Gas chromatography/mass spectrometry analysis of reaction products of sulfur mustards with phenol

Article abstract of DOI:10.1177/1469066719886424

Screening of chemicals related to chemical weapons convention including their all possible degradation and reaction products in environmental samples is important in the organization for prohibition of chemical weapons verification process. Sulfur mustards, commonly known as blistering agents, are included in schedule 1 chemicals of chemical weapons convention. Because of the presence of chlorine atoms in sulfur mustards, they are highly reactive and prone to react with other organic molecules such as phenols to produce corresponding reaction products. Thus, it is important to screen for not only the sulfur mustards but also their reaction products for verification process. The sulfur mustards and their degradation products have been routinely analyzed by gas chromatography/mass spectrometry method, however, the methods are yet to establish for the reaction products. In this study, the reaction products of the sulfur mustards with phenol (compounds 1–7) were studied by gas chromatography/mass spectrometry under electron ionization and chemical ionization conditions. The EI spectra of 1–7 displayed molecular ion and characteristic fragments that provided structure information. Mostly the fragment ions were due to homolytic cleavages involving C–O, C–S, and C–C cleavages. The methane or isobutane CI spectra showed M^+., [M + H]⁺, and [M ? H]⁺ ions including reagent specific adduct ions. The CI spectra also showed other adduct ions formed by association of analyte molecule with its most abundant fragment ion. The gas chromatography/retention index values were also calculated, which support unambiguous identification of targeted molecules in suspected environmental samples. The method was demonstrated for detection of the targeted molecules spiked in soil samples.

Full text of DOI:10.1177/1469066719886424

Original research paper
European Journal of Mass Spectrometry
0(0) 1–12
Gas chromatography/mass spectrometry
analysis of reaction products of sulfur
mustards with phenol
! The Author(s) 2019
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DOI: 10.1177/1469066719886424
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A Veeresham1, M Sandeep2, T Jagadeshwar Reddy1, A Suresh Pal1,
K Srinivas2 and S Prabhakar1
Abstract
Screening of chemicals related to chemical weapons convention including their all possible degradation and reaction
products in environmental samples is important in the organization for prohibition of chemical weapons verification
process. Sulfur mustards, commonly known as blistering agents, are included in schedule 1 chemicals of chemical weapons
convention. Because of the presence of chlorine atoms in sulfur mustards, they are highly reactive and prone to react with
other organic molecules such as phenols to produce corresponding reaction products. Thus, it is important to screen for not
only the sulfur mustards but also their reaction products for verification process. The sulfur mustards and their degradation
products have been routinely analyzed by gas chromatography/mass spectrometry method, however, the methods are yet to
establish for the reaction products. In this study, the reaction products of the sulfur mustards with phenol (compounds 1–7)
were studied by gas chromatography/mass spectrometry under electron ionization and chemical ionization conditions. The
EI spectra of 1–7 displayed molecular ion and characteristic fragments that provided structure information. Mostly the
fragment ions were due to homolytic cleavages involving C–O, C–S, and C–C cleavages. The methane or isobutane CI spectra
showed Mþ., [M þ H]þ, and [M ꢀ H]þ ions including reagent specific adduct ions. The CI spectra also showed other adduct
ions formed by association of analyte molecule with its most abundant fragment ion. The gas chromatography/retention
index values were also calculated, which support unambiguous identification of targeted molecules in suspected environ-
mental samples. The method was demonstrated for detection of the targeted molecules spiked in soil samples.
Keywords
Sulfur mustards, phenol decontamination products, reaction products, gas chromatography/mass spectrometry, electron
ionization, chemical ionization, retention indices, soil samples, organization for prohibition of chemical weapons
verification
Received 22 July 2019; accepted 12 October 2019
Introduction
The organization for prohibition of chemical weap-
Chemical warfare agents (CWAs) are toxic small
organic compounds comprising of hetero atoms
such as phosphorus, nitrogen, sulfur, fluorine, and
chlorine.1 Based on their lethal and incapacitating
effects on human, the CWAs are classified as choking
agents, nerve agents, blood agents, blister agents, tear
agents, vomiting agents, malodarants, and psycholog-
ical agents. In the past, CWAs were used in World
War I and II, Iran–Iraq war etc. and caused more
than million casualties.2–4 In the year 2013, some of
the CWAs were allegedly used in Syria resulting mas-
sive deaths. Chemical weapons convention (CWC)
was implemented in 1997 for eliminating the weapons
of mass destruction, and it aims to prohibit the
production, development, acquisition, retention,
stockpiling, transfer, or use of chemical weapons.
