectrometer. Its

absence from the 1000ppm sample could be explained by either of these processes, as

the derivatising agent has shown to have reacted with the MPA to form the derivative.

Though an excess was still likely present, it may not have been at a sufficient quantity to

form this compound. Regardless, it is certain that the presence of this reagent degradant

does not confirm the presence or absence of MPA in the original samples, due to it being

shown in the blank sample’s chromatogram.

The large-sloped peak shown at the beginning of the 1000ppm sample’s chromatogram

gives a mass spectrum relating to 3-Trifluoroacetoxypentadecane (81% similar, Shimadzu

database). The presence of this compound seems unrelated to the presence of MPA or its

reaction with MTBSTFA due to its alkane nature. Due to the hold time of the GC-MS

before recording, the full peak was not obtained. Because of this and its sole presence in

the 1000ppm sample which was run through the analyser before the other samples, this

compound could merely have been a result of a previous run not completely eluted.

Selected ion monitoring was also performed alongside TIC for the ion of m/z 267 for

Batch 3, as can be seen below in Figure 4.

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Figure 5 Selected ion monitoring chromatograms for the ion m/z 267 for Batch 3, of samples 1000ppm and 100ppm respectively.

Most notable from the SIM analysis of Batch 3 was the presence of a peak on the

chromatograms of the 1000ppm and 100ppm samples, both at 8.223 minutes. Though

they share the same elution times, the mass spectra are different. The spectrum

produced by the 1000ppm sample confirms the MPA bis[(dimethyl)(tert-butyl)silyl] ester

(94% similarity). However, the different spectrum of the 100ppm sample, most notably

the additional ion at m/z 45 results in a spectrum relating to Trimethylsilyl

[(trimethylsilyl)oxy](4-[(trimethylsilyl)oxy]phenyl)acetate (71% similarity). Figure 5 shows

the mass spectra produced by these two samples. Mass spectra given by this method of

SIM anaylsis performed alongside TIC analysis results in the same mass spectra generated

at the corresponding elution times- SIM analysis done in this way only alters the

chromatograms to generate peaks where the 267 ion was detected. Thought there was

no peak shown on the TIC analysis for the 100ppm sample, selection of this elution time

still generated the mass spectrum as shown in Figure 5.

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Figure 6 Mass spectra of the SIM analysis of the 1000ppm and 100ppm sample respectively. These are identical to mass spectra given at the same elution time by the TIC analysis

The SIM analysis of the 10ppm sample of Batch 3 gave a peak at the same time of 8.223,

though the spectrum given in relation was matched to formamide and ethylamine, both

of 99% similarity (Shimadzu database). However, peaks were repeated from then on in

the elution giving the same mass spectrum. This was also exhibited in the 1ppm sample,

though the peak at 8.223 was slightly smaller than those eluted later. The 0.1ppm sample

and blank sample had no mass spectrum related to the ‘peaks’ at 8.223 minutes, though

the peaks of these two samples were indistinguishable from the background noise. The

structure and amine nature of the mass spectra given here through SIM may therefore

not give any indication of MPA present in the original sample, though it is interesting to

note the strong signal given by the 100ppm sample at the 8.223 minutes correlating to

the same time of the derivative eluted in the 100ppm sample, where no such peak was

detected using TIC for the same sample.

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After the success of Batch 3, the same sample preparation technique was utilised,

however GC-MS analysis was undertaken with SIM and TIC carried out separately by

injection of sample done twice such that two runs were performed. As with batch 3,

absolute success was observed in the 1000ppm sample, with the MPA bis[(dimethyl)(tert-

butyl)silyl] ester derivative observed in the TIC analysis run of the 1000ppm sample. The

results of Batch 3 were replicated in Batch 4, as shown previously in figure 3, though the

partial peak eluted at the beginning of the 1000ppm sample on Batch 3 was absent in

Batch 4.

