ISSN 1061-9348, Journal of Analytical Chemistry, 2013, Vol. 68, No. 1, pp. 1–11. © Pleiades Publishing, Ltd., 2013.

Original Russian Text © O.I. Orlova, E.I. Savel’eva, N.S. Khlebnikova, 2013, published in Zhurnal Analiticheskoi Khimii, 2013, Vol. 68, No. 1, pp. 4–14.

REVIEWS

Methods for the Detection of Sulfur Mustard Metabolites

in Biological Materials: An Analytical Review
O. I. Orlova, E. I. Savel’eva, and N. S. Khlebnikova
Research Institute of Hygiene, Occupational Pathology, and Human Ecology, Federal Medical–Biological Agency of Russia,
pos. Kuz’molovskii, Vsevolozhskii region, Leningrad oblast, 188663 Russia
Received April 14, 2011; in final form, March 7, 2012

Abstract—This review surveys published data to generalize methods for the determination of sulfur mustard
and its metabolites in various media. Attention is focused on biological materials. Data on the toxicokinetics
and metabolism of sulfur mustard are cited, and the problem of choosing a biomarker for the indication of
sulfur mustard poisoning is considered.

Keywords: sulfur mustard, biological fluids, biomarker, gas chromatography–mass spectrometry, high-per-
formance liquid chromatography
DOI: 10.1134/S1061934813010103

The development and improvement of methods for
the determination of the metabolites (biomarkers) of
toxic agents in biological materials is a problem of
considerable current interest because of the need for
detecting the action of toxic agents on humans and
animals, as follows from both the requirements of the
Chemical Weapons Convention [1] and the current
threat of chemical terrorism. The correct selection of
a biological matrix (analytical sample) and a biomar-
ker characterizing the level and character of exposure
is especially important with respect to toxic agents,
which affect various targets in the body. Sulfur mus-
tard, which is characterized by an especially compli-
cated metabolism, is a prominent example of these
agents [2].
Sulfur mustard (bis(2-chloroethyl) sulfide, mus-
tard gas, yperite) is the best known vesicant chemical
warfare agent. It was used for the first time in the First
World War and more recently in a conflict between
Iran and Iraq. Up to now, large amounts of sulfur mus-
tard remain stockpiled worldwide; this causes the
potential possibility of its use for terrorist purposes.
In the functioning of enterprises for the destruction
of chemical weapons, there is a danger of exposing the
operating personnel and the population of adjacent
territories in the case of emergencies. Sulfur mustard
predominates among disposed chemical weapons,
whose disposal and dumping sites are not known with
certainty. Because of these risks, effective procedures
should be developed for both rapid and retrospective
detection of the action of yperite on the human body.
In addition to that, these procedures should make it
possible to quantitatively evaluate the exposure level,
which is an extremely important medical aspect for
producing a strategy for the treatment of poisoned
persons and the risk assessment of late effects. An
important application of procedures for the determi-
nation of the bio-markers of toxic substances, includ-
ing sulfur mustard, in the human body can be the
biomonitoring of the personnel of enterprises for the
disposal and destruction of chemical weapons and also
forensic medical examination, in particular, for estab-
lishing the participation of potential terrorists to the
production, storage, and transportation of chemical
warfare agents.
Because of this, a considerable number of publica-
tions were dedicated to methods for the determination
of sulfur mustard metabolites in biological samples.
Recently [3–5], the concepts of the expected levels of
sulfur mustard metabolite concentrations in biological
samples depending on dose and time interval from the
exposure have been revised to a considerable degree
with consideration for toxicokinetic data obtained in
recent years with the aid of instrumentation of a new
generation.
General information on the mechanism of the toxic
action of sulfur mustard. Sulfur mustard renders both
local (on skin) and systemic (resorptive) effects; it is an
alkylating agent, which acts through the cyclization of
the ethylene group with the formation of an extremely
reactive sulfonium center. This electrophile can react
with the numerous nucleophilic centers of cell macro-
molecules [6]. The metabolism of sulfur mustard
occurs via several reaction paths: hydrolysis, oxida-
tion, conjugate formation with glutathione, and β-
lyase reactions.
Thiodiglycol (2,2'-thiodiethanol, TDG) is the pri-
mary product of sulfur mustard hydrolysis. Jakubowski
et al. [7] found in toxicokinetic experiments
that, upon the hypodermic injection of 750 μg/kg

1
2 ORLOVA et al.
(1/3 LD50) of yperite into rats, the concentrations of
TDG in urine were 196, 116, and 17 ng/mL 20, 48,
and 116 h after intoxication, respectively, and the con-
centrations of TDG in urine were 34 and 297 ng/mL
upon the exposure of guinea pigs to yperite vapor. In
this case, the concentrations of TDG in the urine of
the control groups of animals were no higher than
1 ng/mL. Increased TDG concentrations in urine
were detected within a week after intoxication. More
recently, Black and Read [8] found that not free TDG
but the product of its metabolic oxidation at the sulfur
atom, thiodiglycol oxide (bis(2-hydroxyethyl) sulfox-
ide, TDGO) formed the greatest fraction in the prod-
ucts of excretion with urine and proposed a methodol-
ogy including the pretreatment of an urine sample
with titanium trichloride for the reduction of TDGO
to TDG. In this case, the release of TDG and TDGO
(with its subsequent reduction) from conjugates also
occurred because of the high acidity of titanium
trichloride solution. Black and Read [8] hypothesized
that this approach is most reasonable for retrospective
analysis and confirmed this hypothesis by the detec-
tion of thiodiglycol in rat urine at a level of 60 ng/mL
8 days after the cutaneous application of 336 μg of sul-
fur mustard. Black et al. [9] performed a more detailed
analysis of the excretion profiles of TDG, TDGO, and
β-lyase metabolites with the urine of rats after the
cutaneous application of sulfur mustard (2 μmol per
animal). The fractions of free TDG and TDG and
TDGO released from conjugates as a result of acidic
treatment for 8 days after exposure were 0.3, 1–1.5,
and 3.4–4.3% on an initial sulfur mustard dose basis,
respectively. In a series of experiments in which TDG
and TDGO were determined together and β-lyase
metabolites were determined as 1,1'-sulfonylbis[2-
(methylthio)ethane], the common product of their
reduction; the corresponding values were 3.7–13.6%
on an exposure dose basis for TDG + TDGO and 2.5–
5.3% for the β-lyase metabolites. In this case, the
excretion curve of these latter was considerably
sharper (a maximum of ~60 nmol after 24 h and a drop
to zero after 72 h) than that for the products of hydrol-
ysis (a maximum of ~45 nmol in the first hours after
exposure, a drop to ~10 nmol after 3 days, and the
retention of this level up to the 8th day). This was
explained by the slow release of TDG from adducts
with macromolecules.