ons (OPCW), The Hague, The Netherlands is the
implementing body of the CWC. The important
task of the OPCW is the on-site and off-site verifica-
tion processes for CWC-related chemicals in sus-
pected environmental samples. However, most of
the CWC related chemicals, due to their high
1Centre for Mass Spectrometry, Analytical Department, CSIR–Indian
Institute of Chemical Technology, Hyderabad, T.S., India
2Fluoro Agrochemicals Department, CSIR–Indian Institute of Chemical
Technology, Hyderabad, T.S., India
Corresponding author:
S Prabhakar, Centre for Mass Spectrometry, Analytical Department, CSIR–
Indian Institute of Chemical Technology, Hyderabad 500 007, Telangana,
India.
Email: prabhakar@iict.res.in

2
European Journal of Mass Spectrometry 0(0)
reactivity, undergo degradation when they are
exposed to different environmental matrices like
water, soil, and organic matters. Therefore, it is
important for verification process to detect and iden-
tify not only the CWC-related chemicals but also their
degradation and/or reaction products in environmen-
tal matrices.5–7
products/decontamination products). For example,
the sulfur mustards can react with phenols during
decontamination process and form corresponding
phenyl ethers.25,26 Thus, detection of such phenol reac-
tion products of sulfur mustards in environmental sam-
ples becomes significant for the verification process of
the OPCW. To the best our knowledge, no reports are
available in the literature on the analysis of phenolic
reaction products of sulfur mustards. In fact, in the
37th official OPCW PT, bis(2-phenoxyethyl)sulfide
(the phenol reaction product of sulfur mustard) was
spiked in one of the aqueous test sample. The partici-
pating laboratories have to synthesize bis(2-
phenoxyethyl)sulfide for unambiguous identification
because of nonavailability of the analytical data on
such reaction products. Hence, we have aimed at syn-
thesis of the sulfur mustards phenolic reaction products
and development of a GC/MS method for separation
and identification. These analytical methods could be
used in future verification programs as well as PTs.
In this study, we have synthesized the sulfur mustards
reaction products with phenol and characterized by
using GC/MS under electron ionization (EI) and chem-
ical ionization (CI) conditions.
Most of the degradation/reaction products CWAs
are not scheduled chemicals as per CWC; however,
they are relevant for the verification process. Thus,
the OPCW listed probable degradation/reaction
products of CWC-related chemicals as “Additional
non-scheduled reportable chemicals”; this list is
being updated by the OPCW time to time and
annexed to the work instructions of the official
OPCW proficiency tests (PTs) as a quick guide.
Other reportable nonscheduled degradation/reaction
products include those chemicals that are one reac-
tion step from CWC-scheduled chemicals, including
precursor chemicals. The degradation products or
reaction products of CWC-related chemicals could
be used as the marker that provides a hint on the
presence of the original toxic chemical in the sus-
pected sample and its alleged usage. Consequently,
identification of degradation/reaction products of
CWC-related chemicals in suspected samples becomes
crucial for validation. In these lines, we have studied
the degradation and oxidation products of sulfur
mustards, nitrogen mustards, and their related hydro-
lyzed compounds.8–11
Experimental
Materials
Most of the environmental samples consist of dif-
ferent background chemicals or matrix-associated
chemicals that are expected to mask the CWAs and
their reaction/degradation products, especially when
present at low ppm concentrations.12,13 In such cases,
application of suitable methods of sample extraction,
cleanup, and detection technique is crucial, and many
reports are available in literature in these lines. Among
the detection techniques, gas chromatography (GC)
coupled with element-specific detectors with mass spec-
trometry (MS) detector14–18 have been the routine
choice for the screening of CWC-related chemicals
and their degradation/reaction products.
Sulfur mustards are a group of nine chemicals
listed under CWC Schedule 1 chemicals; they also
known as blistering agents that damage the eyes,
skin, internal organs, respiratory system.19–24 Bis
2-chloroethyl sulfide, commonly known as the sulfur
mustard, was widely used during World War II.