The small peak in the 1000ppm samples at 8.233 minutes gives a mass spectrum with

96% similarity to bis[(dimethyl)(tert-butyl)silyl] ester (Shimadzu database). Interestingly in

comparison to the previous batch at the same concentration is the notably smaller

amount of the derivative eluted from the column. With no change in sample preparation

between these two batches, the only explanation for such could be differences in the

stirring of the samples while heated. The 1000ppm sample of Batch 3 was angled slightly

due to the clamping of all the reaction vials together. It may be that as a result of this, the

magnetic stirrer was circulated with a slight vertical movement within the solution

instead of flat rotation on the base of the vial as was done in Batch 4. The observation of

an emulsia in the 1000ppm sample of Batch 3 compared to the smooth vortex of that of

Batch 4 may have increased the surface area between the organic and aqueous

components so greatly that it has allowed for a greater amount of desired product to

result. Future efforts utilising the method presented herein may want to consider this as a

factor in the efficiency of the reaction, or may want to test this theory to optimise the

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reaction. It could well be that detection limits may be improved with more vigorous

stirring of the reaction solution.

In addition, easily observed in Batch 4 was the production of by-products of the

derivatisation reaction at peaks of elution times around 3.925 and 5.108 minutes. These

peaks correspond to N-methyl-N-trimethylsilyl-2,2,2-trifluoroacetamide and 1,3-ditert-

butyl-1,1,3,3-tetramethyldisiloxane respectively. Respective similarities on the Shimadzu

database are given as 75-78% and 97-98%, with no significant difference of these mass

spectra between the samples and blank. As discussed previously, these two peaks are

related to the hydrolysis and degradation of the MTBSTFA derivatising agent (23). The

little difference between the retention times and amounts eluted (as visible on the

chromatograms) between Batch 3 and 4 confirms consistency in the sample preparation

between these batches. A greater amount of the 1,3-ditert-butyl-1,1,3,3-

tetramethyldisiloxane by-product appeared to be eluted in the 1000ppm sample of Batch

4 over the other samples. This is interesting to note, however given its presence also in

the blank and with seeming no correlation to the concentration of MPA in the samples it

cannot be used to give any indication of the presence of absence of MPA to be

derivatised in the original sample. Its greater presence in the 1000ppm sample may also

be a result of the more vigorous stirring of this reaction vial as aforementioned, allowing

not only for more reaction of derivatising agent with MPA, but also with water (23).

Performing SIM separately to TIC analysis for Batch 4 did not give an absolute similarity

match to the desired derivative for mass spectra, rather utilised the sensitivity of the SIM

to direct attention to the elution time of possible derivative or derivative-related

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compounds. Unlike in Batch 3 where mass spectra were given as full profiles as given by

the TIC analysis performed alongside SIM, Batch 4 SIM analysis generated mass spectra

giving only the 267 ion. As a result, all mass spectra given by SIM analysis done in this

manner were matched to the compound 4H-Dibenzo[de,g]quinoline-10,11-diol, 5,6,6a,7-

tetrahydro-6-methyl-aporphine (100%, Shimadzu database), which has this singular ion at

m/z 267. Because of this the SIM analysis could only be used to direct attention to elution

times of possible derivative related compounds. As shown below in Figure I, peaks were

present on the SIM chromatograms at the same elution times for the TIC analysis which

had no peaks. This, along with the presence of such peaks in SIM analysis of the blank

sample shows SIM analysis done this way does not positively identify the MPA

bis[(dimethyl)(tert-butyl)silyl] ester derivative.

As similar to the results of Batch 3 shown in Figure 4, notable is the strong peak at 8.235

minutes of the 1000ppm sample, which corresponds to the weak peak at 8.233 minutes

on the TIC analysis of the same sample which gave a mass spectrum matched to the

derivative. This demonstrates the ability of SIM to make interpretation of chromatograms

easier, particularly when weak signals appear in the alongside TIC analysis. The smaller

peak at 4.940 minutes only corresponds to very small peak on the TIC analysis which gave

a mass spectra similar to cyclopentasiloxane. The height of this peak in relation to others

along the chromatogram, along with mass spectrum generated gives rise to the belief

that such a compound was merely background noise and a result of the sensitivity of SIM.

The peaks of 4.940 shared in all samples, particularly larger in the diluted samples and

blank, do not have a corresponding peak in the TIC analysis. The closest peak in the TIC

analysis is around 5.117 minutes relating to the hydrolysis product 1,3-ditert-butyl-

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1,1,3,3-tetramethyldisiloxane. This however, would not be related to this peak in SIM due

to its greater elution time and lack of the m/z 267 ion. Similarly, the peaks of N-methyl-N-

(trimethylsilyl)trifluoroacetamide detected in the TIC analysis do not match to the peak(s)

around 3.730 and 3.955 minutes due to the lack of the 267 ion produced by the MTBSTFA

degradation product.