Two metabolites, which supposedly resulted
from the reactions of yperite with macromolecules,
were also identified in urine. N7-(2-Hydroxyethyl-
thioethyl)guanine was detected in the urine of guinea
pigs after the intravenous introduction of 1 mg/kg of
sulfur mustard. This compound is formed upon the
destruction of alkylated DNA [10]. The maximum
excretion level of this metabolite was observed in the
first 2–3 h after exposure (50 ng/mL); the excretion
level decreased to 10 ng/mL in a period from 34 to
48 h. N1-(2-hydroxyethylthioethyl)-4-methylimida-
zole, which is supposedly the degradation product of
JOURNAL OF ANALYTICAL CHEMISTRY
an alkylated histidine residue, was detected by fast
atom bombardment mass spectrometry in the urine of
pigs after the cutaneous application of sulfur mustard
[11].
Determination of sulfur mustard. It was assumed
that absorbed sulfur mustard is completely metabo-
lized. However, it was found that sulfur mustard could
be accumulated in adipose tissues because of its high
lipophilicity. There are also data on the detection of
sulfur mustard in hair [12]. The unexpectedly high
concentration of sulfur mustard was detected in differ-
ent organs and tissues, especially in the abdominal fat
extracted from the body of a victim of sulfur mustard
poisoning [13].
The determination of sulfur mustard traces is not
very difficult to perform, although the presence of the
unmetabolized agent in urine or blood samples is
extremely highly improbable in the majority of possi-
ble poisoning scenarios. Extraction from homoge-
nized tissues [13] or blood [14] is carried out with
organic solvents (dichloromethane and ethyl acetate).
Solid-phase extraction can be used for the purification
of extracts, and gas chromatography–mass spectrom-
etry (GC–MS), including tandem (GC–MS–MS)
with nonpolar capillary columns, can be used for the
analysis. A method for the identification of sulfur mus-
tard in blood and tissue homogenates by two-dimen-
sional GC–MS with thermal desorption on tubes with
a sorbent was proposed [15]. The sample was treated
with ethyl acetate; thereafter, the extract was consecu-
tively applied portionwise to the sorption tube in a
weak flow of nitrogen, and the tube was then placed in
the thermal desorber of a gas chromatograph. The
reported limit of detection is 10 pg/mL for blood or
pg/g for tissue homogenates. Within the framework of
an UN investigation of the use of chemical weapons in
conflict between Iran and Iraq [12], sulfur mustard in
a concentration of ~0.5–1 ppm was detected in a
dichloromethane extract from a hair sample. Sulfur
mustard was detected in postmortem human tissue
samples taken seven days after sulfur mustard poison-
ing in concentrations of 0.8–10.7 μg/g in the lungs,
kidneys, and brain and in concentrations to 15.1 μg/g
in adipose tissues (including skin). These results
showed that sulfur mustard accumulated in the adi-
pose tissues could remain in the body for a longer time
than that followed from the tests with animals.
Currently, methods for the determination of six
sulfur mustard metabolites in urine have been devel-
oped, in particular, thiodiglycol; bis(2-hydroxyethyl)-
sulfoxide; 1,1'-sulfonylbis[2-S-(N-acetylcys-
teinyl)ethane]; 1,1'-sulfonylbis[2-(methylsulfi-
nyl)ethane]; 1-methylsulfinyl-2-[(methylthio)ethyl-
sulfonyl]ethane; and N7-(2-hydroxyethylthio-
ethyl)guanine.
Methods for the determination of sulfur mustard
metabolites. The data on methods for the determina-
tion of free sulfur mustard metabolites given in this
survey are dedicated to only urinary metabolites
Vol. 68 No. 1 2013
 METHODS FOR THE DETECTION OF SULFUR MUSTARD METABOLITES
because the efforts of researchers to find sulfur mus-
tard exposure markers and to develop methods for
determining the metabolites in the last 20 years have
been concentrated in this area. Urinary metabolites
are the most convenient test substances as markers of
the action of chemical warfare agents. Urine can be
taken in large quantities using a noninvasive tech-
nique, and it is a comparatively clean matrix because,
as a rule, it does not contain proteins and lipids. The
main amounts of both free and metabolized agent are
excreted with urine. The excretion of urinary markers
is about 90% of the obtained dose of the agent, and its
active phase is 48–72 h since the exposure time.
Therefore, analysis with the use of comparatively sim-
ple procedures and instrumental techniques (for
example, low-resolution GC–MS) can be performed
only at high exposure levels and during the first days
after the action, when the concentrations of metabo-
lites in urine are at a level of 50–100 ng/mL. In suc-
ceeding days, a slower excretion phase occurs mainly
due to metabolites released from covalent adducts with
proteins and DNA. Free metabolites, in particular,
TDG [39], can also be detected in blood, as well as uri-
nary metabolites, for a short time after the exposure.
