Mass spectral fragmentation of perfluoroacyl derivatives of half nitrogen mustards for their detection by gas chromatography/mass spectrometry

Article abstract of DOI:10.1002/rcm.8777

Rationale: Analytical methods for the detection and identification of half nitrogen mustards (halfNMs), i.e., partially hydrolyzed products of nitrogen mustards (pHpNMs), using silyl derivatives are often associated with low sensitivity and selectivity. In order to overcome these limitations, the derivatization of halfNMs was performed using perfluoroacylation. Methods: Two efficient derivatization techniques using trifluoroacetyl (TFA) and heptafluorobutyryl (HFB) groups were developed for the unambiguous identification of halfNMs. A mass spectral database was generated by performing gas chromatography/electron ionization mass spectrometry (GC/EI-MS) and gas chromatography/positive chemical ionization mass spectrometry (GC/PCI-MS). The fragmentation pathways were studied by tandem mass spectrometry (MS/MS) in both EI and PCI mode. Results: The EI-MS spectra of the TFA and HFB derivatives of halfNMs contain intense molecular ions and fragment ions, thus making perfluoroacylation preferable to silylation. In addition, the background-free chromatogram obtained using these derivatives provides unambiguous identification of these compounds in blind samples. The structures of the fragment ions were postulated, and the sources of significant ions were traced by performing MS/MS precursor ion scans. In the PCI-MS spectra, along with the protonated molecule, significant peaks due to neutral losses of HF, HCl, CH₃Cl and CF₃COOH were observed. Conclusions: This is the first report of the elucidation of the fragmentation pathways of perfluoroacyl derivatives of halfNMs. The complementary GC/PCI-MS and GC/PCI-MS/MS data will be helpful in the identification of unknown metabolites in a fast and reliable fashion.

Full text of DOI:10.1002/rcm.8777

Received: 21 November 2019
DOI: 10.1002/rcm.8777
Revised: 22 January 2020
Accepted: 6 March 2020
R E S E A R C H A R T I C L E
Mass spectral fragmentation of perfluoroacyl derivatives of
half nitrogen mustards for their detection by gas
chromatography/mass spectrometry
Buddhadeb Chandra | Kanchan Sinha Roy | Mahabul Shaik |
Chandrakant Waghmare | Meehir Palit
VERTOX – Biochemistry Division, Defence
Research and Development Establishment,
Jhansi Road, Gwalior 474002, Madhya
Pradesh, India
Rationale: Analytical methods for the detection and identification of half nitrogen
mustards (halfNMs), i.e., partially hydrolyzed products of nitrogen mustards
(pHpNMs), using silyl derivatives are often associated with low sensitivity and
selectivity. In order to overcome these limitations, the derivatization of halfNMs was
performed using perfluoroacylation.
Correspondence
M. Palit, VERTOX – Biochemistry Division,
Defence Research and Development
Establishment, Jhansi Road, Gwalior-474002,
Madhya Pradesh, India.
Methods: Two efficient derivatization techniques using trifluoroacetyl (TFA) and
heptafluorobutyryl (HFB) groups were developed for the unambiguous identification
Email: meehirpalit@rediffmail.com
of halfNMs.
A mass spectral database was generated by performing gas
chromatography/electron ionization mass spectrometry (GC/EI-MS) and gas
chromatography/positive chemical ionization mass spectrometry (GC/PCI-MS). The
fragmentation pathways were studied by tandem mass spectrometry (MS/MS) in
both EI and PCI mode.
Results: The EI-MS spectra of the TFA and HFB derivatives of halfNMs contain
intense molecular ions and fragment ions, thus making perfluoroacylation preferable
to silylation. In addition, the background-free chromatogram obtained using these
derivatives provides unambiguous identification of these compounds in blind
samples. The structures of the fragment ions were postulated, and the sources of
significant ions were traced by performing MS/MS precursor ion scans. In the PCI-
MS spectra, along with the protonated molecule, significant peaks due to neutral
losses of HF, HCl, CH3Cl and CF3COOH were observed.
