the development of an in-field rapid derivatisation
TRANSCRIPT
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
iii
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
2
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.
3
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
21
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
7
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).
8
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
9
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
10
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.
11
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.
12
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
13
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
14
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).
15
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)
16
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)
17
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.
18
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.
19
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).
20
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.
24
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.
7. References 1. Meyers R. Encyclopedia of analytical chemistry. Chichester [etc.]: Wiley; 2011.
2. Noort D, Benschop H, Black R. Biomonitoring of Exposure to Chemical Warfare
Agents: A Review. Toxicology and Applied Pharmacology. 2002;184(2):116-126.
3. Munro N, Talmage S, Griffin G, Waters L, Watson A, King J et al. The sources, fate,
and toxicity of chemical warfare agent degradation products. Environmental Health
Perspectives. 1999;107(12):933-974.
4. Sidell F. Medical Aspects of Chemical and Biological Warfare-Textbook of Military
Medicine. Washington D.Cc: Office of the Surgeon General, US Army; 1997.
5. Kroening K, Easter R, Richardson D, Caruso J. Analysis of Chemical Warfare
Degradation Products. Somerset: Wiley; 2016.
6. Greenfield R, Slater L, Bronze M, Brown B, Jackson R, Iandolo J et al. Microbiological,
Biological, and Chemical Weapons of Warfare and Terrorism. The American Journal
of the Medical Sciences. 2002;323(6):326-340.
7. Yokoyama K, Yamada A, Mimura N. Clinical profiles of patients with sarin poisoning
after the tokyo subway attack. The American Journal of Medicine. 1996;100(5):586.
8. CHEMICAL WARFARE: UN confirms use on Iranian troops. Chemical & Engineering
News. 1984;62(14):4.
48
9. OPCW Director-General Shares Incontrovertible Laboratory Results Concluding
Exposure to Sarin [Internet]. Organisation for the Prohibition of Chemical Weapon.
2017 [cited 31 January 2018]. Available from:
https://www.opcw.org/news/article/opcw-director-general-shares-
incontrovertible-laboratory-results-concluding-exposure-to-sarin/
10. Delfino R, Ribeiro T, Figueroa-Villar J. Organophosphorus compounds as chemical
warfare agents: a review. Journal of the Brazilian Chemical Society. 2009;20(3).
11. Ganesan K, Raza S, Vijayaraghavan R. Chemical warfare agents. Journal of Pharmacy
and Bioallied Sciences. 2010;2(3):166.
12. Huebner K. CBRNE - Nerve Agents, G-series - Tabun, Sarin, Soman: Background,
Pathophysiology, Epidemiology [Internet]. Emedicine.medscape.com. 2016 [cited 5
March 2018]. Available from: https://emedicine.medscape.com/article/831648-
overview
13. Keyes D. CBRNE - Nerve Agents, V-series - Ve, Vg, Vm, Vx: Background,
Pathophysiology, Epidemiology [Internet]. Emedicine.medscape.com. 2015 [cited 5
March 2018]. Available from: https://emedicine.medscape.com/article/831760-
overview
14. Vanninen P, Black R, Timperley C, Kiljunen H, Joutsiniemi K, Harju K et al.
Recommended Operating Procedures for Analysis in the Verification of Chemical
Disarmament. Helsinki: The Ministry for Foreign Affairs of Finland; 2017.
15. Cannard K. The acute treatment of nerve agent exposure. Journal of the
Neurological Sciences. 2006;249(1):86-94.
16. Kroening K. Analysis of Chemical Warfare Degradation Products. Chichester: Wiley;
2011.
49
17. Creasy W, Stuff J, Williams B, Morrissey K, Mays J, Duevel R et al. Identification of
chemical-weapons-related compounds in decontamination solutions and other
matrices by multiple chromatographic techniques. Journal of Chromatography A.
1997;774(1-2):253-263.
18. D'Agostino P, Hansen A, Lockwood P, Provost L. Capillary column gas
chromatography—mass spectrometry of tabun. Journal of Chromatography A.