At the same time, blood is a more complex matrix,
which is a concentrated buffer solution containing
suspended cells, proteins, fats, and salts. Fresh whole
blood can be easily separated into red cells and plasma
or serum, which is a more convenient test matrix. The
concentrations of metabolites in urine are higher than
those in blood at any feasible scenarios of the use of
chemical warfare agents. In this case, the analysis of
blood is much more important for the retrospective
detection of long-lived exposure markers—the
adducts of sulfur mustard with blood proteins.
Thiodiglycol. TDG can be determined by gas chro-
matography (GC) without preliminary derivatization.
However, even with the use of columns with a polar
stationary phase, TDG appears as a broad peak under
conditions of direct GC determination; because of
this, TDG at a level of <1 ppm cannot be determined
by direct GC analysis. For reaching lower limits of
detection (about a few ppm or lower), TDG should be
extracted from urine or blood with a nonaqueous sol-
vent, derivatized, and determined by GC–MS or
GC–MS–MS. Trimethylsilyl (TMS) or tert-
butyldimethylsilyl (TBDMS) derivatives are com-
monly used for environmental analysis [16]. These
derivatives are also suitable for the analysis of biologi-
cal fluids after the action of comparatively high sulfur
mustard doses; however, pentafluorobenzoyl and hep-
tafluorobutyryl ethers were most frequently used for
reaching lower limits of determination.
Negative chemical ionization (NCI) GC–MS and
GC–MS–MS were used for determining TDG in the
form of bis(pentafluorobenzoyl) derivatives [17, 18].
Urine, blood, or plasma with [ 2H4]TDG added as an
internal standard was passed through a cartridge with
a sorbent, and the target substances were eluted with
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68
3
ethyl acetate. The eluate was purified on Florisil
(urine) or C18 (blood and plasma). After preconcen-
tration, the dry residue was treated with pentafluo-
robenzoyl chloride (pyridine; room temperature;
5 min). Thiodiglycol bis(pentafluorobenzoate) was
identified by the detection of the molecular ion [M]–
with m/z 510 and 511 at a retention time of 12 min (the
standard substance was scanned for an ion with
m/z 514). The limit of definition of 1 ng/mL was
reached. The limited possibility of confirmatory anal-
ysis is a drawback of the method. The same derivative
was determined more selectively and sensitively
(<0.1 ng/mL) by GC–NCI MS–MS [18] with moni-
toring the daughter ion with m/z 167 ([C6F5]–). Insuf-
ficiently complete derivatization is a disadvantage of
the use of pentafluorobenzoyl chloride for the prepa-
ration of derivatives; as a result of this, the derivative
can be selectively detected only by NCI MS. A modi-
fication of this technique including the stage of deriv-
ative purification on C18 was proposed [16].
Jakubowski et al. [7] proposed a procedure for the
determination of TDG in urine as a bis(heptafluo-
robutyryl) derivative. The procedure included the
enzymatic hydrolysis of TDG conjugates with the aid
of glucuronidase, and [2H8]TDG was introduced as an
internal standard. The hydrolyzate was acidified to
pH 3–4 and evaporated to dryness; the dry residue was
dissolved in ethyl acetate and treated with heptafluo-
robutyric anhydride (60°C; 1 h). Detection by GC–
MS with electron ionization was performed using the
monitoring of selected ions with m/z 300 and 301 (70
and 50%, respectively). The limit of detection of
5 ng/mL was reached. The advantage of this method is
that more accessible low-resolution GC–MS is used
as an analytical tool. The disadvantages include ana-
lyte losses upon the evaporation of urine to dryness
and also the residual trace amounts of water, which
decrease the yield of derivatization.
There is a fundamentally different approach to the
determination of TDG in urine [19, 20] through the
conversion of TDG into sulfur mustard by treatment
with concentrated HCl. Urine with [ 2H8]TDG added
as an internal standard was passed through two car-
tridges with C18. After the treatment with concen-
trated HCl at 120°C, the released sulfur mustard was
transferred from the vapor phase to the sorbent with a
flow of nitrogen with the subsequent thermal desorp-
tion in GC–MS. The method was used for the analysis
of urine samples from the victims of the Iran–Iraq
conflict. The concentration of TDG in urine taken 5–
10 days after an attack varied in a range of 5–
100 ng/mL, but it was 330 ng/mL in a single case (this
patient died immediately after entering into the hospi-
tal). Very low levels of TDG (3–8 ng/mL) were
detected in urine taken 18 days after an attack. A draw-
back of the method is that not only TDG but also other
metabolites can be converted into sulfur mustard upon
the treatment with HCl.
No. 1 2013
4 ORLOVA et al.
The conversion of TDG into TMS ethers occurs
sufficiently rapidly at room temperature as a reaction
with bis(trimethylsilyl) trifluoroacetamide (BSTFA)
or bis(trimethylsilyl)acetamide (BSA) in the presence
of trimethylchlorosilane. Hexamethyldisilazane in a
mixture with trimethylchlorosilane was also used for
the conversion of TDG into volatile derivatives [21,
22]; data on rapid derivatization with trimethylsilyl
cyanide, which is usually little used, were reported
[23]. Derivatization occurs most effectively in an
anhydrous solvent; however, the successful extraction
trimethylsilylation of TDG by interaction with
BSTFA was reported [24, 25]. Conversion into di-
TBDMS derivatives requires more severe derivatiza-
tion conditions with the use of a mixture of methyltri-
fluoroacetamide (MTBSTFA) + 1% trimethylchlo-
rosilane at 100°C and a reaction time of no shorter
than 30 min. For the complete derivatization of trace
TDG in ground water, the test sample was incubated
for 1 h at 105°C [26]; the reaction with TBDMS in
acetonitrile was carried out at 80°C for 1 h [27].