Conclusions: This is the first report of the elucidation of the fragmentation pathways
of perfluoroacyl derivatives of halfNMs. The complementary GC/PCI-MS and
GC/PCI-MS/MS data will be helpful in the identification of unknown metabolites in a
fast and reliable fashion.
1
|
INTRODUCTION
respiratory system and lead to failure of internal organs.3 In addition
to the formation of painful blisters, these vesicant agents can alkylate
Nitrogen mustards (NMs) are schedule 1 chemical warfare agents
(CWAs)1 in the Chemical Weapon Convention (CWC).2 These agents
are highly toxic with strong vesicant properties that corrode mucosal
membranes as well as causing skin and eye damage, affect the
DNA4 and free nucleophilic sites present in proteins,5 leading to
cancer and metabolic disorders. Such CWAs remain a high-priority
concern due to the their possible use against civilian targets, as
occurred in the 1995 subway attack with sarin in Tokyo.6 As mustard
Rapid Commun Mass Spectrom. 2020;34:e8777.
wileyonlinelibrary.com/journal/rcm
© 2020 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/rcm.8777

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CHANDRA ET AL.
agents are easier to synthesize and deploy than nerve agents, the
development of analytical methods for their detection, and for the
study of their toxicology and metabolism, is necessary for public
safety. Although there is much literature available on sulfur mustards,
there is very little on biomarkers for NMs, because NMs have not
been used in a real-life scenario. However, the possibility of their
deployment cannot be ignored. In this investigation we have focused
on the derivatization and mass spectral identification of half nitrogen
mustards (halfNMs) for the retrospective detection of exposure
to NMs.
temperature, and
a
chromatogram with significant background
peaks. These problems can be overcome by substituting
derivatization by perfluoroacylation. Herein, we report on the
advantages of perfluoroacylation for the sensitive and selective
detection of halfNMs using electron ionization mass spectrometry
(EI-MS) and positive chemical ionization mass spectrometry mass
spectrometry (PCI-MS). Tandem mass spectrometry (MS/MS;
precursor ion, product ion and neutral loss spectra) was also helpful
in determining the fragmentation pathways. Furthermore,
understanding the fragmentation pathways will help in selecting
appropriate diagnostic precursor-to-product ion transitions for the
selected reaction monitoring (SRM)/multiple reaction monitoring
(MRM) detection of the target analytes in trace amounts in
complex matrices.
Nitrogen mustards (HN1, HN2 and HN3) are labile and rapidly
degrade under certain environmental and biological conditions to give
completely hydrolyzed products, viz. ethanolamines (EAs)7
(Scheme S1, supporting information), which are less toxic than their
parent compounds. Moreover, the presence of such hydrolyzed
products in environmental and/or biomedical samples can provide
valuable information about exposure to the parent compound.
Because of the medicinal benefits of NMs, especially in cancer
therapy, the corresponding amino alcohols have been extensively
used to manufacture therapeutic cancer drugs/pro-drugs.8 In addition,
EAs can be used in the production of cosmetics.9,10 Thus, these
industries are subject to the CWC verification program conducted by
the Organisation for the Prohibition of Chemical Weapons (OPCW).2
During any OPCW inspection, non-compliance cannot be based on
the identification of EAs, but the presence of partially hydrolyzed
products of NMs, i.e. halfNMs, at those sites will support a non-
compliance finding (Figure 1). Upon exposure to NMs, various
adducts (e.g. DNA and protein adducts) and metabolites
(e.g. partially and completely hydrolyzed products) are formed.
Therefore, any biological samples collected from the victims of
exposure to NMs are likely to contain the halfNMs. Thus, isolation
and identification of halfNMs will provide significant evidence for
the exposure of individuals to NMs.
