Mass spectral characterization of the CWC-related isomeric dialkyl alkylphosphonothiolates/alkylphosphonothionates under gas chromatography/mass spectrometry conditions

Article abstract of DOI:10.1002/rcm.6596

RATIONALE The isomeric dialkyl alkylphosphonothiolates and dialkyl alkylphosphonothionates are listed as scheduled chemicals of the Chemical Weapons Convention (CWC) implemented by the OPCW. The P-S and P-R bond connectivity has to be correctly identified for the verification of the CWC. The present study demonstrates successful identification of the target isomers by selective fragmentation under electron ionization (EI) or chemical ionization (CI) conditions. METHODS All the studied isomeric compounds (27 in total) were synthesized in our laboratory using established methods, then analyzed by EI and CI gas chromatography/mass spectrometry (GC/MS) using an Agilent 6890 gas chromatograph equipped with a HP-5MS capillary column and interfaced to a 5973 N mass-selective detector. The retention index (RI) values of all the compounds were calculated using Van den Dool's formula. GC/MS/MS and GC/HRMS experiments were also performed using a VG-Autospec (magnetic sector) and JEOL-AccuToF (time-of-flight) mass spectrometer, respectively. RESULTS The EI mass spectra of all the compounds had an abundant molecular ion at m/z 182, except in the case of a few selected butyl-substituted compounds, where this ion was of low abundance. The EI fragmentation pathways include α-cleavage, McLafferty rearrangement, McLafferty + 1 rearrangement, O/S-alkyl radical loss, and an alkene loss with a hydrogen shift. The characteristic fragment ions and their relative abundances are significant in elucidating the alkyl group attached to the P/S/O-atoms as well as the P-S/P = S bond connectivity. The EI and CI mass spectra together with RI values enable unambiguous identification of all the studied isomeric compounds. CONCLUSIONS The present study highlights the structural characterization of the isomeric phosphonothiolates and phosphonothionates based on their selective EI fragmentation. The assigned fragmentation pathway helps in the assignment of P-S and P-alkyl connectivity in phosphonothiolates and phosphonothionates, consequently the structure of the unknown compounds. The EI mass spectra (27 compounds) of isomeric compounds are immensely useful in the OPCW official proficiency tests and for off-site analysis. Copyright 2013 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/rcm.6596

Research Article
Received: 21 February 2013
Revised: 8 April 2013
Accepted: 9 April 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 1461–1472
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6596
Mass spectral characterization of the CWC-related isomeric
dialkyl alkylphosphonothiolates/alkylphosphonothionates
under gas chromatography/mass spectrometry conditions
1
1
1
1
1
*
R. Karthikraj , L. Sridhar , S. Prabhakar , N. Prasada Raju , M. R. V. S. Murty
1,2**
and M. Vairamani
1National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad 500 007, A.P., India
2School of Bio-Engineering, SRM University, Chennai 603 203, T.N., India
RATIONALE: The isomeric dialkyl alkylphosphonothiolates and dialkyl alkylphosphonothionates are listed as
scheduled chemicals of the Chemical Weapons Convention (CWC) implemented by the OPCW. The P–S and P–R bond
connectivity has to be correctly identified for the verification of the CWC. The present study demonstrates successful
identification of the target isomers by selective fragmentation under electron ionization (EI) or chemical ionization
(CI) conditions.
METHODS: All the studied isomeric compounds (27 in total) were synthesized in our laboratory using established
methods, then analyzed by EI and CI gas chromatography/mass spectrometry (GC/MS) using an Agilent 6890 gas
chromatograph equipped with a HP-5MS capillary column and interfaced to a 5973 N mass-selective detector. The
retention index (RI) values of all the compounds were calculated using Van den Dool’s formula. GC/MS/MS and
GC/HRMS experiments were also performed using a VG-Autospec (magnetic sector) and JEOL-AccuToF (time-of-flight)
mass spectrometer, respectively.
RESULTS: The EI mass spectra of all the compounds had an abundant molecular ion at m/z 182, except in the case of a
few selected butyl-substituted compounds, where this ion was of low abundance. The EI fragmentation pathways
include a-cleavage, McLafferty rearrangement, McLafferty + 1 rearrangement, O/S-alkyl radical loss, and an alkene loss
with a hydrogen shift. The characteristic fragment ions and their relative abundances are significant in elucidating the
alkyl group attached to the P/S/O-atoms as well as the P–S/P = S bond connectivity. The EI and CI mass spectra together
with RI values enable unambiguous identification of all the studied isomeric compounds.
CONCLUSIONS: The present study highlights the structural characterization of the isomeric phosphonothiolates and
phosphonothionates based on their selective EI fragmentation. The assigned fragmentation pathway helps in the
assignment of P–S and P–alkyl connectivity in phosphonothiolates and phosphonothionates, consequently the structure
of the unknown compounds. The EI mass spectra (27 compounds) of isomeric compounds are immensely useful in the
OPCW official proficiency tests and for off-site analysis. Copyright © 2013 John Wiley & Sons, Ltd.