The traces of water after solid-phase extraction
(SPE) or after sample preconcentration by evapora-
tion to dryness can negatively affect the quantitative
determination of TDG as silylated derivatives. The
sorbent Ambersorb 572 was used for SPE, and losses
upon evaporation were decreased by the introduction
of pyridine into the eluate [26]. As a rule, derivatiza-
tion occurred at both OH groups on the silylation of
TDG. Only Ohsawa et al. [28] detected both mono-
and diethers of TDG upon the measurement of the
mass chromatograms of silylated derivatives.
Silyl derivatives possess good chromatographic
properties. The electron spectrum of TDG, which has
three heteroatoms, contains a large number of frag-
ment ions. Under the conditions of electron impact,
the di-TMS derivative does not afford a molecular ion,
but it gives a weak ion (~5%) with m/z 251, which cor-
responds to [M–Me]+ and other high mass
ions (m/z 191, 176, 161, 147, 133, 130, and 116)
formed upon the elimination of fragments from the α
and β-positions of different heteroatoms. The quasi-
molecular ions MH+ and [M+NH4]+ are formed upon
chemical ionization (CI) with ammonia, but the
main peak of m/z 177 corresponds to
[TMSOCH2CH2SCH2CH2]+. The TBDMS deriva-
tives also give the peak M+ upon EI, but the most
intense peak has m/z 293 ([M–Bu]+); this peak is
commonly used for determination using the selected
ion monitoring mode (comparative intensities change
depending on MS conditions). Tomkins and Sega [26]
reported the intensity of a peak with m/z 293; this is
~40% relative to the main peak with m/z 73). Other
intense signals in the mass spectrum corresponding to
high mass ions are characterized by the mass-to-
charge ratios m/z 233, 190, 189, 149, and 148.
Upon CI with methane, the sufficiently intense ion
[MH–CH4]+ with m/z 335, 293 and the main peak
JOURNAL OF ANALYTICAL CHEMISTRY
with m/z 219 can be used [26]. D’Agostino and Provost
[24] derivatized TDG and other yperite hydrolysis
products under the following conditions: BSTFA +
1% trimethylchlorosilane at 60°C for 20 min. The
resulting MH+ and [M+NH4]+ upon CI with ammo-
nia make it possible to perform identification. The
intensities of signals vary in a range from weak to
100%.
D’Agostino and Provost [24] reported the use of the
TMS and TBDMS derivatization of TDG in the
tests of the Organization for the Prohibition of Chem-
ical Weapons (OPCW) [24]. Derivatization with
MTBSTFA is more preferable, if the target-oriented
determination of TDG is performed; however, the
derivatization with BSTFA is more effective in the case
when the simultaneous determination of TDG and
TDG sulfoxide should be performed [29].
Both acylation with heptafluorobutyric anhydride
(HFBA) or trifluoracetic anhydride (TFAA) and alky-
lation—silylation (TMS and TBDMS) or pentafluo-
robenzylation—are used to obtain TDG derivatives
[16].
A method was developed for the identification of
sulfur mustard decomposition products, including
TDG, by tandem HPLC–MS/MS with electrospray
ionization [30]. Aqueous soil extracts were analyzed
by HPLC with the quadrupole/time-of-flight detec-
tion with a resolution of 9000. Electrospray HPLC–
MS was recommended for the determination of TDG
in aqueous soil extracts [29], and it did not find appli-
cation to the analysis of biological samples up to now.
Thiodiglycol sulfoxide. TDGO is more polar than
TDG. For this reason, TDGO is more difficult to
extract from aqueous matrices and to purify by solid-
phase extraction because it is strongly retained by sil-
ica gel and its elution requires the use of a highly polar
solvent. At the same time, the substance is almost not
retained on reversed-phase sorbents. Underivatized
TDGO can be identified with the aid of HPLC–MS;
however, the limits of detection are insufficiently low,
as in the case of TDG. In particular, upon derivatiza-
tion for GC–MS analysis with the use of pentafluo-
robenzoyl chloride, the same derivative as from TDG
is formed; that is, the sulfoxide function is reduced.
Under these conditions, TDGO cannot be distin-
guished from TDG differently than with the use of
selective extraction. In 1991, a method was proposed
for the simultaneous determination of TDG and
TDGO in urine based on the preliminary reduction of
the latter with a hydrochloric-acid solution of TiCl3
followed by analysis in accordance with the above pro-
cedure for the GC–NCI MS determination of TDG
as a pentafluorobenzoyl derivative [8]. This method
was used for the analysis of urine samples taken from
three victims of the Iran–Iraq conflict eight days after
an injury and stored at –20°C for five years. The con-
centration of TDG varied within the limits of 27–
72 ng/mL against 11 ng/mL in the control sample.
Vol. 68 No. 1 2013
 METHODS FOR THE DETECTION OF SULFUR MUSTARD METABOLITES
More recently, this approach was used for develop-
ing a procedure for the simultaneous determination of
TDG and TDGO and also β-lyase metabolites in
urine with the aid of GC–MS–MS [32].
β-Lyase metabolites. As noted above, the glu-
tathione/β-lyase metabolic pathway of sulfur mustard
leads to the formation of two metabolites: 1,1'-sulfo-
nylbis[2-(methylsulfinyl)ethane] (1) and 1-methyl-
sulfinyl-2-[(methylthio)ethylsulfonyl]ethane (2),
which contain one and two sulfoxide groups, respec-
tively. A method for the simultaneous determination of
these metabolites as 1,1'-sulfonylbis[2-(meth-
ylthio)ethane] after reduction with TiCl3 was proposed
[18, 33].