2
|
EXPERIMENTAL
| Chemicals
2.1
Dichloromethane (DCM), acetone, and acetonitrile (ACN) were
obtained from Merck (Mumbai, India) and N,O-bis(trimethylsilyl)
trifluoroacetamide
(BSTFA),
N-methyl-N-tert-
butyldimethylsilyltrifluoroacetamide (MTBSTFA), N-(trifluoroacetyl)
imidazole (TFAI), N-(heptafluorobutyryl)imidazole (HFBI), N-
methyldiethanolamine, N-ethyldiethanolamine, and triethanolamine
were obtained from Sigma–Aldrich (Mumbai, India). Protonated salts
of
bis(2-chloroethyl)
ethylamine
[HN1],
bis(2-chloroethyl)
methylamine [HN2] and tris(2-chloroethyl)amine [HN3] were
synthesized in microgram quantities in our laboratory when required
and used immediately.
HalfNMs are not amenable to analysis by gas chromatography
(GC) because of the presence of polar hydroxyl groups and hence
they have to be derivatized prior to GC/MS analysis. In a previous
article GC/MS-based identification of halfNMs after derivatization
by silylation was reported.11 However, silylation of halfNMs has
2.1.1
|
Precautions
Nitrogen mustards are DNA alkylating and blister agents. These
chemicals should be handled by trained professionals in an efficient
fume hood equipped with an alkali scrubber and appropriate
protective gear must be worn. A decontamination solution must be
kept at the workplace and the solvent waste must be properly
decontaminated.
disadvantages:
a long derivatization time, a high derivatization
2.2
|
Instrumentation
GC/EI-MS studies were performed using a 6890N gas chromatograph
coupled to a 5975B single quadrupole mass spectrometer controlled
by Chemstation software (all supplied by Agilent Technologies,
Santa Clara, CA, USA). A Trace 1300 gas chromatograph coupled to a
TSQ Duo triple quadrupole mass spectrometer controlled by
Xcalibur software (all supplied by Thermo Fisher Scientific,
Waltham, MA, USA) was used for the EI-MS/MS, PCI-MS and MS/MS
studies.
FIGURE 1 Chemical structures of different partially hydrolyzed
products of NMs (halfNMs)

CHANDRA ET AL.
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2.3
|
GC/MS analysis
[In the remainder of this article, the trifluoroacetyl derivatives of
the halfNMs are abbreviated as halfHN2TFA, halfHN1TFA,
1halfHN3TFA and 2halfHN3TFA, and the heptafluorobutyryl
derivatives as halfHN2HFB, halfHN1HFB, 1halfHN3HFB and
2halfHN3HFB, of the corresponding NMs.]
The transfer-line and injector temperatures were 280ꢀC and 250ꢀC,
respectively. Helium was used as the carrier gas at a flow rate of
1 mL min−1
. A DB-5MS capillary column (30 m × 0.25 mm I.D.,
0.25 μm film thickness; Thermo Fisher Scientific) was used for the
separation. The GC oven temperature was programmed from 50ꢀC
(1 min hold) to 120ꢀC at 40ꢀC min−1, raised to 161ꢀC at 5ꢀC min−1
,
3
|
RESULTS AND DISCUSSION
| Synthesis of halfNMs
and finally to 280ꢀC at 60ꢀC min−1 (3 min hold). The scan range was
from m/z 50 to 800 at a rate of 3.47 scans per second. GC/MS/MS
analysis was carried out using the triple quadrupole instrument fitted
with a CI source operating in positive ion multiple reaction monitoring
(MRM) mode. MRM confirmation of each compound involved the use
of two MRM transitions: the quantifier transition (Q) and the qualifier
transition (q).
3.1
The selective synthesis of halfNMs is challenging as the hydrolysis of
NMs often yields the fully hydrolyzed products. Therefore, in our
previous study11 a controlled hydrolysis process was developed to
yield the partial hydrolysis product. The protonated NMs (I) are highly
soluble in water and remain in equilibrium with the free form (II)
(Scheme S1, supporting information). In pure water, this equilibrium is
shifted towards I due to their polar nature, making the rate of
hydrolysis of I too slow. With the addition of acetone to the water,
the free NMs becomes solubilized and the equilibrium shifts towards
the free form (II), as shown in Scheme S1 (supporting information).