1985;347:257-266.
19. D'Agostino P, Provost L. Determination of chemical warfare agents, their hydrolysis
products and related compounds in soil. Journal of Chromatography A. 1992;589(1-
2):287-294.
20. MacNaughton M, Brewer J. Environmental chemistry and fate of chemical warfare
agents. San Antonio, Tex.: Southwest Research Institute; 1994.
21. D'Agostino P, Provost L, Looye K. Identification of tabun impurities by combined
capillary column gas chromatography—mass spectrometry. Journal of
Chromatography A. 1989;465(3):271-283.
22. Marrs T, Maynard R, Sidell F. Chemical warfare agents. Chichester: Wiley; 2007.
23. Sanches M, Russel C, Randolf C. Chemical Weapons convention signature analysis.
Alexandria: Defense Technical Information Center; 2018.
24. Richardson D, Caruso J. Derivatization of organophosphorus nerve agent
degradation products for gas chromatography with ICPMS and TOF-MS detection.
Analytical and Bioanalytical Chemistry. 2007;388(4):809-823.
25. Oklahoma State University. Toxic Chemicals in the Soil Environment. Volume 2.
Interactions of Some Toxic Chemicals/Chemical Warfare Agents and Soils.
Stillwater: Defense Technical Information Center; 1985..
50
26. VERWEIJ A, BOTER H, DEGENHARDT C. Chemical Warfare Agents: Verification of
Compounds Containing the Phosphorus-Methyl Linkage in Waste Water. Science.
1979;204(4393):616-618.
27. United States Army. Newport Chemical Depot, Construction and Operation, Pilot
Testing of Neutralization/Supercritical Water Oxidation of VX Agent: environmental
impact statement. 1998.
28. U.S Army. Potential Military Chemical/ Biological Agents and Compounds. 1990.
29. NMFA. Verification of a chemical weapons convention. Geneva: UN; 1982.
30. Johnsen B, Blanch J. Analysis of snow samples contaminated with chemical warfare
agents. Archives Belges. 1984;22.
31. Howells D, Hambrook J, Utley D, Woodage J. Degradation of phosphonates II. The
influence of theO-alkyl group on the breakdown of someO-alkyl
methylphosphonofluoridates in wheat plants. Pesticide Science. 1973;4(2):239-
245.
32. Larsson L, Siitonen S, Skrifvars B, Schliack J, Reio L. The Alkaline Hydrolysis of
isoPropoxy-methyl-phosphoryl Fluoride (Sarin) and some Analogues. Acta Chemica
Scandinavica. 1957;11:1131-1142.
33. Clark D. Review of Chemical Agents in Water. Frederick: U.S. Army Biomedical
Research and Development Laboratory; 1989.
34. Keay L. THE PREPARATION AND HYDROLYSIS OF ALKYL HYDROGEN
METHYLPHOSPHONATES. Canadian Journal of Chemistry. 1965;43(9):2637-2640.
35. Yang Y, Baker J, Ward J. Decontamination of chemical warfare agents. Chemical
Reviews. 1992;92(8):1729-1743.
51
36. Schowanek D, Verstraete W. Hydrolysis and Free Radical Mediated Degradation of
Phosphonates. Journal of Environment Quality. 1991;20(4):NP.
37. Kingery A, Allen H. The environmental fate of organophosphorus nerve agents: A
review. Toxicological & Environmental Chemistry. 1995;47(3-4):155-184.
38. Epstein J, Callahan J, Bauer V. The kinetics and mechanisms of hydrolysis of
phosphonothiolates in dilute aqueous solution. Phosphorus. 1974;4:157-163.
39. Gustafson R, Martell A. A Kinetic Study of the Copper(II) Chelate-catalyzed
Hydrolysis of Isopropyl Methylphosphonofluoridate (Sarin). Journal of the American
Chemical Society. 1962;84(12):2309-2316.