Chemical warfare agents (CWAs) include nerve agents,
vesicants, and choking compounds, which can lead to mass
destruction of humans, animals, and plants in a short span
of time and contaminate the environment.[1,2] Over the past
two decades, a great deal of attention has been focused on
the development of analytical techniques for the rapid
identification and analysis of CWAs, their precursors,
degradation products and reaction products. Most
CWAs are volatile and relatively non-polar and, hence, gas
chromatography (GC) coupled with various selective and
sensitive detectors such as flame ionization detector (FID),
nitrogen phosphorus detector (NPD), flame photometric
detector (FPD), Fourier transform infrared (FTIR)
spectroscopy, and mass spectrometry (MS) is widely used
for the analysis of non-polar CWAs.[3–8] Of these, the MS
detector has been extensively used because of its high
sensitivity and versatility. Compounds of polar nature can
be analyzed directly by liquid chromatography (LC)/MS or
by GC/MS after derivatization.[9–15] GC/MS analysis is
typically performed under electron ionization (EI) or
chemical ionization (CI) conditions.[16–20] EI is more
routinely used than CI, because it provides extensive
* Correspondence to: S. Prabhakar, National Centre for Mass
Spectrometry, Indian Institute of Chemical Technology,
Hyderabad 500007, A.P., India.
details about
a compound through its characteristic
fragmentations. In addition, EI mass spectra of many
CWAs and their degradation products are available in
the OPCW Central Analytical Database[21] and in other
commercial library databases, thus helping in the
identification of target compounds.
E-mail: prabhakar@iict.res.in
**Correspondence to: M. Vairamani, School of Bio-Engineering,
SRM University, Chennai 603203, T.N., India.
E-mail: mvairamani@hotmail.com; dean.biotech@ktr.srmuniv.ac.in
Rapid Commun. Mass Spectrom. 2013, 27, 1461–1472
Copyright © 2013 John Wiley & Sons, Ltd.

R. Karthikraj et al.
The Chemical Weapons Convention (CWC) is an
international treaty implemented in 1997, banning the
production, stockpiling, and use of chemical weapons.[22,23]
A part of the CWC involves on-site analysis (by the OPCW
inspectors) or off-site analysis (by OPCW-designated
laboratories) of CWAs and their degradation products in
suspected environmental matrices. Since the verification
process is an internationally sensitive issue, the analytical
performance of the participating laboratories (the designated
laboratories and the laboratories seeking designation) is
periodically evaluated by the OPCW by conducting
proficiency tests (PTs) twice a year.[24] The tests involve
unambiguous identification of spiked scheduled chemicals
and/or their degradation/reaction products in different
environmental matrices.
EXPERIMENTAL
Materials
All the compounds (1–27) were synthesized in-house
according to a reported procedure (Scheme 1).[33,34] Briefly,
the alkylphosphonic dichloride (0.02 mmol) was treated
with the corresponding alkyl alcohol/thiol (0.02 mmol) in
the presence of triethylamine (0.025 mmol in dry
tetrahydrofuran) at room temperature (25 ꢀC) for 30 min.
The residue was extracted with ethyl acetate and the extracts
were directly analyzed by GC/MS. The O,O’-diethyl
methylphosphonothionate was procured from Sigma-Aldrich
(Steinheim, Germany) and other chemicals and solvents
used for the synthesis were purchased from Sd-Fine
Chemicals (Mumbai, India). Analytical grade solvents for
chromatographic and mass spectrometric analysis were
obtained from E-Merck (Mumbai, India).
Nerve agents constitute one of the most important groups
of the CWAs and they are categorized as some of the most
toxic species; the alleged use of these nerve agents in the
Iran-Iraq conflict and the Tokyo subway incident has been
well documented.[25,26] Most of the precursor/degradation
products of nerve agents such as phosphonofluoridates,
phosphonic acids, cyclic and acylic phosphonates have
been studied extensively for their unambiguous
identification.[27–29] Dialkyl alkylphosphonothiolates and the
corresponding phosphonothionates (schedule 2.B.4), which
are precursors of the nerve agents and/or are by-products
during the synthesis of the nerve agents,[30] are less toxic
and are stable in normal environmental samples. Thus, they
are often used as the spiking chemicals in the samples of the
official PTs conducted by the OPCW. During the PT, the
participating laboratories must identify the P-alkyl groups
and establish the correct connectivity between P and S
(phosphonothiolates and phosphonothionates). The O/S-alkyl
groups may be reported using the generic name of the alkyl,
without identification of the exact structure. The scheduled
list shows that thousands of phosphonothiolates and
phosphonothionates are possible under schedule 2.B.4. A few
reports have been published on the isomeric differentiation of
dialkylphosphonothiolates, but not of phosphonothionates.[31]
Moreover, to the best of our knowledge, there is no detailed
study emphasizing the differentiation of phosphonothiolates
and phosphonothionates based on their EI fragmentation
patterns. This prompted us to synthesize a series of isomeric
O,S-dialkyl alkylphosphonothiolates and O,O’-dialkyl
alkylphosphonothionates and investigate their EI/CI mass
spectra. As a first approach, we synthesized 27 isomeric
compounds fixing the molecular formula as C6H15O2PS
(molecular weight 182), where the alkyl group attached
to P is C1–C3, and that attached to O or S is C1–C4; and
the alkyl groups, i.e., methyl, ethyl and propyl, are
interchanged among the three heteroatoms (O, P and S)
whereas the butyl group is interchanged between the
oxygen and sulfur atoms. A few phosphonothionates are
known to undergo thiono-thiolo rearrangement under EI
and electrospray ionization (ESI) conditions[32] and this may
pose a difficulty in differentiation of phosphonothionates
from their rearranged products, phosphonothiolates.
