Mass spectral studies of silyl derivatives of partially hydrolyzed products of nitrogen mustards: Important markers of nitrogen mustard exposure

Article abstract of DOI:10.1002/rcm.8586

Rationale: Nitrogen mustards (NMs) are vesicant class of chemical warfare agents. From the viewpoint of the Chemical Weapons Convention partially hydrolyzed products of nitrogen mustards (pHpNMs) are considered as important markers of nitrogen mustard exposure. The detection of pHpNMs from biological or environmental samples is highly useful for obtaining forensic evidence of exposure to NMs. Methods: Gas chromatography interfaced with tandem mass spectrometry (GC/MS/MS) is a widely used tool for the identification and sensitive detection of metabolites of NMs in complex matrices. The pHpNMs were derivatized using silylating agents as they are highly polar and non-amenable to GC. The mass spectral studies of these silyl derivatives of pHpNMs were performed using GC/MS/MS in both electron ionization (EI) and chemical ionization (CI) mode. Results: Various approaches have been proposed to assess the fragmentation pathways of the trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS) derivatives of pHpNMs. All the proposed fragmentation pathways were based on the product and/or precursor ion scanning of corresponding ions in both EI and CI mode. In the case of EI, most of the fragmentation pathways involved either α-cleavage or inductive cleavage. Conclusions: This is the first report on the MS study of the silyl derivatives of pHpNMs. The study of the two different derivatives of pHpNMs using both EI- and CI-MS provides a reliable, unambiguous identification of pHpNMs in complex environmental and biomedical matrices (such as plasma and urine) during any verification activities.