Earlier, Black et al. [33] detected with the use of
GC–MS with positive chemical ionization (reagent
gas, NH3) using the selective detection of the ion
[M+NH4]+, m/z 232; the limit of detection was
2 ng/mL. Black and Read [18] reached a lower limit of
detection (0.1 ng/mL) for the same test substance with
the use of GC–MS–MS with positive chemical ion-
ization and the monitoring of the disintegration
m/z 232 → [MeSCH2CH2]+. The use of titanium(III)
chloride for the reduction of sulfoxide groups made it
possible to propose an exceptionally promising
method for the joint determination of hydrolytic and
β-lyase metabolites in the samples of urine [32]. In the
cited work, the background levels of TDG (TDG +
TDGO) and 1,1'-sulfonylbis[2-(methylthio)ethane]
(β-lyase metabolites) in 105 urine samples were most
representatively examined. In 80% of the samples, the
concentrations of TDG varied from 0.5 to 20 ng/mL
(on the average, 3.43 ng/mL); in this case, the
repeated analysis of samples with concentrations of 3–
19 ng/mL without the stage of reduction using TiCl3
showed that the concentrations of TDG were no
higher than 2.5 ng/mL. This result convincingly dem-
onstrated that the major portion of TDG in the urine
of unexposed people occurred in an oxidized form.
According to the results of the determination of 1,1'-
sulfonylbis[2-(methylthio)ethane], β-lyase metabo-
lites were not detected in the samples of urine from
unexposed volunteers.
Currently, HPLC–MS–MS is considered an opti-
mum method for the separate determination of β-
lyase metabolites in urine. The use of tandem
HPLC–MS–MS instruments made it possible to suc-
cessfully separately determine bis- and monosulfoxide
β-lyase metabolites in the urine of victims [34]. The
limits of detection were 0.1 ng/mL for monosulfoxide
and 0.5 ng/mL for bis-sulfoxide in complex samples at
a signal-to-noise ratio of 3 : 1. These limits of detec-
tion are comparable with that achieved for the product
of the coreduction of these metabolites determined by
GC–MS–MS. In this case, it is important that
HPLC–MS–MS makes it possible to avoid the stages
of oxidation and derivatization, which not only
decreases the time and labor intensity of analysis but
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68
5
also decreases errors. A combined method of analysis
was proposed based on the simultaneous determina-
tion of metabolites as 1,1'-sulfonylbis[2-(meth-
ylthio)ethane] after reduction with titanium(III)
chloride by HPLC–MS–MS with electrospray ion-
ization [35] with the limit of detection of 0.1 ng/mL.
The background concentrations of TDG in urine at
a level of 1–12 ng/mL were also detected in earlier
studies [19, 20]. In 24% of cases, the concentrations of
TDG in the urine of exposed people did not exceed
25 ng/mL. Thus, TDG and TDGO can be considered
as the markers of the action of sulfur mustard on the
human body only upon the detection of high concen-
trations (according to [36], >100 ng/mL). It is consid-
ered that paints, polymers, inks, and photographic
materials can be independent sources of TDG in
human biological fluids.
β-Lyase metabolites are the unambiguous markers
of sulfur mustard exposure, and their detection at any
leve l (to 0.1–0.3 ng/mL) convincingly indicates the
⎯
fact of exposure. At the same time, as mentioned
above, β-lyase metabolites were detected in the body
in a shorter period. Thus, comparable concentrations
of andinβ-lyase
(42TDG (52–80were
57 ng/mL) ng/mL)
detected metabolites
urine sampled after
2–3 days from two persons occasionally exposed to
sulfur mustard from projectiles of the times of the First
World War. At the same time, in a retrospective study
of urine sampled 13 days after exposure, the concen-
tration of β-lyase metabolites was 0.1–5 ng/mL,
although the concentration of TDG still remained suf-
ficiently high (11–72 ng/mL). Thus, for the most
complete characteristic of sulfur mustard exposure
based on the results of urine analysis, the determina-
tion of metabolites formed by both of the metabolic
mechanisms should be performed.
Bis(N-acetylcysteine) conjugate of sulfur mustard
sulfone. Attempts at the GC–MS determination of
1,1'-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] were
unsuccessful because of its thermal instability. The
limit of detection of 25 ng/mL for a dimethyl deriva-
tive was reached using HPLC–MS–MS with thermo-
spray ionization [37]. A much lower limit of detection
(1 ng/mL) was obtained with the use of HPLC–MS–
MS with electrospray ionization for the underivatized
conjugate [38]. In the latter case, the analyte was pre-
concentrated from acidified urine on polymer car-
tridges for solid-phase extraction and detected in the
negative ion detection mode with the monitoring of
multiple reactions. In this work, the only example of
No. 1 2013
6the detection of the bis(N-acetylcysteine) conjugate
ORLOVA et N7-(2-Hydroxyethylthioethyl)guanine.
al. The adduct
of sulfur mustard sulfone in the real samples of urine of guanine can be separated from urine using solid-
was described: it was detected at a level of about 1 phase extraction on C18. The GC–MS technique was
ng/mL in two urine samples from the victims of found unsuitable for analysis because of the fact that
occasional sulfur mustard poisoning.

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68 No. 1 2013

 METHODS FOR THE DETECTION OF SULFUR MUSTARD METABOLITES
the substance could not be derivatized with heptafluo-
robutyric anhydride and pentafluorobenzoyl bromide
and TBDMS derivatives exhibited poor gas-chro-
matographic properties. A sensitive method for deter-
mining underivatized conjugate was proposed with the
use of HPLC–MS–MS with electrospray ionization
and the monitoring of the fragmentation m/z 256 →
[CH2CH2SCH2CH2OH]+, m/z 105. The limit of
detection of this adduct was 0.2 ng/mL upon its intro-
duction into urine [10]. No examples of the detection
of this adduct in real urine samples have been currently
described.