Thus, we have also focused on the impact of this
rearrangement in the selected set of compounds. Herein,
we report the characterization of the isomeric
phosphonothiolates and phosphonothionates using GC/EIMS
and GC/CIMS.
Mass spectral analysis
The GC/EIMS analyses were performed using an Agilent
6890 gas chromatograph (Agilent Technologies, Palo Alto,
CA, USA) equipped a HP-5MS capillary column (length
30 m, 250 mm i.d. and 0.25 mm film thickness) and interfaced
to a 5973 N mass-selective detector (a single quadrupole
instrumeꢀnt). The column oven was programmed initially
from 50 C with a 2 min hold time to the final temperature
of 280 ꢀC with 10 ꢀC/min ramp. The final temperature
hold time was 5 min (total run: 30 min). Helium was used
as the carrier gas in constant flow mode at a flow rate of
1.0 mL/min. The inlet and GC/MS interface temperatures
were 250 ꢀC and 280 ꢀC, respectively. The samples were
introduced in split injection mode at a split ratio of 10:1.
The temperatures of the EI source and quadrupole analyzer
were 230 ꢀC and 150 ꢀC, respectively. Similar experimental
conditions were used for the GC/CIMS experiments using
methane as the reagent gas. The mass spectrometer was
scanned from m/z 29 to 600 and from m/z 60 to 600 for EI and
CI, respectively. All the spectra were recorded under identical
conditions. The RI values were calculated for all the isomeric
compounds by using Van den Dool’s formula[5] with respect
to standard n-alkane hydrocarbons (Supplementary Table S1,
Supporting Information).
Collision-induced dissociation (CID) spectra were recorded
using a VG Autospec M triple-sector (EBE geometry) mass
spectrometer (Manchester, UK) equipped with
Masslynx data system, using a dedicated EI source. The
source conditions for electron ionization were: accelerating
a VG
Scheme 1. Synthetic routes of (i) dialkyl alkylphosphonothiolates
and (ii) dialkyl alkylphosphonothionates.
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MS of dialkyl alkylphosphonothiolates/alkylphosphonothionates
voltage 7 kV, electron energy 70 eV, trap current 200 A and
source temperature 250 ꢀC. Spectra were acquired using a
scan time of 1 s/decade and 0.3 s inter-scan delay. All the
samples (1–27) were introduced through a HP 5890 Series II
gas chromatograph using similar conditions to those
described above. The O,O’-diethyl methylphosphonothionate
was introduced into the EI source through a direct insertion
probe. Tandem mass spectrometry (MS/MS) experiments
were performed using linked scan techniques, where the
first electric sector (E) and magnet sector (B) were
scanned simultaneously so as to preserve a predetermined
relationship between scan parameters to produce product
ion and precursor ion spectra. CID product ion spectra were
recorded at constant B/E under computer control, with air
as the collision gas admitted into the first field-free region
so that the main beam transmission was reduced to 20–30%
of its original intensity. Precursor ion spectra were obtained
at constant B2/E.
GC/EIMS analysis
The EI mass spectra of the studied phosphonothiolates (1–18)
and phosphonothionates (19–27) are summarized in
Supplementary Tables S2 and S3 (see Supporting
Information), respectively. Some typical spectra are shown
in Figs. 1, 2 and 5. All the compounds show an abundant
+
•
molecular ion (M , a) at m/z 182 (13–78%), except for dialkyl
alkylphosphonothionates bearing a butyl group (21–23, 0.3–2%).
The isotopic patterns of M+ ions matched well with the
•
simulated isotopic pattern for the formula, C6H15O2PS. EI
mass spectra of nine of the 27 compounds were recently
added in the OPCW Central Analytical Database (OCAD)
(v14); however, there was no investigation of their mass
spectral fragmentation.