Once the free form II is produced it undergoes partial hydrolysis via a
reactive azeridinium intermediate (III). As the reaction progresses, the
released HCl slows down the partial hydrolysis and it thus takes more
than 20 h to obtain the desired halfNMs (IV). To speed up the
reaction, stoichiometric amounts of very dilute (1 mM) NaOH solution
were added, which consumes the released HCl, and the desired
product was obtained within 2 h. The progress of the reaction was
monitored by GC/MS after derivatizing the halfNMs with HFBI.
2.4
|
Synthesis of partially hydrolyzed products of
nitrogen mustards
The halfNMs were synthesized using our previously reported
method11 with some modification. Briefly, 1 mg of the salt of the
NMs was dissolved in 1 mL of acetone/water (2:1 v/v) and
incubated at room temperature with vigorous stirring. To this, an
equimolar amount of 1 mM NaOH aqueous solution was added
and the solution was stirred for 2 h. The evaporation of the
mixture under a gentle nitrogen stream resulted in a solid residue
(≥90% yield).
2.5
|
Perfluoroacylation of halfNMs
3.2
|
Selection of perfluoroacylation over silylation
Two different perfluoro-acyl derivatizing agents were used for
derivatization: TFAI for trifluoroacetylation (TFA) and HFBI for
heptafluorobutyrylation (HFB). The derivatizing agent (5 μL) was
The halfNMs (halfHN1, halfHN2, 1halfHN3 and 2halfHN3) were
monitored by GC/EI-MS after derivatization by four different
derivatizing agents (BSTFA, MTBSTFA, TFAI and HFBI, as shown in
Scheme 1). High temperature (>70ꢀC) is required for silylation and at
this elevated temperature degradation of the silylating agents occurs.
The degradation products or byproducts12 produced during the
dissolved in 95 μL DCM, and to this 20 μg mL−1 of
a mixed
standard solution of halfNMs was added. After 5 min of incubation,
a 1 μL sample from the DCM layer was subjected to GC/MS
analysis.
SCHEME 1 Derivatives of partially
hydrolyzed products of NMs (TMS, TBDMS, TFA
and HFB) [Color figure can be viewed at
wileyonlinelibrary.com]

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CHANDRA ET AL.
derivatization create a high GC background and mask the analytes,
especially the early eluting compounds, as shown in Figures 2A and
2B. It is clear from Figure 2 that the halfHN1TBDMS derivative is
heavily masked by the degradation products/byproducts generated by
the MTBSTFA. Several additional peaks can be seen (Figure 2) in the
total ion chromatogram (TIC) of the TMS and TBDMS derivatives
while using pure analytes at higher concentration than with the TFA
or HFB derivatives. On the other hand, trifluoroacetylation and
heptafluorobutyrylation occur at room temperature and a clean
chromatogram is obtained, as shown in Figures 2C and 2D. Thus, this
reduction in background noise enhances the sensitivity.
Moreover, the molecular ion and abundant high-mass fragment
ions were observed in the mass spectra of the perfluoroacyl
derivatives of halfNMs, whereas the corresponding silyl derivatives
provided abundant fragment ions at low m/z values only. The
presence of the molecular ion and the high-mass fragment ions for
the perfluoroacyl derivatives improves the selectivity of the
detection. In addition, these abundant high-mass ions open the
opportunity to develop a MS/MS method for the detection of
these analytes at trace level. For the TMS or TBDMS derivatives
the abundance for most of the fragment ions was less than 30% of
the base peak, whereas, for the TFA and HFB derivatives, two or
more ions have an abundance of ≥50% of the base peak. Hence,
the perfluoroacyl derivatives are also useful for the identification of
these compounds in complex matrices using extracted ion
chromatograms.
The derivatization of halfNMs using perfluoroacylation is almost
instantaneous, whereas the silylation reaction takes at least 30 min
for complete derivatization. Thus, the reduction in derivatization time
is very useful for high-throughput sample analysis.
FIGURE 2 Total ion chromatograms
obtained during GC/EI-MS analysis of A,
TMS; B, TBDMS; C, TFA; and D, HFB
derivatives of halfNMs [Color figure can
be viewed at wileyonlinelibrary.com]

CHANDRA ET AL.
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CHANDRA ET AL.