40. Epstein J, Bauer V, Saxe M, Demek M. The Chlorine-catalyzed Hydrolysis of Isopropyl
Methylphosphonofluoridate (Sarin) in Aqueous Solution. Journal of the American
Chemical Society. 1956;78(16):4068-4071.
41. Howells D, Hambrook J. The phytotoxicity of some methylphosphonofluoridates.
Pesticide Science. 1972;3(3):351-356.
42. Durst H, Sarver E, Yurow H, Beaudry W, D'Eramo P. Support for the Delisting of
Decontaminated Liquid Chemical Surety Materials as Listed Hazardous Waste from
Specific Sources (STATE) MD02 in COMAR 10.51.02.16-1. Ft. Belvoir: Defense
Technical Information Center; 1988.
43. Epstein J, Rosenblatt D. Kinetics of Some Metal Ion-catalyzed Hydrolyses of
Isopropyl Methylphosphonofluoridate (GB) at 25°. Journal of the American
Chemical Society. 1958;80(14):3596-3598.
44. Epstein J, Mosher W. Magnesium ion catalysis of hydrolysis of isopropyl
methylphosphonofluoridate. Charge effect in metal ion catalysis. The Journal of
Physical Chemistry. 1968;72(2):622-625.
52
45. Epstein J. Rate of Decomposition of GB in Seawater. Science. 1970;170(3965):1396-
1398.
46. Ward J, Szafraniec L, Beaudry W, Hovanec J. On the mechanism of phosphono or
phosphorofluoridate hydrolysis catalyzed by transition metal ions. Journal of
Molecular Catalysis. 1990;58(3):373-378.
47. Leslie D, Long G, Pantelidis S. Investigation of the reaction order for nucleophilic
substitution of dialkyl methylphosphonates by alkoxides. International Journal of
Chemical Kinetics. 1992;24(10):851-859.
48. Sass S, Fisher T, Steger R, Parker G. Gas chromatographic methods for the analysis
of trace quantities of isopropyl methylphosphonofluoridate and associated
compounds, in situ and in decontamination effluent. Journal of Chromatography A.
1982;238(2):445-456.
49. D'Agostino P. Recent advances and applications of LC-MS for the analysis of
chemical warfare agents and their degradation products – A review. Trends in
Chromatography. 2005;1:23-42.
50. Bogusz (Ed) M. Handbook of Analytical Separations. 6th ed. Amsterdam: Elsevier;
2005.
51. Hammond P, Forster J. A polymeric amine–copper(II) complex as catalyst for the
hydrolysis of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman) and bis (1-
methylethyl) phosphorofluoridate (DFP). Journal of Applied Polymer Science.
1991;43(10):1925-1931.
52. Harris L, Braswell L, Fleisher J, Cliff W. Metabolites of pinacolyl
methylphosphonofluoridate (Soman) after enzymatic hydrolysis in vitro.
Biochemical Pharmacology. 1964;13(8):1129-1136.
53
53. Somani S. Chemical warfare agents. San Diego: Academic Press; 1992.
54. Michel H, Epstein J, Plapinger R, Fleisher J. EA 2192. A Novel Anticholinesterase.
Edgewood,: U. S. Army Chemical Research and Development Laboratories; 1962.
55. Yang Y, Szafraniec L, Beaudry W, Rohrbaugh D. Oxidative detoxification of
phosphonothiolates. Journal of the American Chemical Society. 1990;112(18):6621-
6627.
56. Szafraniec L, Szafraniec L, Beaudry W, Ward J. On the Stoichiometry of
Phosphonothiolate Ester Hydrolysis. Aberdeen: U.S. Army Chemical Research,
Development and Engineering Center; 2018.
57. Organisation for the Prohibition of Chemical Weapons. EVALUATION OF THE
RESULTS OF THE THIRTY-FIFTH OFFICIAL OPCW PROFICIENCY TEST. 2014.
58. Xu B, Chen J, Wu J, Tang J, Lin Y, Zhao Y et al. Can BZ react with normal extraction
solvent DCM?. Presentation presented at; 2017; OPCW Proficiency Test Meeting.