Degradation of sulfur mustards in various environ-
mental samples has been well studied. In our earlier
study, GC/MS analysis of the vinylic degradation
products of sulfur mustards was demonstrated.9
However, studies on the reaction products of sulfur
mustards have not been explored. Sulfur mustards
consist of two highly reactive chlorine atoms in
their structure, hence they are prone to react with
other organic molecules with hydroxy functional
group to produce corresponding derivatives (reaction
Thiodiglycol, dithiodiglycol, 2-mercaptoethanol,
ethane-1,2-dithiol, propane-1,3-dithiol, butane-1,4-
dithiol, pentane-1,5-dithiol, (2-bromoethoxy)ben-
zene, cesium carbonate, sodium carbonate, potassi-
um carbonate, phosphorus tribromide, phenol, and
hydrocarbon mixture were procured from Sigma
Aldrich (Steinheim, Germany). The compounds 1–7
were synthesized from appropriate starting materials
with known procedure as summarized in Scheme 1.
The reaction mixture was worked up and passed
through fast column chromatography and the
eluent was analyzed by GC/MS. The product com-
pounds were well separated from starting materials
and/or possible bio-products, if any, under the used
GC conditions. The by-products included the reac-
tion products among the unwanted starting materi-
als, elimination reactions of the final products, mixed
trisulfides, partially S-alkylated products, and/or
intramolecular cyclized products. The purity of
each product was verified from its GC/MS chro-
matogram, and they were found to be 80–90%.
The compounds of better purity (ꢁ90%) were select-
ed for the spiking experiments and the purity of com-
pound is considered for calculating the levels of
spiking. HPLC grade solvents were used for all the
experiments, and they were procured from Sd. Fine
chemicals (Mumbai, India).

Veeresham et al.
3
Scheme 1. Synthetic scheme for the compounds 1–7.
GC/MS interface at 280ꢂC, EI source at 230ꢂC, and
quadrupole analyzer at 150ꢂC. The electron energy
used in EI source was 70 eV. The scan range of the
quadrupole was set from m/z 29 to 600, and the scan
speed was 1.3 scans/s for recording EI mass spectra.
The GC/chemical ionization mass spectrometry
(CIMS) experiments were performed using methane
as the reagent gas using similar experimental condi-
tions as that of GC/electron ionization mass
spectrometry (EIMS), except keeping the electron
energy at 220 eV and scanning the analyzer from
m/z 60 to 600 (at scan speed 1.4 scans/s) for CIMS
analysis.
The EI-high resolution mass spectrometry (HRMS)
data were acquired on a JEOL GC-AccuTOF (Tokyo,
Japan) mass spectrometer interfaced with 7890A gas
chromatograph. The experimental conditions of GC
and MS were identical to that used for GC/EIMS
single quadrupole mass spectrometer. The resolution
of the GC-TOF MS was 8000 full width at half max-
imum (FWHM) (at m/z 219). The internal reference
compound was perflurokerosene and it was introduced
in to the EI source through the septum reservoir.
HRMS data were acquired using Mass Centre System
(version 2.5.1a; M/s Jeol) software. The elemental com-
position and mass errors were estimated using the
Elemental composition estimation software (version
2.3.0.0.; M/s Jeol).
Spiking of soil sample
Soil sample was collected from a local river side and
used as the matrix for spiking experiment, and this
consisted of clean sand with other matter. Soil matrix
(5 g) was spiked with compound 1 and 6 by the addi-
tion of 1 mL of 50 ppm in dichloromethane (50 mg
each). Hydrocarbon mixture was also spiked at
higher concentration (1000 ppm) as the masking
agent. Excess solvent was added and stirred to
homogenize the sample. The solvent was evaporated
by gentle nitrogen stream to get the dry soil matrix,
and final concentration of 1 and 6 in the soil was
10 mg/g (10 ppm). The spiked soil sample was stored
in refrigerator till further analysis.
Extraction from soil sample
The spiked soil sample (1 g) was extracted twice with
1 mL dicholoromethane by hand shaking (5 min) and
sonication for 1 min. The dichloromethane fraction
was immediately separated from soil and centrifuged
for 5 min at 5000 r/min. The supernatant was concen-
trated to 100 mL by nitrogen purge, and 1 mL of the
solution was used for GC/MS analysis.