3.3
|
Retention indices
The retention indices of each derivatized compound were calculated,
as shown in Table 1 for halfHNxTFA derivatives and Table 2 for
halfHNxHFB derivatives (according to the Van Den Dool and Kartz
formula13), to provide an additional degree of specificity for the
detection and identification of targeted metabolites by EI-MS.
3.4
|
Mass spectral fragmentation analysis of
perfluoroacyl derivatives
3.4.1
|
EI-MS interpretation
The fragmentation of the EI molecular ions involves two important
fragmentation reactions: (a) α-homolytic cleavage and (b) inductive
heterolytic cleavage. In the spectra of the halfHNxTFA (M+• at m/z
233, 247, 281 and 359, for halfHN2TFA, halfHN1TFA, 1halfHN3TFA
and 2halfHN3TFA, respectively) and halfHNxHFB (M+• at m/z
333, 347, 381 and 559 for halfHN2HFB, halfHN1HFB, 1halfHN3HFB
and 2halfHN3HFB, respectively) derivatives, series of even-electron
fragment ions (EE+) were observed. Most of the fragmentation
pathways were common to all halfHNxTFA and halfHNxHFB
derivatives, but they differed significantly from those of the
respective silyl derivatives.
The mass spectra of the halfHNxHFB derivatives follow a similar
fragmentation pathway to the halfHNxTFA compounds and also
provide similar fragment ions, with differences in their masses due to
the difference in the perfluoroalkyl group, i.e. CF3 in TFA and C3F7 in
HFB. The spectra of the perfluoroacyl derivatives of the halfNMs are
similar to those of the corresponding EAs.14–16 The EI mass spectra of
all the derivatives of the halfNMs are shown in Figure 3 (3A –
halfHN1TFA, 3B – halfHN2TFA, 3C – 1halfHN3TFA and 3D –
2halfHN3TFA) and Figure 4 (4A – halfHN1HFB, 4B – halfHN2HFB,
4C
– 1halfHN3HFB and 4D – 2halfHN3HFB). The common
fragmentation pathways and the corresponding fragment ions are
depicted in Scheme 2. As a result of the α-cleavage, intense ions
([M − R2COOCH2]+ (1) and [M − 49]+ (2)) are formed, with the
assumption that the radical cation is formed on the nitrogen atom
(as shown in Scheme S2, supporting information). Ion (1) results from
the elimination of a methyl perfluoroalkanoate radical (CF3COOCH2
for the TFA derivative and C3F7COOCH2 for HFB) from the ester
moiety side and ion (2) from the loss of a chloromethyl radical (49 u)
from the 2-chloroethyl group, as depicted in Scheme 2. Another
abundant ion resulted from the loss of [R1NCH2CH2Cl] (3) from the
molecular ion (at m/z 141 for TFA and m/z 241 for HFB) by inductive
cleavage at the C–N bond next to the ester group after the
elimination of an amine radical (Scheme S3, supporting information).
When radical cation formation occurred on the chlorine atom,
inductive cleavage led to the formation of a low-abundance [M − 35]+
ion (4) by elimination of a chlorine atom (35 u) (Scheme S3, supporting
information). When radical cation formation occurred on the oxygen
atoms of the ester group, inductive cleavage yielded a low-abundance

CHANDRA ET AL.
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FIGURE 3 EI-MS spectra of A,
halfHN1TFA; B, halfHN2TFA; C, 1halfHN3TFA;
and D, 2halfHN3TFA [Color figure can be viewed
at wileyonlinelibrary.com]
[M − R2COO]+ ion (5) after the elimination of the perfluoroalkanoate
radical (CF3COO from the TFA derivative and C3F7COO from HFB)
(Scheme S3, supporting information).
from the β carbon of the R1 group to the N atom, as shown in
Scheme S4 (supporting information). Inductive cleavage of the
[M − R2COOCH2]+ ion at the C–N bond provides a characteristic ion
at m/z 63 (7) (+CH2CH2Cl) after expulsion of a neutral imine. A
precursor ion scan of m/z 63 (Figure S1B, supporting information)
shows that this ion can also arise from m/z 92 and 134, i.e. from ions
Another characteristic ion (6) is formed at m/z 92, which results
from the EE+ fragment ion [M − R2COOCH2]+ (this pathway is
supported by the precursor ion scan of m/z 92 depicted in
Figure S1D, supporting information) after βH-rearrangement17
containing the -CH2CH2Cl group.