59. D'Agostino P, Provost L. Capillary column isobutane chemical ionization mass
spectrometry of mustard and related compounds. Biological Mass Spectrometry.
1988;15(10):553-564.
60. Black R, Clarke R, Read R, Reid M. Application of gas chromatography-mass
spectrometry and gas chromatography-tandem mass spectrometry to the analysis
of chemical warfare samples, found to contain residues of the nerve agent sarin,
sulphur mustard and their degradation products. Journal of Chromatography A.
1994;662(2):301-321.
61. Black R, Read R. Application of liquid chromatography-atmospheric pressure
chemical ionisation mass spectrometry, and tandem mass spectrometry, to the
54
analysis and identification of degradation products of chemical warfare agents.
Journal of Chromatography A. 1997;759(1-2):79-92.
62. Kataoka M, Seto Y. Discriminative determination of alkyl methylphosphonates and
methylphosphonate in blood plasma and urine by gas chromatography–mass
spectrometry after tert.-butyldimethylsilylation. Journal of Chromatography B.
2003;795(1):123-132.
63. D'Agostino P, Provost L, Hancock J. Analysis of mustard hydrolysis products by
packed capillary liquid chromatography–electrospray mass spectrometry. Journal
of Chromatography A. 1998;808(1-2):177-184.
64. D’Agostino P, Chenier C, Hancock J. Packed capillary liquid chromatography–
electrospray mass spectrometry of snow contaminated with sarin. Journal of
Chromatography A. 2002;950(1-2):149-156.
65. Sample Preparation for Analysis of Chemicals Related to the Chemical Weapons
Convention in an Off-site Laboratory. Encyclopedia of Analytical Chemistry. 2010.
66. Desoubries C, Chapuis-Hugon F, Bossée A, Pichon V. Three-phase hollow fiber
liquid-phase microextraction of organophosphorous nerve agent degradation
products from complex samples. Journal of Chromatography B. 2012;900:48-58.
67. Black R, Muir B. Derivatisation reactions in the chromatographic analysis of
chemical warfare agents and their degradation products. Journal of
Chromatography A. 2003;1000(1-2):253-281.
68. Noort D, Benschop H, Black R. Biomonitoring of Exposure to Chemical Warfare
Agents: A Review. Toxicology and Applied Pharmacology. 2002;184(2):116-126.
55
69. Black R, Read R. Detection of trace levels of thiodiglycol in blood, plasma and urine
using gas chromatography—electron-capture negative-ion chemical ionisation
mass spectrometry. Journal of Chromatography A. 1988;449:261-270.
70. Jakubowski E, Woodard C, Mershon M, Dolzine T. Quantification of thiodiglycol in
urine by electron ionization gas chromatography—mass spectrometry. Journal of
Chromatography B: Biomedical Sciences and Applications. 1990;528:184-190.
71. Riches J, Read R, Black R. Analysis of the sulphur mustard metabolites thiodiglycol
and thiodiglycol sulphoxide in urine using isotope-dilution gas chromatography–ion
trap tandem mass spectrometry. Journal of Chromatography B. 2007;845(1):114-
120.
72. Shih M, Smith J, McMonagle J, Dolzine T, Gresham V. Detection of metabolites of
toxic alkylmethylphosphonates in biological samples. Biological Mass Spectrometry.
1991;20(11):717-723.
73. Fredriksson S, Hammarström L, Henriksson L, Lakso H. Trace determination of alkyl
methylphosphonic acids in environmental and biological samples using gas
chromatography/negative-ion chemical ionization mass spectrometry and tandem
mass spectrometry. Journal of Mass Spectrometry. 1995;30(8):1133-1143.
74. Riches J, Morton I, Read R, Black R. The trace analysis of alkyl alkylphosphonic acids
in urine using gas chromatography–ion trap negative ion tandem mass
spectrometry. Journal of Chromatography B. 2005;816(1-2):251-258.