A peak is observed at 8.235 minutes in the SIM analysis of the 100ppm, 1ppm, 0.1ppm

and blanks sample. This peak is near the 8.235 minute peak corresponding to the

derivative shown in the 1000ppm sample. However, due to there being no corresponding

peaks in TIC analysis, and with this peak also present in the blank sample, the presence of

this peak in the diluted samples does not give a true indication of MPA in the original

sample converted into its derivative. These peaks were surrounded by a lot of noise,

which was also seen in Batch 3 though at a larger extent here. The increase of the

baseline, particularly at the less concentrated samples and blank may also indicate

column or septa bleed, which have given rise to these interferent peaks. As such it

remains that SIM analysis ought to only be utilised in this method to reinforce and aid

interpretation of TIC analysis.

A derivatisation reaction of dry MPA in acetonitrile was undertaken to ensure the correct

retention and identification of the derivative through GC-MS. The chromatogram and

mass spectrum generated by the peak at 8.275 minutes can be observed in Figure 6.

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Figure 7 Chromatogram and mass spectrum of the derivative formed by dry derivatisation of MPA, corresponding to the peak at 8.275 minutes.

The mass spectrum generated resulted in a 79% similarity to the MPA bis[(dimethyl)(tert-

butyl)silyl] ester derivative. The peak at 3.967 corresponds to the hydrolysis product

formed from MTBSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide, as

aforementioned. Derivatisation done in this way is set out as the recommended method

(8), and so confirms the retention time for the target derivative for the method that is

tested.

A possibility for future research to quantify the efficiency of this method is to evaporate

the remaining aqueous layer content of the reaction vials to dryness and derivatise any

possible MPA that remained unreacted in the aqueous component. Quantifying both the

derivative formed from this reaction as well as the derivative formed from the sample

preparation method proposed herein could allow for a comparative ratio of MPA

derivatised for both reactions, provided the same conditions are met (that being 60˚C for

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half an hour) for the derivatisation reaction. Due to limitations of time and budget for this

research, such could not be undertaken.

Future efforts developing this method may also use an additional 1% catalyst of Tert-

Butyldimethylsilyl chloride (TBDMSCl) with the MTBSTFA derivatising agent. Doing so may

increase efficiency of this derivatisation method and increase detection limits. Pure

MTBSTFA was used in this research as it was already available at the university and so

allowed a budget saving measure. An alternative to MTBSTFA is be tert-butyldimethylsilyl

cyanide, which reportedly derivatises acid degradants efficiently at ambient temperature

(13), however the research investigating this has yet to be published. This method could

be tested using this reagent in future research, with a possible increase in efficiency, and

also less resource intensive due to there being no requirement for heating of the

reaction.

As mentioned previously, no literature has reported success using the BPX-5 column. It

was shown that there was a great increase in column bleed between Batch 3 and 4. There

was little time between the running of these samples, however the running of samples

belonging to other researchers and their possible detrimental effects upon the column

cannot be commented on. Regardless, the rapid deterioration of the column between

these batches is good cause for future efforts to choose the recommended SE-54: (5%-

phenyl)(1%-vinyl)-methylpolysiloxane fused silica column should budget allow so.

Silylated glassware is also recommended so as to avoid adsorption of MPA onto the glass

surface. It has been shown that silylated glassware increased detection of MPA by 20%

85

(12). Utilising silylated glassware in future testing of this method will therefore likely

increase its detection limits.

Conclusion The results of this research show that derivatisation of MPA is possible without the

removal of water. Limits of detection were to 1000ppm, which shows that so far this

method is applicable to environments where MPA may be found in a high concentration,

such as recent nerve agent use or disposal. The inefficiency of this method provides

avenues for future research to improve this two-phase derivatisation method.

While low detection limits where achieved, with typical OPCW testing using analytes at a

concentration of 1-10ppm (24), the less labour-intensive and rapid generation of results

provided by the method presented herein allow for an alternative method for forensic

analysts. As with all forensic testing, the detection limits must be taken into account to

deem whether utilisation of this method is an appropriate course of action.

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