Methods for the Determination of the Covalent Adducts
of Sulfur Mustard
Sulfur mustard adducts with DNA. Sulfur mustard
can alkylate DNA in the N7-position of the deoxygua-
nosine residue with the formation of N7-(2-hydroxy-
ethylthioethyl)-2'-deoxyguanosine. The depurination
of the latter gives N7-(2-hydroxyethylthioethyl)gua-
nine (N7-HETE-Gua) and also the di-adduct of N7-
guanine and the adduct of N3-adenine [40, 41]. Sev-
eral methods for the determination of N7-HETE-Gua
were proposed. As noted above, this adduct cannot be
identified by GC–MS because corresponding deriva-
tives are difficult to obtain. For the analysis of urine, a
method was proposed that includes the separation of
the adduct by solid-phase extraction on C18 and
detection with the aid of HPLC–MS–MS with elec-
trospray ionization [42]. N7-HETE-Gua was discov-
ered by this method in the urine of guinea pigs exposed
to sulfur mustard (the concentration of this adduct
considerably decreased 36–48 h after the exposure)
and also in skin and blood samples. Based on a similar
methodology, N7-HETE-guanine was detected in the
spleen and liver of rats after the cutaneous application
of sulfur mustard [43]. Procedures for the determina-
tion of N7-HETE-Gua by HPLC [44] and a radio-
chromatographic procedure based on the preparation
of N7-HETE-Gua labeled with 32P after the separa-
tion of the fraction of N7-guanine adducts from the
DNA of the thymus of a calf incubated with sulfur
mustard [45].
Immunochemical methods [46] and an ELISA
method were developed for the determination of N7-
HETE-Gua in blood; this latter was successfully used
for detecting this adduct in the samples of blood taken
22 and 26 days after poisoning from two victims of the
Iran–Iraq conflict. The concentrations of the adduct
in the lymphocytes and granulocytes of the victims
corresponded to a level of 0.015–0.43 μM sulfur mus-
tard in blood treated in vitro [47].
Sulfur mustard adducts with hemoglobin. The iden-
tification and the development of methods for the
determination of sulfur mustard adducts with hemo-
globin is given considerable attention. These adducts
are stable. Their lifetime is about 120 days, whereas
adducts with DNA are destroyed for several days. In
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68
7
the case of acute poisonings, they can be detected for
a long time after poisoning, and they are accumulated
in blood under chronic exposure. Furthermore, the
blood contains a large amount of hemoglobin
(140 mg/mL), which can be easily isolated in the form
of globin.
The studies of the distribution of an 32S marker in
rats after the cutaneous application of marked sulfur
mustard and also in human blood treated in vitro with
marked sulfur mustard showed that a part of radioac-
tivity was associated with hemoglobin, and it was
retained for the entire lifetime of red blood cells [48].
More recently, six different histidine residues, three
glutamic acid residues, and both N-terminal valines
were identified by HPLC–MS–MS in the peptides of
hemoglobin alkylated by radioactive sulfur mustard
after trypsinization [49, 50]. Cysteine, aspartic acid,
lysine, and tryptophan alkylated by yperite were iden-
tified after the decomposition of hemoglobin with the
use of pronase. The adducts of histidine at N1 and N3
were detected in the greatest amounts.
Although the fraction of the product of hemoglobin
alkylation at the N-terminal valine is only 1–2%
among all of the hemoglobin alkylation products, this
product is a promising biomarker because it is easily
eliminated from hemoglobin with the use a modified
Edman degradation technique [51]. Globin isolated
from blood by a standard procedure is dissolved in for-
mamide and treated with pentafluorophenyl isothio-
cyanate at 60°C for 2 h. N-alkylated valine is extracted
with diethyl ether, the solvent is removed, and the res-
idue is dissolved in toluene. The resulting pentafluo-
rophenyl thiohydantoine can be determined by GC–
MS–MS after further derivatization with heptafluo-
robutyric anhydride or heptafluorobutyryl imidazole
[52, 53]. The limit of detection in blood in vitro
treated with yperite is 0.1 μM. As found in experi-
ments in vivo performed in monkeys, the adducts of
hemoglobin are relatively long-lived biomarkers of
sulfur mustard poisoning. After a single introduction
of 4.1 mg/kg of sulfur mustard, alkylated N-terminal
valine was detected even 94 days after poisoning [54].
With the use of the above procedure, alkylated
N-terminal valine was detected in blood samples taken
22–26 days after poisoning from the victims of the
Iran–Iraq war [47]. The level of the adduct in blood
corresponded to a dose of 0.9 μM sulfur mustard in in-
vitro tests. The analysis of blood from four victims
sampled 5–10 days after poisoning also gave positive
results [53]. The concentration of the valine adduct
was 0.3–0.8 ng/mL.
The histidine adducts of hemoglobin were detected
in the greatest amounts in in-vitro tests. It was shown
that these adducts remained even upon the hydrolysis
of globin with the use of 6 M HCl. However, it was
impossible to analyze them by GC–MS because of the
high polarity and thermal instability of corresponding
volatile derivatives. A sensitive method for the deter-
mination of these adducts as N-9-fluorenylmethoxy-
No. 1 2013
8 ORLOVA et al.
carbonyl derivatives was developed based on HPLC–
MS–MS with electrospray ionization [55]. However,
note that the procedure of the determination of histi-
dine adducts is labor-intensive and less sensitive than
the procedure of the determination of N-terminal
valine with the use of the modified Edman method.
Sulfur mustard adducts with albumin. Noort et al.
[56] found that sulfur mustard alkylates the cysteine
residue Cys34 of human serum albumin. The alkyla-
tion site was determined by the introduction of
[14C]yperite into the tryptic digest of albumin. Cys34 is
the only free cysteine residue in albumin, and it has a
relatively low value of pKa because of the intermolecu-
lar stabilization of the thiolate anion.