Sample analysis by GC/MS
All the samples were analyzed by a GC/MS (EI) using
a single quadrupole mass spectrometer, where 6890
gas chromatograph was interfaced with 5973 N
mass-selective detector (Agilent Technologies, Palo
Alto, CA, USA). The samples were introduced into
GC through splitless injection mode and the analytes
were separated in HP-5MS capillary column (5%
phenyl methylpolysiloxane; length–30 m, inner diam-
eter–250 mm, and film thickness–0.25 mm). The initial
GC oven temperature was set at 50ꢂC for 2 min and
heated at the rate of 10ꢂC/min to reach 280ꢂC and
this temperature was held for 15 min. The carrier gas
(helium) flow was set to be 1 mL/min at constant flow
mode. The typical temperatures were: inlet at 250ꢂC,
Results and discussion
The reaction products of sulfur mustards with phenol
(compounds 1–7 shown in Scheme 1) were synthe-
sized from suitable precursors. The products were
subjected for GC/EIMS and GC/CIMS analyses.
The peaks corresponding to the compounds 1–7
were dominant in the total ion chromatograms
obtained from GC/MS analyses and these peaks
were well resolved from the respective starting mate-
rials and/or byproducts in the GC/MS analysis.
Hence, the mass spectra (both EI and CI) obtained

4
European Journal of Mass Spectrometry 0(0)
for compounds 1–7 were found to be pure and free
from the background or interfering peaks. Among the
homologues series 2–7, as expected, there was a grad-
ual increase in GC retention times with the increase of
–CH2– spacer chain length in between the two sulfur
atoms. The GC/EI and CI spectra of 1–7 include
molecular weight information and structure indicative
fragment ions (data rich spectrometric techniques)
that enables detection and identification of the
target compounds; the GC/retention index (RI)
values further supports identification. The mass spec-
tral data and GC/RI data are discussed below.
7 is shown in Figures 1 and 2, respectively, as typical
example (the EI spectra of 2–6 are given in Figures
S1–S5, Supplementary information). The molecular
ions (Mþ•) (4–12%) were appeared in the EI spectra
of all the compounds (1–7), thus provide molecular
weight information straight away. The EI fragmenta-
tion of 1–2 was different when compared to that of 3–
7 due to difference in their structures (with or without
spacer methylenes in between sulfur atoms). It was
easy to identify the number of carbons and sulfur
atoms in molecular ion and the fragment ions based
on the þ1 and þ2 isotopic peak abundances.
Most of the structure indicative fragment ions in the
compounds 1–7 are due to homolytic cleavages.27All
the possible fragments due to homolytic cleavages are
summarized in Scheme 2. The cleavages indicated as k,
l, m, and n (Scheme 2) are expected to be common in
GC/EIMS of compounds 1–7
The EI mass spectral data of the compounds 1–7 are
summarized in Table 1, and the EI spectrum of 1 and
Table 1. EI mass spectra of reaction products of sulfur mustards with phenol (1–7).
Ion (m/z), Relative abundance (%)
Comp.
No.
Mþ.
a
b
b0
b0-H
c
d
d0
e
–
e0
–
Other ions
1
274 (4) 77 (40) 93 (11) 181 (100) –
107 (6) 121 (18) 153 (60)
39(9), 45(5), 47(1), 51(7), 53(2), 59
(7), 60(4), 61(1), 65(22), 79(2),
87(8), 91(10), 94(8), 97(7), 103
(4), 119(3), 120(2), 125(62), 137
(1), 165(1)
2
3
306 (12) 77 (31) 93 (7) 213 (8)
320 (4) 77 (23) 93 (5) 227 (45)
–
–
107 (14) 121 (3) 185 (2) 153 (100) –
39(8), 45(4), 47(1), 51(5), 53(2), 61
(2), 65(20), 79(4), 91(10), 94
(11), 97(8), 103(1), 119(3), 120
(4), 125(44), 139(1), 157(22)
107 (100) 121 (3) 199 (8) 153 (4) 167 (27) 94(8), 39(4), 41(1), 45(4), 47(3), 51
(4), 53(2), 59(2), 60(2), 61(5), 65
(10), 73(3), 79(6), 91(5), 97(2),
103(1), 119(2), 120(2), 125(9),
139(4)
4
5