75. Palit M, Gupta A, Jain R, Raza S. Determination of pentafluorobenzyl derivatives of
phosphonic and phosphonothioic acids by gas chromatography–mass
spectrometry. Journal of Chromatography A. 2004;1043(2):275-284.
56
76. Lin Y, Chen J, Yan L, Guo L, Wu B, Li C et al. Determination of nerve agent metabolites
in human urine by isotope-dilution gas chromatography-tandem mass
spectrometry after solid phase supported derivatization. Analytical and
Bioanalytical Chemistry. 2014;406(21):5213-5220.
77. Albo R, Valdez C, Leif R, Mulcahy H, Koester C. Derivatization of pinacolyl alcohol
with phenyldimethylchlorosilane for enhanced detection by gas chromatography–
mass spectrometry. Analytical and Bioanalytical Chemistry. 2014;406(21):5231-
5234.
78. M.-L. Kuitunen, in: Meyers R. Encyclopedia of analytical chemistry. Chichester:
Wiley; 2011.
79. Blau K. Handbook of derivatives for chromatography. Chichester: Wiley; 1997.
80. V. Taguchi in: Clement R. (Ed) Gas chromatography. New York: Wiley; 1990.
81. Wells R. Recent advances in non-silylation derivatization techniques for gas
chromatography. Journal of Chromatography A. 1999;843(1-2):1-18.
82. Saha M, Saha J, Giese R. 4-(Trifluoromethyl)-2,3,5,6-tetrafluorobenzyl bromide as a
new electrophoric derivatizing reagent. Journal of Chromatography A.
1993;641(2):400-404.
83. Dasgupta A, Macaulay R. Microwave-induced rapid synthesis of 4-
carbethoxyhexafluorobutyryl derivatives of fatty alcohols —a novel derivative for
gas chromatography-chemical ionization mass spectrometric study. Journal of
Chromatography A. 1995;695(1):136-141.
84. Rosenfeld J. Solid-phase analytical derivatization: enhancement of sensitivity and
selectivity of analysis. Journal of Chromatography A. 1999;843(1-2):19-27.
57
85. Pan L, Pawliszyn J. Derivatization/Solid-Phase Microextraction: New Approach to
Polar Analytes. Analytical Chemistry. 1997;69(2):196-205.
86. Tang P, Ho J. Liquid-solid disk extraction followed by supercritical fluid elution and
gas chromatography of phenols from water. Journal of High Resolution
Chromatography. 1994;17(7):509-518.
87. Winberry W, Murphy N, Riggin R. Compendium of methods for the determination
of toxic organic compounds in ambient air. Research Triangle Park, N.C.:
Atmospheric Research and Exposure Assessment Laboratory, Office of Research
and Development, U.S. Environmental Protection Agency; 1988.
88. Cummins M, Wells R. In situ derivatisation and extraction of volatile fatty acids
entrapped on anion-exchange resin from aqueous solutions and urine as a test
matrix using pentafluorobenzyl bromide in supercritical carbon dioxide. Journal of
Chromatography B: Biomedical Sciences and Applications. 1997;694(1):11-19.
89. Miki A, Tsuchihashi H, Yamano H, Yamashita M. Extractive pentafluorobenzylation
using a polymeric phase-transfer catalyst: a convenient one-step pretreatment for
gas chromatographic analysis of anionic compounds. Analytica Chimica Acta.
1997;356(2-3):165-175.
90. Jedrzejczak K, Gaind V. Polymers with reactive functions as sampling and
derivatizing agents—Part 2. Polymeric pentafluorobenzoylating reagent for gas
chromatographic–mass spectrometric determination of aliphatic amines. The
Analyst. 1993;118(11):1383-1387.
91. Hušek P. Chloroformates in gas chromatography as general purpose derivatizing
agents. Journal of Chromatography B: Biomedical Sciences and Applications.
1998;717(1-2):57-91.
58
92. Minero C, Vincenti M, Lago S, Pelizzetti E. Determination of trace amounts of highly
hydrophilic compounds in water by direct derivatization and gas chromatography ?
mass spectrometry. Fresenius' Journal of Analytical Chemistry. 1994;350(6):403-
409.