General EI fragmentation
High-resolution (HR) mass spectra were recorded using
JEOL GC-AccuTOF (Tokyo, Japan), a time-of-flight
The general EI fragmentation patterns of the
phosphonothiolates and phosphonothionates, based on
HRMS and linked scan (MS/MS) data, are given in Schemes 3
and 4, respectively. The HRMS data for the significant ions of
the compound 2 is presented in Supplementary Table S4 (see
Supporting Information). The alkyl (methyl, ethyl, propyl/
isopropyl, and butyl/isobutyl/tert-butyl) groups attached to
O, S and P influenced the overall fragmentation. The alkyl
groups are lost either as radicals or as neutrals after
proton migration. The alkene losses through McLafferty
rearrangement are dominant in higher alkyl group-containing
compounds (e.g., ions c, e and h). The fragment ions due to
[Mclafferty + 1] rearrangement (ions d and g) are also observed
in the spectra. Further loss of an alkene from the McLafferty
rearrangement fragment ion is also found when that ion
contains a higher alkyl group (other than a methyl group). Other
ions include those due to homolytic fission of the P–R, P–OR
a
mass spectrometer, equipped with Agilent 7890A gas
chromatograph. The GC and MS conditions were similar to
those described above. The instrument mass resolution was
8000 (full width at half maxima (FWHM)) and
perfluorokerosene was used as the internal reference.
RESULTS AND DISCUSSION
The structures of the studied isomeric compounds of O-alkyl,
S-alkyl alkylphosphonothiolates (1–18) and O,O’-dialkyl
alkylphosphonothionates (19–27) are shown in Scheme 2.
All the studied compounds are isomeric and, hence,
distinction of one isomer from another is a challenging task.
With a view to differentiating the studied set of isomers, we
analyzed their GC/EIMS, linked scan, GC/CIMS and GC-RI
data. The impurities and/or by-products present in the
reaction mixture were well resolved in the GC method; hence,
the EI or CI mass spectra of target compounds thus obtained
are pure without any interfering peaks from other products.
+
•
+
or P–SR bond (a-cleavage) from the M . The [M–OR] and
[M–(OR-H)]+ ions are diagnostic of the alkyl group attached
•
to the oxygen, while the [M–SR]+ and [M–(SR-H)]+ ions
•
indicate the alkyl group attached to the sulfur. Fragment ions
formed by thiono/thiolo rearrangements are also observed
in both alkylphosphonothiolates and alkylphosphonothionates
Scheme 2. Structures of the studied dialkyl alkylphosphonothiolates (1–18) and dialkyl
alkylphosphonothionates (19–27).
Rapid Commun. Mass Spectrom. 2013, 27, 1461–1472
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R. Karthikraj et al.
79
100
80
60
40
20
O
A)
P S
O
141
43
95
113
62
29
121
123
182
153
167
20
40
60
80
80
100
120
140
160
180
200
220
240
O
100
80
60
40
20
B)
P S
O
112
140
43
95
62
124
182
89
107
29
167
154
20
40
60
80
100
120
140
160
180
200
220
240
m/z
Figure 1. EI mass spectra of (A) compound 1 and (B) compound 2.
(discussed in a separate section at the end), but they are low
abundance and do not affect the structure indicative
fragmentation pattern (discussed below). Apart from the ions
discussed above, the spectra include a set of low-mass fragment
ions at m/z 65 [P(OH)2]+ (r), 63 (P ꢁ S+) (s), 47 (P ꢁ O+) (t), 43
rearrangement ions are always higher in O-propyl group-
containing compounds than in those with an O-isopropyl group,
and the reverse trend is observed for McLafferty rearrangement
products [Supplementary Table S2 (Supporting Information),
compounds 1 and 2 (Fig. 1)]. This is in good agreement with
the previous report, where the McLafferty + 1 ion was found to
be more prominent in the n-alkyl-group-containing
compounds than in those with branched alkyl groups.[34]
Similarly, O-propyl-group-containing compounds showed
a relatively more dominant [M–OC3H7]+ ion than the
O-isopropyl-group-containing compounds, while this trend
•
•
( C3H7) (u), and 29 ( C2H5) (v) that reflect the connectivity and
nature of the alkyl group attached to the heteroatoms.
Phosphonothiolates
The phosphonothiolates contain alkyl groups on three
heteroatoms (P, O and S) and it is possible to identify the
alkyl group attached to each heteroatom based on the
observed selective fragmentation (Scheme 3).
is reversed in the relative abundances of the [M–OC3H6]+
•
ions. Among the O-butyl-group-containing compounds,
the McLafferty + 1 rearrangement ions are dominant over
McLafferty rearrangement ions in all O-C4H9-alkyl-group-
containing compounds, irrespective of the butyl structure.
In a similar manner, the [M–OC4H9]+ ion is always dominant
O-Alkyl differentiation
over the [M–OC4H8]+ ion and there are no significant
•
The [M–R2/R3]+ and [M–(R2/R3-H)]+ ions provide information
•
about the size of the alkyl group; however, the [M–OR2/R3]+
differences among the isomeric butyl groups.
and [M–(OR2/R3-H)]+ ions confirm the attachment of the
•
alkyl group (other than a methyl) to the oxygen atom. The
O-methyl-group-containing compounds did not show the
[M–OCH3]+ ion. As expected, the O-ethyl-, O-C3H7-alkyl-,
and O-C4H9-alkyl-group-containing compounds yielded
[M–C2H4] , [M–C3H6] , and [M–C4H8] ions, respectively
(McLafferty rearrangement ion). In addition, the compounds
possessing O-C3H7-alkyl and O-C4H9-alkyl groups yielded
McLafferty + 1 rearrangement ions. The McLafferty + 1
S-Alkyl differentiation
It is easy to identify the alkyl group attached to the sulfur by
observing the presence of the [M–SR]+ and/or [M–(SR-H)]+
•
+
•
+
•
+
•
ions. All the S-methyl-containing compounds (5, 6, 11, 12,
16, and 18) specifically show the [M–SCH3]+ ion (m/z 135).