334 (5) 77 (45) 93 (13) 241 (52) 240 (8) 107 (15) 121 (24) 213 (56) 153 (68) 181 (100) 39(3), 41(1), 45(3), 47(1), 51(3), 53
(1), 59(5), 60(4), 61(3), 65(18),
79(2), 87(12), 91(12), 94(12), 97
(5), 103(2), 119(5), 120(2), 125
(61), 137(1), 147(5), 157(10)
348 (12) 77 (28) 93 (5) 255 (2) 254 (5) 107 (22) 121 (40) 227 (100) 153 (2) 195 (2) 39(5), 41(6), 45(4), 47(1), 51(4), 53
(1), 59(4), 60(2), 61(4), 65(8), 73
(7), 79(2), 81(6), 87(2), 91(4), 94
(5), 97(1), 101(3), 103(2), 113
(2),119(1), 120(2), 125(3), 133
(8), 135(3), 157(2)
362 (6) 77 (31) 93 (7) 269 (2) 268 (5) 107 (7) 121 (42) 241 (100) 153 (2) 209 (36) 39(5), 41(1), 45(6), 47(2), 51(3), 53
6
7
(2), 55(12), 59(3), 60(8), 61(4),
65(12), 73(4), 79(2), 87(14), 88
(24), 91(5), 94(5), 97(3), 103(4),
115(6), 119(3), 120(1), 125(5),
135(2), 147(6)
376 (4) 77 (28) 93 (10) 283 (24) 282 (13) 107 (11) 121 (40) 255 (100) 153 (11) 223 (64) 39(6), 41(11), 45(4), 47(2), 51(4),
53(2), 59(5), 60(4), 61(9), 65
(12), 69(13), 79(2), 87(9), 91(8),
94(6), 95(22), 97(2), 101(46),
103(10), 107(10), 119(1), 120(1),
125(12), 129(9), 135(2), 161(5),
181(1), 189(8)

Veeresham et al.
5
Figure 1. EI mass spectrum of compound 1.
Figure 2. EI mass spectrum of compound 7.
Scheme 2. The possible homolytic cleavages and the expected fragment ions during the EI fragmentation of compounds 1–7.
all the compounds 1–7, and such each cleavage is
expected to yield two different fragment ions, i.e., a,
a0; b, b0; c, c0, and d, d0 fragment ions, respectively. As
the extra S and methylene spacers are introduced in the
structure, other new homolytic cleavages are possible
(referred as o, p, and q cleavages in Scheme 2), which
can result in the formation of the fragment ions e, e0; f,
f0, and g, g0. However, the expected fragment ions due
to a0, c0, f, f0, g, and g0 were absent in the respective EI
spectra of 1–7. Apart from the homolytic cleavages,

6
European Journal of Mass Spectrometry 0(0)
Scheme 3. General EI fragmentation of the compounds 1–7.
Scheme 4. The retro Diels–Alder reaction products from the fragment ions of m/z 153 and 167.
the spectra also included other fragment ions due to
proton transfer during homolytic cleavages and also
due to retro-Diels–Alder (RDA) reactions from the
fragment ions. The general EI fragmentation of 1–7
is summarized in Scheme 3 (Some of the fragment
ions, to some extent, might be formed by the further
fragmentation of major fragment ions, apart from
expected simple cleavages). The GC/HRMS data fur-
ther supported the elemental composition of the
resulted fragment ions (Table S1; Supplementary infor-
mation). In addition to the ion at m/z 93 (ion b), the
m/z 94 due to proton transfer to the phenoxy group
during the l-cleavage (Scheme 2) was also observed in
1–7. The fragment ions at m/z 65 and 39 resulted from
further fragmentation of the ion m/z 91 were consis-
tently found in the spectra of 1–7.
The mass spectrum of compound 1 displayed the
homolytic cleavagþe fragment ion m/z 181 correspond-
ing to [M-C6H5O] (b0 ion) as the base peak. The frag-
ment ions resulted by other homolytic cleavages (m/z
77, 93, 107, 121, 153, and 197) also appeared in the
spectrum. The ion m/z 153 (ion d0) upon further loss
of C2H4 through RDA reaction resulted the fragment
ion m/z 125 (Scheme 4). The mass spectrum of 1 well
matched with the spectrum of bis(2-phenoxyethyl)
sulfide available in the OCAD library. The other frag-
ments ions appeared in low mass region at m/z 97, 91,
87, 59, 51, and 45 corresponding to [C5H5S]þ,
[C7H7]þ, [C4H7S]þ, [C2H3S]þ, [C4H3]þ, and [HCS]þ,
respectively, are resulted by further fragmentation of
the high mass fragment ions through expected clea-
vages with or without rearrangements. The EI mass

Veeresham et al.