A sensitive method was developed for determining
the cysteine adduct of sulfur mustard with
albumin based on the cleavage of the alkylated albu-
min by the enzyme pronase with the formation of the
tripeptide S-[2-(dihydroxyethyl)thio]ethyl-Cys-Pro-
Phe, which was detected by microcolumn HPLC in
combination with tandem mass spectrometry [56].
With the use of this method, the adduct was detected
in blood in vitro treated with 1 nM sulfur mustard; that
is, the sensitivity of this method is higher by two orders
of magnitude than that in a procedure for the determi-
nation of the N-terminal valine adduct of hemoglobin
according to Edman. The capabilities of this method
were demonstrated by the analysis of blood samples
taken from nine victims of the Iran–Iraq conflict in
1986 eight to nine days after poisoning and stored for
more than 10 years at –70°C. The tripeptide was
detected in all cases. According to the results of the
analysis, the concentrations of the adduct in the sam-
ples corresponded to the levels of the adduct in blood
in vitro treated with sulfur mustard at a level of 0.4–
1.8 μM. The advantage of the methods based on the
determination of albumin adducts is the possibility of
the fast separation of albumin from blood by affine
chromatography. At the same time, albumin adducts
rank below hemoglobin adducts in terms of retrospec-
tive analysis because of a shorter lifetime of albumin
(20–25 days against 120 days for hemoglobin). Thus,
the albumin adduct as a marker of the action of sulfur
mustard can be effectively used only in the first weeks
after exposure.
Adducts with aspartic and glutamic acid residues.
An alternative approach to the determination of the
adducts of sulfur mustard with blood proteins based on
the determination of TDG released from the proteins
by hydrolysis was proposed [57]. Globin and albumin
contain a number of aspartic and glutamic acid resi-
dues at which sulfur mustard can be added [50].
The method is based on the precipitation of blood
proteins and the hydrolysis of esterified aspartic and
glutamic acids with the use of 1 M NaOH (2 h; 70°C).
After neutralization, thiodiglycol was extracted from
the aqueous phase with ethyl or propyl acetate and
determined as a bis(pentafluorobenzoyl) derivative by
JOURNAL OF ANALYTICAL CHEMISTRY
GC–MS. The method exhibited high sensitivity. In
particular, TDG was determined in porcine blood in
vitro treated with 2 mM sulfur mustard and in the
blood of monkeys 45 days after the intravenous intro-
duction of 1 mg/kg sulfur mustard. The background
values of thiodiglycol were determined as 0.5 pg per
1 mg of protein.
An analysis of published data on the identification
and methods for the determination of sulfur mustard
adducts with DNA and proteins showed that the
adducts are long-lived markers suitable for retrospec-
tive analysis. An important circumstance is that,
unlike metabolites such as TDG and TDGO, none of
the adducts was detected in the biological fluids of
human beings unexposed to sulfur mustard. Thus, the
detection of these markers can serve as unambiguous
evidence for the action of sulfur mustard. Unfortu-
nately, the most effective detection techniques are
based on the application of complex and expensive
instrumental methods (gas chromatography and, pre-
dominantly, high-performance liquid chromatogra-
phy in combination with tandem mass spectrometry)
mastered only at large research centers. Standard pro-
cedures for the analysis of the covalent adducts of sul-
fur mustard should be developed. Biochemical meth-
ods of analysis are also used in the studies of the toxi-
cology and the mechanisms of action of sulfur
mustard.
The activity levels of enzymes (alanine ami-
notransferase (ALT), aspartate aminotransferase
(AST), lactate dehydrogenase (LDH), γ-glutamyl
transpeptidase (GGT), and alkaline phosphatase
(ALP)) and also the levels of glucose, total protein,
cholesterol, creatinine, and urea are used as biochem-
ical characteristics for the diagnostics of sulfur mus-
tard poisoning and the evaluation of the status of the
main systems of the body in the analysis of blood
serum. The set of immunological tests includes the
determination of the phagocytic activity of neutro-
phils (PAN), the amount of T lymphocytes (CD3,
CD4, and CD8), the amount of B lymphocytes, and
the level of serum immunoglobulins (IgA, IgG, and
IgM).
It is well known [58–64] that a number of bio-
chemical characteristics change under the action of
yperite. Biochemical studies made it possible to much
better understand the mechanisms of the toxic action
of this agent on the body, and they are highly valuable
for the development of means for the treatment of sul-
fur mustard poisoning. At the same time, variations in
biochemical indices (changes in the levels of NAD+,
glutathione, intracellular calcium, lactate dehydroge-
nase, glutamyl transferase, albumin, leukocytes, and
neutrophils) are not selective indicators, and they can-
not be used for the specific diagnostics of yperite poi-
soning. Note that the monitoring of biochemical char-
acteristics was mainly performed under the conditions
of an acute experiment and at short time intervals after
the action (1–4 h). In this context, taking into account
Vol. 68 No. 1 2013
 METHODS FOR THE DETECTION OF SULFUR MUSTARD METABOLITES
the capability of sulfur mustard to interact and form
adducts with DNA and body proteins, immunochem-
ical methods are of much greater interest.
Among the advantages of the immunochemical
methods of analysis are their high sensitivity and spec-
ificity and also the possibility of developing commer-
cial sets. At the same time, note that the procedures of
sample preparation for analysis are labor-intensive
and time-consuming.
However, the main problem of all immunochemi-
cal methods is the need for obtaining antiserums and
antibodies, which retain activity and specificity for a
long time, for each type of adducts. In publications
[46, 47, 65–67], it was noted that a search for stable
antibodies is still far from completion. Note that the
results of immunochemical analysis should be con-
firmed by spectroscopic and chromatographic–spec-
troscopic techniques, which ensure unambiguous
structural identification.
With consideration for the above circumstances,
chemicoanalytical methods for the detection of the
action of sulfur mustard on the body are currently
preferable.