The S-ethyl-containing compounds (1, 2, 15, and 17)
consistently show a distinct [M–SC2H5]+ ion (m/z 121) in
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MS of dialkyl alkylphosphonothiolates/alkylphosphonothionates
127
O
100
A)
P S
O
80
60
40
57
79
41
109
20
29
135
95
167
182
20
40
60
80
100
120
140
160
180
200
220
240
93
O
100
80
60
40
20
126
B)
P O
S
153
79
182
63
109
47
29
140
149
167
160
20
40
60
80
100
120
140
180
200
220
240
m/z
Figure 2. EI mass spectra of (A) compound 5 and (B) compound 8.
addition to a [M–SC2H4]+ ion (m/z 122). Similarly, S-C3H7-
stability of the leaving SCH3 radical than of the OCH3
•
•
•
•
alkyl- (3, 4, 13 and 14) and S-C4H9-alkyl-group-containing
compounds (8–10) show ions at m/z 107/108 and 93/94,
radical. The dominant SCH3 radical loss from O,S-dimethyl
methylphosphonothiolate was reported in theoretical
studies.[35,36] In the case of O-ethyl- and S-ethyl-group-
containing compounds, the [M–SC2H5]+ ion (j) is always
more abundant in S-ethyl-containing compounds than is
the [M–OC2H5]+ ion in O-ethyl-containing compounds. The
O-C3H7-alkyl-group-containing compounds show both the
respectively, due to [M–SR]+/[M–(SR-H)]+ ions. All the
•
S-butyl-containing compounds (8–10) show a dominance of
McLafferty rearrangement over McLafferty + 1 rearrangement,
irrespective of the structure of the butyl group. However, some
compounds (2, 3, 11, 12, 15, 16, and 18) without a propyl or
butyl group on the sulfur atom also show the ions at m/z 93,
94 or 107, where these result from other routes of fragmentation
as confirmed from the MS/MS and HRMS data.
[M–OC3H7]+ (i) and [M–OC3H6]+ ions, but their abundances
•
vary with the propyl/isopropyl groups attached to oxygen;
whereas the S-C3H7-alkyl-group-containing compounds
always show the [M–SC3H6]+ ion as dominant over the
•
[M–SC3H7]+ ion. This suggests that the stability of the
O-Alkyl vs. S-alkyl group
•
leaving SC3H7 radical is less than that of the SC3H6
molecule, probably through attaining a stable trimethylene
sulfide (or thietane) structure. The [M–OC4H9]+ ion is
consistently dominant in all O-C4H9-alkyl-group-containing
compounds over the [M–SC4H9]+ ion in the corresponding
S-C4H9-alkyl-group-containing compounds. As expected,
The fragmentation of the S-alkyl group is similar to that of the
O-alkyl group, but there are pronounced differences in
the McLafferty/McLafferty + 1 rearrangement processes. The
compound with
a larger O-alkyl group favors the
McLafferty + 1 rearrangement, whereas the same alkyl group
on the sulfur atom exclusively yields the McLafferty
rearrangement ion (spectra of 5 and 8 are shown in Fig. 2 as
an example). The lack of a McLafferty + 1 rearrangement in
the higher S-alkyl groups (R = C3–C4 alkyl) may be because
of the reduced tendency of the sulfur atom to accept
the second hydrogen, as similarly reported in the case
of O-ethyl S-alkyl methylphosphonothiolates.[31] All
the S-methyl-containing compounds show a prominent
[M–SCH3]+ ion (f), whereas O-methyl compounds do
not show [M–OCH3]+ ions. This could be due to the higher
the [M–SC4H8]+ ion from S-C4H9-alkyl-group-containing
•
compounds is more abundant than the corresponding
[M–OC4H8]+
ion from O-C4H9-alkyl-group-containing
•
compounds, but these ions are not present in compounds
where there is a tertiary butyl group on the oxygen/sulfur-atom.
The fragmentation pattern pertinent to the P-alkyl groups is
different from that observed in the case of O-alkyl and S-alkyl
groups. Moreover, the P-alkyl group fragmentation is almost
similar between phosphonothiolates and phosphonothionates
and this point is further discussed later.
Rapid Commun. Mass Spectrom. 2013, 27, 1461–1472
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R. Karthikraj et al.
Scheme 3. Plausible EI fragmentation pathway of dialkyl alkylphosphonothiolates.
Scheme 4. Plausible EI fragmentation pathway of dialkyl alkylphosphonothionates.