A
three-membered cyclic

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CHANDRA ET AL.
FIGURE 4 EI-MS spectra of A,
halfHN1HFB; B, halfHN2 HFB; C, 1halfHN3 HFB;
and D, 2halfHN3 HFB [Color figure can be
viewed at wileyonlinelibrary.com]
N-methylene aziridinium ion is produced at m/z 56 (8) which can
result from any of the nitrogen-containing intermediate ions such as
(1), (2), (4), (5) and (6), as depicted in Scheme S5 (supporting
information), and this pathway is supported by the precursor ion scan
of m/z 56 (Figure S1A, supporting information).
supporting information) confirms that it arises from intermediate ions
containing the perfluoroalkyl moiety (Scheme S6, supporting
information), such as m/z 97 (R2CO+), 113 (R2COO+),
141 (R2COOCH2CH2+), and [M − 35]+ and [M − 49]+. The HFB
derivative yields two additional ions, at m/z 169 and 100, that result
from the heptafluorobutyl cation and the 1,1,2,2-tetrafluoroethylene
radical cation, respectively, by loss of the trifluoromethyl radical, as
depicted in Scheme S7 (supporting information).
A moderately abundant peak observed at m/z 69 (9) corresponds
to the trifluoromethyl cation, resulting from C–C bond cleavage in the
perfluoroalkyl chain. A precursor ion scan of this ion (Figure S1C,

CHANDRA ET AL.
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SCHEME 2 Common fragmentation
pathways in EI-MS spectra for HFB and
TFA derivatives of partially hydrolyzed
products of HNs
The perfluoroacyl derivatives of the partially hydrolyzed products
of HN3 show some additional fragment ions formed through H-
rearrangement and βH-elimination: (A) [M − C2H3Cl]+ (at m/z 170 or
270), (B) [M − HX]+ (at m/z 196 or 296) and (C) [M − R2COOH]+
(at m/z 118), as shown in Scheme S8 (supporting information). In
halfHN3 the expulsion of a chloromethyl radical from M+• via
α-cleavage resulted in the [M − 49]+ ion (2) (at m/z 232 and 310, or
m/z 332 and 510, respectively, for the TFA and HFB derivatives). This
ion can be further fragmented to give ions (A), (B) and (C). Ion (A) was
produced after the elimination of a vinyl chloride radical via H-
rearrangement from the β-C to N. Ion (B) was formed by loss of HX
(X can be the heteroatom (Cl) or the perfluoroacyl group) via a βH-
elimination. Similarly, ion (C) is formed after the loss of the
perfluoroacyloic acid (trifluoroacetic acid or heptafluorobutyric acid),
as depicted in Scheme S9 (supporting information).
information), respectively. Each [M + H]+ ion (C1) follows a unique
fragmentation pattern in PCI. The [M + H]+ ions provide fragment
ions (C2) at [M + H − HF]+, (C3) at [M + H − HCl]+ and (C4) [M + H −
CH3Cl]+ for all the TFA derivatives of halfNMs (Figure S2, supporting
information). Another fragment ion (C5) [M + H − CF3COOH]+ is
obtained for each molecule (except halfHN1TFA), as depicted in
Figures S2B, S2C and S2D (supporting information). All the fragment
ions are summarized in Scheme 3. During the PCI-MS/MS or MS/MS
analysis of the protonated molecule a wide range of fragmentation
reactions was observed (Figure 4). Collision-induced dissociation (CID)
fragmentation of the [M + H]+ ion occurs either via inductive cleavage
of the C–heteroatom (N, O, Cl, etc.) bond with charge migration to
the α-C-atom, as shown in Scheme S10 (supporting information), or
via a four-centered H-rearrangement (acyclic EE+ cleavage) with
charge retention on the heteroatom as depicted in Scheme S11
(supporting information). The MS/MS fragmentation pathways were
investigated using 1halfHN3TFA as an example (Figure S3A,
supporting information). The protonated molecule (1) at m/z
282 (Figure S3B, supporting information) provides eight (C5 to C12)
product ions (Scheme 4). When protonation occurs on the N atom,
the inductive cleavage fragmentation follows two pathways, path-a
and path-b, as depicted in Scheme S10 (supporting information).