93. Phototransformations of nitrogen containing organic compounds over irradiated
semiconductor metal oxides. Coordination Chemistry Reviews. 1993;125(1-2):183-
193.
94. Simpson J, Torok D, Markey S. Pentafluorobenzyl chloroformate derivatization for
enhancement of detection of amino acids or alcohols by electron capture negative
ion chemical ionization mass spectrometry. Journal of the American Society for
Mass Spectrometry. 1995;6(6):525-528.
95. Maurino V, Minero C, Pelizzetti E, Angelino S, Vincenti M. Ultratrace determination
of highly hydrophilic compounds by 2,2,3,3,4,4,5,5-octafluoropentyl
chloroformate-mediated derivatization directly in water. Journal of the American
Society for Mass Spectrometry. 1999;10(12):1328-1336.
96. Subramaniam R, Åstot C, Juhlin L, Nilsson C, Ostin A. Direct Derivatization and Rapid
GC-MS Screening of Nerve Agent Markers in Aqueous Samples. Analytical
Chemistry. 2010;82(17):7452-7459.
97. Subramaniam R. Simplified routines for sample preparation and analysis of
chemical warfare agent degradation products. Umea: Department of Chemistry,
Umea University; 2012.
98. Fowler W, Smith J. Indirect determination of O-ethyl S-(2-diisopropylaminoethyl)
methylphosphonothioate in air at low concentrations. Journal of Chromatography
A. 1989;478:51-61.
59
99. Tingfa D. Gas Chromatographic Determination of O-Ethyl S-(N, N-Diisopropylamino)
Ethyl Methylphosphonothiolate and O, O-Diisopropyl S-Benzyl Phosphorothiolate
as Corresponding Phosphonofluoridate and Phosphorofluoridate. International
Journal of Environmental Analytical Chemistry. 1986;27(1-2):151-158.
100. Valdez C, Leif R, Alcaraz A. Effective methylation of phosphonic acids related
to chemical warfare agents mediated by trimethyloxonium tetrafluoroborate for
their qualitative detection and identification by gas chromatography-mass
spectrometry. Analytica Chimica Acta. 2016;933:134-143.
101. Enqvist J, Rautio M. Identification of degradation products of potential
organophosphorus warfare agents. Helsinki: Ministry for Foreign Affairs of Finland;
1980.
102. Berg D. Proceedings of the 1998 ERDEC Scientific Conference on Chemical
and Biological Defense Research, 17-20 November 1998. [United States]: Edgewood
chemical biological center aberdeen proving ground md; 1999.
103. Tørnes J, Johnsen B. Gas chromatographic determination of
methylphosphonic acids by methylation with trimethylphenylammonium
hydroxide. Journal of Chromatography A. 1989;467:129-138.
104. Sega G, Tomkins B, Griest W. Analysis of methylphosphonic acid, ethyl
methylphosphonic acid and isopropyl methylphosphonic acid at low microgram per
liter levels in groundwater. Journal of Chromatography A. 1997;790(1-2):143-152.
105. Amijee M, Cheung J, Wells R. Direct on-column derivatisation in gas
chromatography II. Comparison of various on-column methylation reagents and the
development of a new selective methylation reagent. Journal of Chromatography
A. 1996;738(1):43-55.
60
106. Creasy W, Rodríguez A, Stuff J, Warren R. Atomic emission detection for the
quantitation of trimethylsilyl derivatives of chemical-warfare-agent related
compounds in environmental samples. Journal of Chromatography A.
1995;709(2):333-344.
107. Purdon J, Pagotto J, Miller R. Preparation, stability and quantitative analysis
by gas chromatography and gas chromatography—electron impact mass
spectrometry of tert.-butyldimethylsilyl derivatives of some alkylphosphonic and
alkyl methylphoshonic acids. Journal of Chromatography A. 1989;475(2):261-272.