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MS of dialkyl alkylphosphonothiolates/alkylphosphonothionates
154
100
A)
%
140
122
153
155
79
80
112
0
m/z
40
60
100
120
140
160
112
100
%
B)
79
111
121
126
139
0
m/z
40
60
80
100
120
140
79
100
%
C)
79
80
94
0
m/z
30
40
50
60
70
80
90
100
110
120
Figure 3. B/E spectra of (A) m/z 182, (B) m/z 154, and (C) m/z 122 of compound 15.
Phosphonothionates
phosphonothionates (22, 23 and 25–27) show the expected
[M–OCH3]+ ion (m/z 151) to confirm the presence of an
O-methyl group. In the case of the O-ethyl-group-containing
phosphonothionates (19, 20, 26 and 27), the expected ethylene
loss (McLafferty rearrangement) is not observed but they
do exhibit a [M–OC2H5]+ ion at m/z 137. All the O-C3H7-alkyl-
and O-C4H9-alkyl-group-containing phosphonothionates
In O,O’-dialkyl alkylphosphonothionates (19–27), alkyl
groups are attached to both the oxygen and the
phosphorus atom, and the other heteroatom (S) exists as
P = S. Thus, in this case, it is important to determine
the size of the alkyl group attached to oxygen and
phosphorus (Scheme 4).
show [M–OR]+ and [M–(OR-H)]+ ions, and the ions due
•
O-Alkyl differentiation
The alkyl groups attached to oxygen atoms are methyl,
ethyl, propyl/isopropyl, and butyl/isobutyl/tert-butyl.
Unlike the phosphonothiolates, the O-methyl-group-containing
n-OBu
n-SBu
n-O/SPr
n-PPr
i-OBu
i-SBu
i-SPr
i-PPr
P=O
i-OPr
t-OBu of P=O
t-SBu of P=O
P=S
i-OPr
i-OBu
t-OBu
Figure 4. The GC-RI values of compounds 1–27.
Scheme 5. Thiono-thiolo rearrangement.
Rapid Commun. Mass Spectrom. 2013, 27, 1461–1472
Copyright © 2013 John Wiley & Sons, Ltd.
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R. Karthikraj et al.
to McLafferty and McLafferty + 1 rearrangements. The
McLafferty + 1 rearrangement process is always dominant
over the McLafferty rearrangement process, irrespective of
the structure of the alkyl group.
observed in O/S-C3H7-group-containing compounds, is
absent in the P-C3H7 compounds; however, these
compounds show a low-abundance [M–C3H7]+ ion at m/z
139 formed through a-cleavage (Supplementary Fig. S1,
Supporting Information). The above data enables
easy recognition of the C3H7-alkyl group attached to
P; however, the data cannot provide any direct
evidence about the structure of the C3H7-alkyl group
(propyl/isopropyl). However, the spectra of the
phosphonothiolates that are isomeric with either P-propyl
or P-isopropyl show distinct differences in the relative
abundances of other characteristic fragment ions. For
example, the ion at m/z 126 is more abundant in
compound 15 than in compound 17. The MS/MS
P-Alkyl differentiation
It is important to correctly identify the alkyl group attached
to the phosphorus atom for all the schedule 2.B.4
chemicals that include dialkyl alkylphosphonothiolates/
phosphonothionates, where the alkyl group can be a methyl,
an ethyl, or a propyl/isopropyl group. All the P-ethyl-group-
containing compounds (11–14, 24 and 25) show loss of
an ethyl radical from the molecular ion.
A
low-
abundance but distinct [M–C2H4]+ ion (c) is found in
dialkyl ethylphosphonothiolates, but not in dialkyl
ethylphosphonothionates. In addition, the P-ethyl-group-
containing compounds consistently exhibit an ion at m/z
111 that results from the molecular ion by McLafferty
•
spectrum of the [M–C2H4]+ ion (m/z 154) from 15 showed
•
an abundant peak at m/z 126, which suggests that the loss
of 28 Da from m/z 154 could be from the P-propyl group;
this process is insignificant in compound 17 due to the
presence of the P-isopropyl group (Fig. 3).
•
rearrangement followed by the loss of the C2H5 radical.
The spectra of P-methyl-group-containing compounds
include the [M – CH3]+ ion, but there are other possible
routes for the loss of methyl radical from the alkyl groups
attached to O/S. The P-C3H7-alkyl-group-containing
Thiono-thiolo rearrangement
The typical EI-induced gas phase ’thiono-thiolo
rearrangement’,
i.e.,
conversion
of > P(S)-O-R
into
compounds show a [M–C3H6]+ ion at m/z 140 that is
> P(O)-S-R, has been observed in many classes of
phosphorus-containing compounds such as phosphines,
phosphates, phosphonates and phosphoramidates,
etc.[37–39] Cooks et al. studied substituted phosphorothioates
•
formed by a [1,4] C–P hydrogen shift (propyl) or a [1,3]
C–P hydrogen shift (isopropyl).[37] The [M–C3H5]+ ion
(McLafferty + 1 rearrangement), which is characteristically
127
O
100
A)
80
P S
O
60
57
40
109
41
40
20
80
80
167
150
160
29
182
180
95
100
20
60
120
140
141
200
220
240
S
100
80
60
40
20
P O
O
B)
79
182
109
123
120
95
63
60
47
29
153
160
20
40
80
100
140
180
200
220
240
m/z
Figure 5. EI mass spectra of compounds with the same RI values: (A)
compound 7 and (B) compound 24.