7
spectrum of 2, similar to that of 1, showed the homo-
lytic cleavages fragment ions m/z 77, 93, 107, 121,
185, 213, 153, and 229. The fragment ion m/z 153
due to S–S cleavage (ion e) was found to be the
base peak in the mass spectrum of 2, and the next
abundant ion at m/z 125 was due to subsequent loss
of C2H4 through RDA reaction. The other abundant
ion was appeared at m/z 157, and its HRMS data
suggested possible elemental composition as
C6H5OS2 and þ2 isotope peak confirmed presence
of two sulfur atoms. The ion m/z 157 might corre-
spond to the loss of C2H4 from m/z 185. The low
mass fragment ions in the spectrum of 2 were found
to be similar to those found in 1.
94 Da from d0 ions was absent in the compounds 1–3,
which gives a clue that the [d0-94] fragment ions attain
stable structures in 4–7 due to the spacer –CH2–
chain. Apart from these, the compound 4 showed
m/z 214 (d0þH ion) that further fragment by the con-
sequent loss of 29 and 28 Da to results in the ions m/z
185 and 157, respectively. In the compound 6, the ion
at m/z 147 further fragmented by the loss of S, C2H3S,
and C2H4S to form ions m/z 115, 88, and 87, respec-
tively. The compound 7 showed the loss of 94 Da
from m/z 283 (b0 ion) resulting the ion at m/z 189 as
supported by HRMS. In addition, the ion m/z 161 of
compound 7 further fragmented to result in m/z 129,
102, and 101 by the loss of S, C2H3S, and C2H4S,
respectively.
The compounds 3–7, have –(CH2)n– spacer chain
(n ¼ 1–5) between two sulfur atoms, opened up a few
other fragmentation pathways. The base peak in the
spectrum of compound 3 was ion c (m/z 107), while
ion d0 and ion e0 (due to C–S cleavages was the base
peak or abundant ion in 4–7. The other fragmenta-
tions in 3–7 varied depending on the –CH2– spacer
chain length between two sulfur atoms.28–30 In com-
pound 3, the fragment ions of homolytic cleavage
were appeared at m/z 77, 93, 107, 121, 153, 167,
199, and 227. There was one structure specific frag-
ment ion at m/z 139 appeared in the spectrum of 3.
The m/z 139 could be resulted by the loss of C2H4
from the ion e0 (m/z 167) through an RDA reaction as
depicted in Scheme 4; this fact was confirmed by ele-
mental composition of m/z 139 and abundances of
isotope peaks.
The anticipated homolytic cleavage b0 ions
appeared in the spectra of compounds 4, 5, 6, and 7
at m/z 241, 255, 269, and 283, respectively; besides
these, the spectra also included [b0-Hþ]þ ions (m/z
240, 254, 268, and 282). The [b0-H] ions were
absent in 1–3, which suggests that a minimum of
two spacer –CH2– groups are necessary to form [b0-
H]þ ion. The possible formation of the [b0-H]þ ion
is depicted in Scheme 5, where initial b0 ion attains
a stable cyclic dithia structure that after loss of a
proton leads to [b0-H]þ ion that gets stabilized
through resonance (1,3-H shift).
GC/CIMS of compounds 1–7
GC/CIMS is a complementary technique to GC/
EIMS, and is useful to confirm the molecular
weight of a compound; especially when EI spectra
fail to exhibit molecular ions.31 The GC/MS tech-
nique has been routinely used as a secondary analyt-
ical technique during official OPCW PTs to support
the identification by the GC/EIMS. The molecular
weight confirmation by CIMS is required even
though the EIMS data show molecular ions. In this
study, the EI mass spectra of compounds 1–7 showed
molecular ions. However, it is important to confirm
the molecular weight by GC/CIMS; hence, all the
compounds were subjected for GC/CIMS analysis
(positive ion mode) using methane and isobutane as
the CI reagent gas.