Generalization of the Experience of the First
International Comparative Test of OPCW
for the Determination of Sulfur Mustard Metabolites
As has noted above, TDG exhibits background
concentration levels in urine and plasma from the
body unexposed to yperite. Furthermore, the source of
its detection in the body can be varnishes, paints, and
other substances contact with which occurs in the nor-
mal mode. Nevertheless, TDG remains the main clas-
sical marker of sulfur mustard. Therefore, the quanti-
tative detection of TDG in urine was a goal in the first
trial interlaboratory test of OPCW performed in
December 2009.
We managed to successfully determine TDG in two
urine samples at concentration levels of 10 and
100 ng/mL in the form of heptafluorobutyryl and pen-
tafluorobenzoyl derivatives by GC–MS with electron
and negative chemical ionization, respectively.
The absolutely selective markers of the action
sulfur mustard on the body are the products of the glu-
tathione/β-lyase metabolic pathway of sulfur mus-
tard—1,1'-sulfonylbis[2-(methylsulfinyl)ethane] and
1-methylsulfinyl-2-[(methylthio)ethylsulfonyl]ethane,
which contain one and two sulfoxide groups, respec-
tively.
Within the framework of the international test,
these compounds were determined by GC–MS after
reduction with titanium(III) chloride and also by
direct HPLC–MS analysis with electrospray ioniza-
tion. Note that two independent chromatographic–
spectroscopic techniques were used for the reliable
identification and more precise quantitative evalua-
tion in the determination of each particular com-
pound.
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 68
9
The analysis of the results of the first international
comparative test for the analysis of urine samples
clearly demonstrated the current status of the problem
of the determination of thiodiglycol and the β-lyase
metabolites of sulfur mustard. In the OPCW test,
22 analytical laboratories took part. Synthetic urine
was used as a biomatrix. Table 1 summarizes data on
the identification of thiodiglycol by the participants of
this test.
Note that only 2 of 13 laboratories that detected
TDG in control samples used HPLC–MS–MS;
moreover, an inadequate signal-to-noise ratio was
obtained in one case, and not TDG but its oxidation
product TDGO was identified in the other case. Good
results were obtained only by high-resolution HPLC–
MS. TDG was detected at good signal-to-noise ratios
using GC–MS–MS (pentafluorobenzoyl derivatives
in the regime of negative chemical ionization and hep-
tafluorobutyryl derivatives in the regime of positive
chemical ionization).
Table 2 summarizes generalized data on the deter-
mination of β-lyase metabolites.
Eighteen laboratories successfully managed the
identification of β-lyase metabolites in the samples of
synthetic urine. The GC–MS–MS technique
requires time-consuming sample preparation includ-
ing the reduction of two derivatives with a hydrochlo-
ric acid solution of titanium(III) chloride to one prod-
uct and its derivatization. According to the generalized
results of the test, HPLC–MS–MS manifested itself
as the most reliable and convenient technique for
determining metabolites of this type, which makes it
possible to considerably simplify sample preparation
and to shorten the duration of analysis.
***
Note that tandem and hybrid analytical techniques
are more widely introduced to reach the minimum
limits of detection of sulfur mustard metabolites with
the lowest labor intensity and time consumption; this
is of particular current interest in the analysis of bio-
medical samples (blood, urine, saliva, and tissue
homogenates). Optimum biomarkers for the unam-
biguous retrospective detection of the action of sulfur
mustard in low concentrations are not determined
until now. The current development of analytical
methods and instrumentation makes it possible to
identify the traces of sulfur mustard biomarkers
(metabolites and adducts with macromolecules).
However, the further development and adaptation of
analytical techniques is required for quantitative
determination (especially, at low exposure levels).
No. 1 2013
1 ORLOVA et al.
0
Table 1. Methods for the determination of thiodiglycol
Determination
Derivatization Ionization Detection mode
method
HPLC–MS/MS None Electrospray in the positive ion mode Multiple reaction monitoring; full
daughter ion scan
High-resolution The same Positive-ion mode electrospray Full scan; selected ion scan
HPLC–MS (1 lab.)*
GC–MS/MS Pentafluorobenzyl Negative chemical ionization (CH4); Multiple reaction monitoring
derivatives electron impact
Heptafluorobutyryl Electron impact; positive chemical ioni- Multiple reaction monitoring; full
derivatives zation (CH4) daughter ion scan
TMS Positive chemical ionization (CH 4) Multiple reaction monitoring
TBDMS Electron impact; positive chemical ioni- Multiple reaction monitoring
zation (CH4)
GC–MS Pentafluorobenzyla- Negative chemical ionization (CH4); Selected ion monitoring; full scan
tion electron impact
Heptafluorobutyryl Electron impact Selected ion monitoring
derivatives
TMS Electron impact Selected ion monitoring; full scan
TBDMS Electron impact; positive chemical ioni- Selected ion monitoring; full scan
zation (CH4)
* Only one of the laboratories that participated in the test used the specified method of analysis.

Table 2. Methods for the determination of the β-lyase metabolites of sulfur mustard
Method of analysis Derivatization Ionization Detection mode
HPLC–MS/MS None Electrospray in the positive ion Multiple reaction monitoring
mode
High-resolution Reduction* The same The same
HPLC–MS (1 lab.)*
High-resolution None The same Full scan; selected ion scan
HPLC–MS (1 lab.)*
HPLC–MS The same The same Selected ion monitoring
GC–MS/MS Reduction Positive chemical ionization (NH 3; Multiple reaction monitoring; full daughter
CH4); electron impact ion scan
GC–MS The same Positive chemical ionization (NH 3; Selected ion monitoring; full scan
iBu); electron impact
GC/GC–MS (time- The same Electron impact Full scan
of-flight detector)
* Only one of the laboratories that participated in the test used the specified method of analysis.

Long-term toxicological studies can contribute to the
development of new potential biomarkers [68].
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