wileyonlinelibrary.com/journal/rcm
Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 1461–1472

MS of dialkyl alkylphosphonothiolates/alkylphosphonothionates
and demonstrated the effect of electronegativity of the
substituent on this rearrangement.[39] Santoro reported this
rearrangement occurring under EI fragmentation of some alkyl
thiophosphate esters.[40] Recently, Saeidian et al. reported
the thiono-thiolo rearrangement in O-alkyl-N,N-dimethyl
alkylphosphonothionoamidates, based on observation of
the same structure (elemental composition); thus HRMS data
alone is not sufficient to confirm the fragmentation pathway
for formation of the [M–SR]+ ion. We thus carried out
linked-scan experiments on a model compound, diethyl
methylphosphonothionate, and found a relatively abundant
[M–SC2H5]+ ion (m/z 107). The precursor ion spectrum of
the ion at m/z 107 shows two peaks corresponding to M+
•
•
unexpected loss of the SR radical, which was equal to or
more abundant than the expected OR radical loss. They
(m/z 168) and [M–C2H4]+ (m/z 140), where the former ion is
•
•
further supported the occurrence of the rearrangement
with theoretical calculations.[41] Barr et al. studied a few
dimethyl methylphosphonothionates (isotopically labelled)
under ESI conditions and found that their protonated
molecules undergo thiono-thiolo rearrangement.[32] Although
many examples of thiono-thiolo rearrangement have been
reported, it has not been explored in the case of dialkyl
alkylphosphonothionates.
dominant. The product ion spectrum of m/z 140 showed a
small peak at m/z 107. This reveals that direct loss of SC2H5
from the molecular ion is the preferred fragmentation that
can be explained by thiono-thiolo rearrangement (Scheme 5,
Supplementary Fig. S2, Supporting Information).
•
Theoretically,
the
reverse
of
the
thiono-thiolo
rearrangement, which may be named as the ’thiolo-thiono’
rearrangement, i.e., conversion of > P(O)-S-R into > P(S)-
O-R, is possible in dialkyl phosphonothiolates. In such a case,
the presence of the [M–OR]+ ion is expected in the EI spectra
of dialkyl phosphonothiolates in addition to the expected
[M–SR]+ ion. Among the studied dialkyl alkylphosphonothiolates
(1–18), the [M–OR]+ ion appeared only in compounds where
a propyl or a butyl group was attached to sulfur (3–4, 8–10,
13–14). This reveals that the dialkyl phosphonothiolates
undergo a ’thiolo-thiono’ rearrangement, especially when a
higher alkyl group is attached to sulfur. Although the
studied compounds undergo thiono-thiolo or thiolo-thiono
In the present study, the EI spectra of all the
•
phosphonothionates (19–27) show the expected OR radical
loss from the molecular ion; however, the spectra also include
•
the ion due to the loss of SR radical from the molecular ion,
[M–SR]+, which is always of lower abundance (<5%, except
for 24) than the [M–OR]+ ion. Formation of the [M–SR]+ ion
can be explained by a thiono-thiolo rearrangement. There is,
however, another plausible fragmentation pathway that can
+
•
•
lead to the same ion, i.e., SH radical loss from [M–(R-H)] ,
the McLafferty rearrangement ion. Both pathways result in
Scheme 6. Plausible CI fragmentation pathways of (i) dialkyl
alkylphosphonothiolates (1–18) and (ii) dialkyl alkylphosphonothionates (19–27).
Rapid Commun. Mass Spectrom. 2013, 27, 1461–1472
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

R. Karthikraj et al.
rearrangement, the resulting fragment ions from the
rearranged products are found to be of low abundance.
Consequently, this process does not affect the regular
fragmentation pathways of the studied compounds that
provide structural information.
On the contrary, the RI values of a few isomers are the same or
very close (ꢂ1), but they show distinct EI spectra. For example,
the RI value of 7 and 24 is 1176.8, whereas the EI spectrum of 7
is different from that of 24 (Fig. 5).