The CI (methane) spectra of 1–7 showed Mþ,
[M þ H]þ, and [M ꢀ H]þ ions at low abundance,
ꢃ8% (Table 2), out of which mostly Mþ is dominat-
ing over the other two ions. The CI (methane) spectra
of 1 and 4 are shown in Figures 3 and 4, respectively,
as examples. Formation of [M þ H]þ, in addition to
Mþ and [M ꢀ H]þ is expected for a few nonpolar or
aromatic compounds, especially when methane was
used as the reagent gas. Proton transfer to the analyte
yielding [M þ H]þ ion is the common process in CI,
however, in some cases hydride abstraction by
reagent forms [M ꢀ H]þ ions. The Mþ ions are
formed by the charge transfer reactions, where
the ionic charge from reagent ion is transferred to
the neutral analyte. In general, it is difficult to recog-
nize the Mþ or [M ꢀ H]þ ions, especially in the case of
The compounds 4–7 also showed other expected
homolytic cleavage ions, viz. d0 ions (m/z 213, 227,
241, and 255). The fragment ions due to further loss
C6H5OH (94 Da) from these d0 ions also appeared at
m/z 119, 133, 147, and 161, respectively. Such loss of
Scheme 5. Possible pathway for the formation of the [b0-H]þ ion from b0 ion.

8
European Journal of Mass Spectrometry 0(0)

Veeresham et al.
9
Figure 3. Methane/CI mass spectrum of compound 1.
Figure 4. Methane/CI mass spectrum of compound 4.
unknown analytes. In such cases, the adduct ions
[M þ 29]þ and [M þ 41]þ or any other selective
adduct ion in high mass region would be crucial in
confirming the molecular weight. Indeed, the adduct
ions due to [M þ C2H5]þ, [M þ C3H5]þ, or [M þ bp]þ
(where bp ¼ base peak fragment ion), e.g., [M þ 181]þ
(m/z 455) in 1, were also appeared in the spectra
of 1–7 (Table 2). Even then, the CI (methane) spectra
of 1–7 were dominated by fragment ions, and most of
major ones were same as those found in their EI
spectra.
We further performed GC/MS analysis using
isobutane as the reagent gas to understand the ion-
ization behavior of the compounds under study
with isobutane/CI plasma. The CI (isobutane)
spectra of the compounds 1–7 showed [M þ H]þ
ions in addition to the Mþ and [M ꢀ H]þ ions, how-
ever, abundance of the [M þ H]þ ions was higher
than other two, except 7 (Table 2). The isobutane/
CI mass spectrum of 7 is shown in Figure 5 as an
example. The spectra also included the expected
[M þ 39]þ, [M þ 41]þ, and [M þ bp]þ in the high
mass region after molecular ion peaks. Like meth-
ane/CI, the isobutane/CI spectra also showed dom-
inant fragment ions similar to those found in EI
mode. Thus, the isobutane/CI mass spectral data
were better than the methane/CI in providing the
molecular weight and structure information. The
[M þ bp]þ ions for the compounds 6 and 7,
though they might exist, could not be detected
because the spectra were recorded up to m/z
600 (the m/z values of the expected adducts ions
are m/z > 660).
For the official OPCW PTs, the criteria to
accept the CI data includes that the intensity of
quasi-molecular ion viz., [M þ H]þ, [M ꢀ H]þ,
[M þ alkali]þ ion etc. must be above 10%. The cur-
rent CI data (both methane and isobutane) for the

10
European Journal of Mass Spectrometry 0(0)
Figure 5. Isobutane/CI mass spectrum of compound 7.
Table 3. Gas chromatography (GC)–RI values for the compounds 1–7 along with the RI values of the parent sulfur mustards from
organization for prohibition of chemical weapons (OPCW) libraries.
Compound
RI value ꢄSD (n ¼ 4)
Sulfur mustard
RI value
Bis(2-phenoxyethyl)sulfide (1)
Bis(2-phenoxyethyl)disulfide (2)
2340 ꢄ 0.30
2572 ꢄ 0.31
2747 ꢄ 0.15
2872 ꢄ 0.09
2988 ꢄ 0.30
3103 ꢄ 0.10
3217 ꢄ 0.25
Bis(2-chloroethyl) sulfide
Bis(2-chloroethyl) disulfidea
1178b
1401c
1569b
1703b
1811b
1933b
2045b
Bis(2-phenoxyethylthio)methane (3)
1,2-Bis(2-phenoxyethylthio)ethane (4)
1,3-Bis(2-phenoxyethylthio)-n-propane (5)
1,4-Bis(2-phenoxyethylthio)-n-butane (6)
5-Bis(2-phenoxyethylthio)-n-pentane (7)
Bis(2-chloroethylthio)methane
1,2-Bis(2-chloroethylthio)ethane
1,3-Bis(2-chloroethylthio)-n-propane
1,4-Bis(2-chloroethylthio)-n-butane
1,5-Bis(2-chloroethylthio)-n-pentane
aBis(2-chloroethyl) disulfide does not belong to the sulfur mustard group, it is a degradation product of bis(2-chloroethyl) sulfide.
bOPCW Central Analytical Database (OCAD) (v17) Library.
cValidation Group Working Database (VGWD) (2015) Library.
compounds 2 and 6 do not meet the OPCW criteria.