108. Kataoka M, Tsunoda N, Ohta H, Tsuge K, Takesako H, Seto Y. Effect of cation-
exchange pretreatment of aqueous soil extracts on the gas chromatographic–mass
spectrometric determination of nerve agent hydrolysis products after tert.-
butyldimethylsilylation. Journal of Chromatography A. 1998;824(2):211-221.
109. M. Rautio (Ed). Recommended Operating Procedures for Analysis in the
Verification of Chemical Disarmament. Helsinki: The Ministry for Foreign Affairs of
Finland; 1994.
110. Kataoka M, Tsuge K, Seto Y. Efficiency of pretreatment of aqueous samples
using a macroporous strong anion-exchange resin on the determination of nerve
gas hydrolysis products by gas chromatography–mass spectrometry after tert.-
butyldimethylsilylation. Journal of Chromatography A. 2000;891(2):295-304.
111. D'Agostino P, Provost L. Capillary column electron impact and ammonia
chemical ionization gas chromatographic-mass spectrometric and gas
chromatographic-tandem mass spectrometric analysis of mustard hydrolysis
products. Journal of Chromatography A. 1993;645(2):283-292.
61
112. Lemarie L, Sokolowski M, Wickramage C. Proceedings of the 3rd SISPAT
International Symposium on Protection against Toxic Substances. 2002.
113. Rohrbaugh D, Sarver E. Detection of alkyl methylphosphonic acids in
complex matrices by gas chromatography–tandem mass spectrometry. Journal of
Chromatography A. 1998;809(1-2):141-150.
114. Minami M, Hui D, Katsumata M, Inagaki H, Boulet C. Method for the analysis
of the methylphosphonic acid metabolites of sarin and its ethanol-substituted
analogue in urine as applied to the victims of the Tokyo sarin disaster. Journal of
Chromatography B: Biomedical Sciences and Applications. 1997;695(2):237-244.
115. Enqvist J, Rautio M. (Eds) Trace Analysis of Chemical Warfare Agents. 1. An
Approach to the Environmental Monitoring of Nerve Agents. Helsinki: Ministry for
Foreign Affairs of Finland; 1981.
116. FMC Corporation. Flame Retardants. US; 5,198,483, 2018.
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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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147 281
22134120745
A
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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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147281221
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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.
References 1. OPCW Director-General Shares Incontrovertible Laboratory Results Concluding
Exposure to Sarin [Internet]. Organisation for the Prohibition of Chemical Weapon.
2017 [cited 31 January 2018]. Available from:
https://www.opcw.org/news/article/opcw-director-general-shares-
incontrovertible-laboratory-results-concluding-exposure-to-sarin/
86
2. Munro N, Talmage S, Griffin G, Waters L, Watson A, King J et al. The sources, fate,
and toxicity of chemical warfare agent degradation products. Environmental
Health Perspectives. 1999;107(12):933-974
3. Meyers R. Encyclopedia of analytical chemistry. Chichester [etc.]: Wiley; 2011.
4. Noort D, Benschop H, Black R. Biomonitoring of Exposure to Chemical Warfare
Agents: A Review. Toxicology and Applied Pharmacology. 2002;184(2):116-126.
5. Sidell F. Medical Aspects of Chemical and Biological Warfare-Textbook of Military
Medicine. Washington D.Cc: Office of the Surgeon General, US Army; 1997.
6. Kingery A, Allen H. The environmental fate of organophosphorus nerve agents: A
review. Toxicological & Environmental Chemistry. 1995;47(3-4):155-184.
7. Richardson D, Caruso J. Derivatization of organophosphorus nerve agent
degradation products for gas chromatography with ICPMS and TOF-MS detection.
Analytical and Bioanalytical Chemistry. 2007;388(4):809-823.
8. Vanninen P, Black R, Timperley C, Kiljunen H, Joutsiniemi K, Harju K et al.
Recommended Operating Procedures for Analysis in the Verification of Chemical
Disarmament. Helsinki: The Ministry for Foreign Affairs of Finland; 2017.