GC/CIMS analysis
RI values
All the compounds (1–27) were analyzed under GC/CIMS
conditions. Methane was chosen as the reagent gas because
methane-CI is likely to yield more fragment ions than
isobutane; the fragment ions can provide additional
structural information. The positive ion methane-CI spectra
of all the studied compounds showed an abundant [M + H]+
ion at m/z 183 in addition to [M + C2H5]+ (m/z 211) and
[M + C3H5]+ (m/z 223) ions (Supplementary Table S5,
Supporting Information). The CI data is particularly useful
for the butyl group compounds, which showed low-
abundance molecular ions in their EI mass spectra. In addi-
tion to [M + H]+ ions, the CI spectra included a few fragment
ions that were characteristic for the O- and S-alkyl groups. The
general CIMS fragmentation is depicted in Scheme 6. The major
fragment ions of dialkyl alkylphosphonothiolates are due to
McLafferty rearrangement from the [M + H]+ and [M + C2H5]+
ions, in addition to low-abundance [MH–R2OH]+ and
[MH–R3OH]+ or [MH–R3SH]+ ions. The CI data reveals
that the McLafferty rearrangement is more feasible through
the O-alkyl group than through the S-alkyl group; for
The RI values were calculated for all the studied
compounds (Supplementary Table S1, Supporting
Information). It is known that lower homologs (fewer
carbon atoms) will elute faster than the higher ones
(methyl < ethyl < propyl < butyl).[42] For the propyl and
butyl groups, the elution order is isopropyl < propyl and
t-butyl < isobutyl < butyl,
respectively.
The
studied
compounds, however, contain different alkyl groups on
the heteroatoms (O, S and P) and hence show combined effects
in their RI values (Fig. 4). The phosphonothionates eluted faster
than the corresponding isomeric phosphonothiolates.
Thus, the RI values are important for the differentiation
of phosphonothiolates from phosphonothionates.
Two pairs of the studied isomers, i.e., 15 and 17; 26 and 27,
displayed similar EI spectra. When the spectrum of one
compound is searched in the in-house library (that includes
the spectra of 1–27), the other compound of the pair appears
in the search result with a close match factor value. In such
cases, the RI value is crucial for assigning the correct structure.
141
100
O
[M+H]+
P
S
A)
90
183
O
80
70
60
50
40
-C3H6
-CH 3SH
30
20
10
0
135
169
-C3H6
[M+C2H5]+
[M+C3H5]+
223
-C3H7OH
153
93 102
86
211
123
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240
m/z
183
O
100
90
P
S
O
[M+H]+
B)
80
70
60
50
40
30
20
10
0
-C3H6
[M+C2H5]+
-C3H7SH 141
[M+C3H5]+
211
-CH 3OH
-C3H6
223
107
169
151
79
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240
m/z
Figure 6. Methane-CI mass spectra of (A) compound 11 and (B) compound 13.
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Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 1461–1472

MS of dialkyl alkylphosphonothiolates/alkylphosphonothionates
example, the [MH–C3H6]+ ion is more abundant in
compound 1 (O-propyl) than in compound 3 (S-propyl). As
expected, compound 19 that has O-ethyl and O-propyl groups
shows a dominant [MH–C3H6]+ ion over the [MH–C2H4]+ ion.
The n-alkyl groups (propyl and butyl) can easily be
differentiated from their branched isomers (isopropyl and
isobutyl/tert-butyl) because the McLafferty rearrangement ion
is always of higher abundance from a branched alkyl group
than from a n-alkyl group. The ion at m/z 155 is found in all
the O-/S-butyl-group-containing compounds (5–10, 21–23),
and it appears to be formed by the loss of C2H4 from
[M + H]+, although this ion also corresponds to [(M + 29)–
C4H8]+. The presence of [MH–R2OH]+ and [MH–R3OH]+ or
[MH–R3SH]+ ions, although of low abundance, gives
information about the alkyl group attached to the heteroatoms.
As expected, the [MH–RSH]+ ion is absent in dialkyl
alkylphosphonothionates. The isomeric compounds, which
have similar EI spectra, show a better differentiation under CI
conditions; for example, compounds 11 and 13 show distinct
CI spectra (Fig. 6). The McLafferty rearrangement ions formed
from the [M + H]+ and [M+ C2H5]+ ions (m/z 141 and 169,
respectively) are more dominant in compound 11 than in
compound 13. The ion at m/z 135 ([MH–CH3SH]+) in 11 and
the m/z 151 ion ([MH–CH3OH]+) in 13 confirm the presence of
S-methyl and O-methyl groups, respectively. In a similar
manner, the ion at m/z 123 in 11 reveals the presence of an
O-propyl group and the ion at m/z 107 in 13 confirms the
presence of an S-propyl group. Thus, the methane-CI data clearly
divulges the alkyl groups attached to the heteroatom (O or S).
are crucial. This study also provide an EI mass spectral
database for the scheduled compounds (2.B.04) that is useful
for their unambiguous identification during OPCW
proficiency tests. The methane-CI mass spectra show
[M + H]+ ions for all the studied compounds. The CI fragment
ions are characteristic of the alkyl groups attached to O and S,
thus making structural characterization easy. The studied
compounds undergo thiono/thiolo rearrangements to some
extent, but the fragment ions resulting from the rearranged
products are of low abundance, and do not interfere with
the structure-indicative fragment ions.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
Acknowledgements
The authors thank the Director, IICT, for the facilities and
encouragement. Two of the authors (RK and LS) thank CSIR,
New Delhi, and UGC, New Delhi, respectively, for the award
of senior research fellowships.
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Rapid Commun. Mass Spectrom. 2013, 27, 1461–1472