Path-a gives the most abundant product ion (C9) at m/z
141 generated after the neutral loss of the secondary amine
(ClCH2CH2NHCH2CH2Cl). Ion (C9) then undergoes another inductive
cleavage by the expulsion of an ethylene molecule to provide the
3.4.2
|
Interpretation of CI-MS spectra
As was observed in the EI-MS spectra, the PCI-MS spectra of the TFA
and HFB derivatives of halfNMs are similar. We report herein only on
the TFA derivatives, since both derivatives follow similar
fragmentation pathways. The fragment ion observed in PCI-MS for
halfHN1TFA ([M + H]+ at m/z 248), halfHN2TFA ([M + H]+ at m/z
234), 1halfHN3TFA ([M + H]+ at m/z 282), and 2halfHN3 ([M + H]+ at
m/z 360) are shown in Figures S2A, S2B, S2C and S2D (supporting
SCHEME 3 PCI-MS fragmentation
pathways of the TFA derivatives of
halfNMs

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CHANDRA ET AL.
SCHEME 4 Different product ions of the
protonated molecule of 1halfHN3TFA after MS2
trifluoroacetate cation (CF3COO+) (C10) at m/z 113. Neutral loss of
CO2 from m/z 113 gives an intense CF3+ ion (C11) at m/z 69.
ACKNOWLEDGEMENTS
The authors thank DRDE, Gwalior, and DRDO, New Delhi, for funding
and fellowship. They also thank Dr D.K. Dubey, Director, DRDE,
Gwalior, for his keen interest and encouragement, and all their
colleagues and lab mates for their unconditional support. This
manuscript is assigned DRDE accession no. DRDE/VTX/42/2019.
Path-b provides the characteristic 2-chloroethyl cation (C12) at
m/z 63 after the expulsion of the secondary amine (Scheme S10,
supporting information). If protonation occurs at the alkoxy oxygen
atom of the ester linkage, after C–O bond cleavage charge
migration occurs on the carbonyl C to form the trifluoroacetyl
cation (C8) at m/z 97 (Scheme S10, supporting information).
Cleavage at the alkoxy O–C bond of the ester moiety in the
protonated molecule (C1) by β-elimination releases trifluoroacetic
ORCID
Meehir Palit
https://orcid.org/0000-0003-0648-239X
acid to yield
a product ion (C5) at m/z 168 (Scheme S11,
REFERENCES
supporting information). This ion can then eliminate vinyl chloride
by H-rearrangement to give the product ion (C6) at m/z 106.
Final β-elimination of HCl from (C6) provides the characteristic
di-vinyl ammonium ion (C7) (Scheme S11, supporting information)
at m/z 70.
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4
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CONCLUSIONS
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To the best of our knowledge, this is the first report of the mass
spectral study of perfluoroacyl derivatives of half nitrogen mustards.
The unequivocal identification of halfNMs was demonstrated by
GC/MS/MS analysis after perfluoroacyl derivatization. The proposed
fragmentation pathways are supported by precursor ion scanning of
crucial product ions. Most of the product ions obtained in the PCI-
MS/MS spectra of protonated molecules were similar to the
fragment ions obtained in the EI-MS spectra. The combination of
these mass spectral techniques added an extra dimension to the
identification of unknown compounds. Moreover, the two types of
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the identification of unknown metabolites in a fast and reliable
fashion.
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How to cite this article: Chandra B, Sinha Roy K, Shaik M,
Waghmare C, Palit M. Mass spectral fragmentation of
perfluoroacyl derivatives of half nitrogen mustards for their
detection by gas chromatography/mass spectrometry. Rapid
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