9. Black R, Read R. Application of liquid chromatography-atmospheric pressure
chemical ionisation mass spectrometry, and tandem mass spectrometry, to the
analysis and identification of degradation products of chemical warfare agents.
Journal of Chromatography A. 1997;759(1-2):79-92.
10. D’Agostino P, Chenier C, Hancock J. Packed capillary liquid chromatography–
electrospray mass spectrometry of snow contaminated with sarin. Journal of
Chromatography A. 2002;950(1-2):149-156.
87
11. Liu Q, Hu X, Xie J. Determination of nerve agent degradation products in
environmental samples by liquid chromatography–time-of-flight mass
spectrometry with electrospray ionization. Analytica Chimica Acta.
2004;512(1):93-101.
12. Subramaniam R, Åstot C, Juhlin L, Nilsson C, Ostin A. Direct Derivatization and
Rapid GC-MS Screening of Nerve Agent Markers in Aqueous Samples. Analytical
Chemistry. 2010;82(17):7452-7459.
13. Black R, Muir B. Derivatisation reactions in the chromatographic analysis of
chemical warfare agents and their degradation products. Journal of
Chromatography A. 2003;1000(1-2):253-281.
14. Driskell W, Shih M, Needham L, Barr D. Quantitation of Organophosphorus Nerve
Agent Metabolites in Human Urine Using Isotope Dilution Gas Chromatography-
Tandem Mass Spectrometry. Journal of Analytical Toxicology. 2002;26(1):6-10.
15. Subramaniam R, Åstot C, Nilsson C, Östin A. Combination of solid phase extraction
and in vial solid phase derivatization using a strong anion exchange disk for the
determination of nerve agent markers. Journal of Chromatography A.
2009;1216(48):8452-8459.
16. Palit M, Gupta A, Jain R, Raza S. Determination of pentafluorobenzyl derivatives of
phosphonic and phosphonothioic acids by gas chromatography–mass
spectrometry. Journal of Chromatography A. 2004;1043(2):275-284.
17. TSUNODA N. The Sarin Incidents in Japan and Mass Spectrometry. Journal of the
Mass Spectrometry Society of Japan. 2005;53(3):157-163.
88
18. Rood D. Gas Chromatography Problem Solving and Troubleshooting. Journal of
Chromatographic Science. 1995;33:347
19. English C. Column Bleed & Septa Bleed – Same Old Thing! « ChromaBLOGraphy:
Restek's Chromatography Blog [Internet]. Blog.restek.com. 2013 [cited 10 June
2018]. Available from: https://blog.restek.com/?p=10706
20. Cochran J. Silicone Autosampler Vial Septa Cause Endrin Breakdown and Sample
Contamination « ChromaBLOGraphy: Restek's Chromatography Blog [Internet].
Blog.restek.com. 2011 [cited 10 June 2018]. Available from:
https://blog.restek.com/?p=3604
21. Rigdon A. The Forgotten Septum: How to Correctly Diagnose the Source of Bleed
Contamination [Internet]. 1st ed. GC Accessories; 2008 [cited 10 June 2018].
Available from: http://www.restek.com/pdfs/adv_2008_01_12.pdf
22. Richardson D, Caruso J. Derivatization of organophosphorus nerve agent
degradation products for gas chromatography with ICPMS and TOF-MS detection.
Analytical and Bioanalytical Chemistry. 2007;388(4):809-823.
23. Glavin D, Freissinet C, Miller K, Eigenbrode J, Brunner A, Buch A et al. Evidence for
perchlorates and the origin of chlorinated hydrocarbons detected by SAM at the
rocknest aeolian deposit in gale crater. Journal of Geophysical Research: Planets.
2013;118(10):1955-1973.
24. Pal Anagoni S, Kauser A, Maity G, Upadhyayula V. Quantitative determination of
acidic hydrolysis products of Chemical Weapons Convention related chemicals
from aqueous and soil samples using ion-pair solid-phase extraction and in situ
butylation. Journal of Separation Science. 2017;41(3):689-696.