It is well known in the CI analysis that the quasi-
molecular ions and their relative abundances are
expected to vary with the reagent gas pressure, CI
source conditions, and the quantity of analyte. If
these parameters are optimized and the scan range
of the mass spectrometer is set to cover all the possi-
ble adduct ions, it might be possible to meet the
OPCW criteria. Alternatively, provision of the spec-
trum from the reference compound obtained from
identical experimental conditions is sufficient to
meet the criteria.
mustard. The RI values are crucial in official PTs to
support and identify the CWC-related chemicals in
suspected samples.
Analysis of soil sample
As per the standard operating procedures, the non-
polar and neutral CWC-related chemicals in a soil
sample are extracted with dichloromethane solvent.
Thus, the soil sample spiked with 10 ppm of com-
pound 1 and 6 was extracted with dichloromethane
and the extract was subjected for GC/MS analysis.
The total ion chromatogram obtained from the
GC/MS (EI) analysis of the dichloromethane extract
of soil sample was shown in Figure S6
(Supplementary Information). Though the total ion
current (TIC) is dominated by the background hydro-
carbon peaks, the peaks were clearly seen extracted
ion chromatogram. The spiked compounds were
easily identified by searching their EI spectra against
in-house library that include the reaction products of
all sulfur mustards with phenol.
GC/RI values
Vanden Dool’s formula was applied to calculate the
GC/RI values of the compounds 1–7 by using
n-alkane hydrocarbons (C23–C32) as the internal
standards.32,33 The RI values of 1–7 calculated from
triplicate experiments are summarized in Table 3.
The RI values of parent sulfur mustards from
OCAD database are also included in Table 3 for com-
parison purpose. The RI values of 1–7 were found
to be always higher than the corresponding parent

Veeresham et al.
11
ORCID iD
Conclusions
S Prabhakar
https://orcid.org/0000-0003-0922-6762
In concurrence with CWC verification (on-site/
off-site), it is imperative for the unambiguous identi-
fication of CWC-scheduled chemicals and their
respective reaction/degradation products in varied
sample matrices. By virtue of high reactivity, CWC-
scheduled chemicals can yield a number of reaction/
degradation products when exposed to diverse chem-
ical and environmental conditions. Nevertheless, lim-
ited analytical inputs are available with respect to
reaction/degradation products of CWC-scheduled
chemicals. The current study encompasses mass spec-
tral analysis of the reaction products sulfur mustards
with phenol (1–7) using GC/EIMS and GC/CIMS
techniques. The EI mass spectra provided molecular
weight as well as structure informative fragment ions
to enable structural characterization. EI fragmenta-
tion mainly attributed to homolytic cleavages accom-
panied with or without migration of hydrogen. The
fragment ions due to homolytic cleavages of all types
of C–O, C–S bonds were appeared in the spectra.
Whereas, the ions due to C–C cleavages in the
spacer alkyl chain between two sulfur atoms were
not observed. The EI-HRMS data supported the pro-
posed fragmentation. The CI mass spectra of com-
pounds 1–7 recorded using methane or isobutane as
the reagent gas showed Mþ, [M þ H]þ, and [M ꢀ H]þ
ions to confirm the molecular weight. In addition, the
CI spectra displayed expected reagent specific adduct
ions and the adduct ions due to the addition of base
peak fragment ion to the neutral analyte, [M þ bp]þ.
The calculated GC/RI values of the compounds 1 to 7
could be used as supporting data for the unequivocal
identification of target compounds in suspected sam-
ples. The sample extraction and detection of the stud-
ied phenol reaction products of sulfur mustards in
environmental samples was successfully demonstrated
by spiking experiments in soil sample.
Supplemental material
Supplemental material for this article is available online.
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