Full text of DOI:10.1002/rcm.8586

Palit Meehir (Orcid ID: 0000-0003-0648-239X)
Mass Spectral Studies of Silyl Derivatives of Partially Hydrolyzed Products
of Nitrogen Mustards: Important Markers of Nitrogen Mustards Exposure
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.
*Corresponding Author. Email: meehirpalit@rediffmail.com, Fax: (+) 0751-2341148.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/rcm.8586
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Abstract:
Rationale: Nitrogen Mustards (NMs) are vesicant class of chemical warfare agents. From the
viewpoint of the Chemical Weapons Convention partially hydrolyzed product of nitrogen
mustards (pHpNMs) are considered as important markers of nitrogen mustards exposure. The
detection of pHpNMs from biological or environmental samples is highly useful for obtaining
forensic evidence of exposure of NMs.
Methods: Gas chromatography interfaced with tandem mass spectrometry (GC/MS/MS) is a
widely used tool for the identification and sensitive detection of metabolites of nitrogen
mustards in complex matrices. The pHpNMs were derivatized using silylating agents as they
are highly polar and non-amenable to GC. The mass spectral studies of these silyl derivatives
of pHpNMs were performed using GC/MS/MS in both electron ionization (EI) and chemical
ionization (CI) mode.
Results: Various approaches have been proposed to assess the fragmentation pathways of the
trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS) derivatives of pHpNMs. All the
proposed fragmentation pathways were based on the product and / or precursor ion scanning
of corresponding ions in both EI and CI mode. In the case of EI, most of the fragmentation
pathways involved either α-cleavage or inductive cleavage.
Conclusion: This is the first report on the MS study of the silyl derivatives of pHpNMs. The
study of the two different derivatives of pHpNMs using both EI and CI mass spectrometry
provides a reliable, unambiguous identification of pHpNMs in complex environmental and
biomedical matrices (such as plasma and urine) during any verification activities.
Key Words: Nitrogen mustard; Half nitrogen mustard; Partially hydrolyzed products;
Derivatization; Trimethylsilyl; tert-butyldimethylsilyl; GC/MS; GC/MS/MS
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1. Introduction
Mustard agents, such as sulphur mustard (SM) and nitrogen mustards (NMs), comprise
the vesicant class of chemical warfare agents (CWAs).1 Environmental and biological systems
are both dangerously affected by the hazardous effect of these highly toxic chemicals.2,3 .The
Organization for the Prohibition of Chemical Weapons (OPCW) was established to impose a
global ban on the use of CWAs (other than for peaceful purposes).4,5 . Despite this ban being
in place, CWAs have been deliberately used in several instances, such as in Syria, Malaysia
and the United Kingdom.6-8
NMs were designed as military agents and the compounds are abbreviated as HN1,
HN2 and HN3.5 The chemical structures of these agents are given in Table 1. In the Chemical
Weapons Convention (CWC) list of compounds NMs are listed as schedule – 1A chemicals,
which cause various physiological abnormalities or even death. Although NMs have not yet
been used in real war situations or for terrorism purpose the possibility of their deployment
cannot be overlooked. As NMs are strong alkylating agents, they can readily alkylate
nucleophilic sites of biomolecules (such as DNA, proteins, etc.) present in biological systems.9
Moreover, the protonated salts of NMs which are not scheduled in the Convention are highly
stable and can easily be synthesized. Hence, NMs can easily stockpiled, transported and finally
can be deployed by simple generation from their salts, indicating that NMs could readily be
uses as a weapon in terrorist activities.10 In response to these possible risks, the development
of reliable analytical method is essential for providing forensic evidence in the event of the
alleged use of NMs as chemical weapon.
The mechanistic understanding of NMs used as chemotherapy pro-drugs
(antineoplastics and cytotoxic drugs) is mostly accompanied by the quantitative evaluation of
the active NMs released within the target cells.11 Usually, NMs have more than one 2-
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chloroethyl groups, which can undergo sequential nucleophilic substitution of one chlorine
atom and then other chlorines. Therefore, in the course of its continuous release process, these
compounds can be trapped either as an active form of the NM or as its partial hydrolysed
products, as depicted in Table 1. Hence, quantitative identification of partially hydrolysed
products of nitrogen mustards (pHpNMs) may also help to study the pharmacokinetics of drugs
in further detail.
Owning to medicinal benefits of NMs, the corresponding amino-alcohols have been
extensively used by many pharmaceutical companies to manufacture therapeutic cancer
drugs/pro-drugs. These pharmaceutical companies are likely to undergo inspection and
verification of their production process under the verification program of CWC conducted by
the OPCW. During this inspection and verification process, the identification of pHpNMs will
provide evidence for compliance with the CWC while their detection at sites where these
chemical have not veen declared will indicate divergence from the CWC.
As a result of exposure to NMs, various NM adducts can be formed along with their
complete as well as partial hydrolysed products. Therefore, any biological sample collected
form victims of NMs exposure during the intervening period (i.e. before appearance of
symptoms) are also likely to contain the pHpNMs. Thus, isolation and identification of
pHpNMs will provide important evidence on the potential exposure of individuals to NMs.
In spite of great significance of pHpNMs, to date there is no analytical method available
for their identification. It is worth mentioning that the analytical methods which are reported
for this class of compounds are mainly focused upon either the intact NMs or its completely
hydrolysed products. The absence of any published report in the open literature for the analysis
of pHpNMs has prompted us to undertake this study. We are thus the first group to report the
study of silyl derivatives of pHpNMs and their detailed fragmentation studies by both Electron
Ionisation Mass Spectrometry (EI-MS) and Chemical Ionisation Mass Spectrometry (CI-MS).
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MS/MS precursor ion and product ion scanning enabled us to establish their fragmentation
pathways.
2. Materials and Methods
2.1 Chemicals: Dichloromethane, acetone, acetonitrile, sodium bicarbonate, sodium carbonate
were obtained from Merck (Darmstadt, Germany), while N,O-bis(trimethylsilyl)-
trifluoroacetamide
(BSTFA),
N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide
(MTBSTFA), methyldiethanolamine, ethyldiethanolamine and triethanolamine were obtained
from Sigma–Aldrich (Mumbai, India). The protonated salts of bis-(2-chloroethyl)ethylamine
[HN1], bis-(2-chloroethyl)methylamine [HN2] and tris-(2-chloroethyl)amine [HN3] were
synthesized in microgram quantities as per the reported procedures10 and immediately
consumed to produce partially hydrolysed products of nitrogen mustards.
(Caution: Nitrogen mustards (HN1, HN2 and HN3) are DNA alkylating and blister agents.
These chemicals should only be handled in the presence of trained professionals in an efficient
fume hood equipped with an alkali scrubber and operators must wear appropriate protective
clothing. Decontamination solution (approximately 15% w/v solution of bleach and alkali)
must be kept at the workplace and the solvent waste must be decontaminated in a proper way.)
2.2 Instrumentation for analysis: All the EI-MS mass spectral fragmentation studies were
performed using an Agilent 6890N gas chromatograph coupled with an Agilent 5975B
quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) controlled by
Agilent Chemstation software. A Thermo Trace1300 gas chromatograph coupled with a
Thermo TSQ Duo triple quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham,
MA, USA) was used for finding product and precursor ions in EI mode and all CI-MS and
MS/MS analyses were performed using this mass spectrometer employing Xcalibur software
(Thermo Fisher Scientific).
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2.2.1 GC/MS analysis: The transfer-line and injector temperatures were maintained at 280 °C
and 250 °C, respectively. Helium was used as the carrier gas at a flow rate of 1 mL min-1. A
Thermo Fisher Scientific DB-5MS capillary column (length: 30 m, internal diameter: 0.25 mm,
film thickness: 0.25 µm) containing 5% diphenylpolysiloxane and 95% dimethylpolysiloxane
was used for the analysis. The GC oven temperature was programmed from 50°C (hold for 1
min.) to 120 °C @40°C min-1, then to 160°C @ 5°C min-1, and finally to 280°C @ 60°C min-
1 (hold for 3 min.). A 10-µL micro syringe was used to inject the samples and the injection
volume was 1 µL. The EI source was kept at 230°C, the ionization energy was 70 eV, and the
quadrupole temperature was 150°C. All the studies were performed in full scan mode and the
scan range was from m/z 50 to 800 (3.47 scans per second).
2.2.2 GC/MS/MS analysis: The parameters (temperatures, column, GC oven program) for the
GC analysis were similar to those mentioned above. The EI source was kept at 200°C;, the
ionization energy was 70 eV and the quadrupole temperature was 150°C, while the CI source
was kept at 200°C, the ionization energy was 70 eV and the quadrupole temperature was
150°C.Methane and nitrogen were used as reagent and collision gas, respectively, for CI
analysis. Different collision energies were used for MS/MS analysis and the collision gas flow
rate was 1.5 mL min-1. All the studies were performed in full scan mode over a scan range of
m/z 60 to 800 with a 0.2 s dwell time.
2.3 Synthesis of salts of nitrogen mustards: All the salts of the nitrogen mustards were
synthesized following the reported procedure10 with some minor modifications. In brief, 7.5µM
of the respective amino alcohol (methyldiethanolamine, ethyldiethanolamine and
triethanolamine) was dissolved in dichloromethane (DCM) and the obtained solution was
cooled to 0- 5°C. An excess amount of thionyl chloride (75µM) was slowly added to the above
solution under vigorous stirring. After completion of the thionyl chloride addition, the reaction
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mixture was refluxed for 30 min. to give complete precipitation of the nitrogen mustard salt.
The progress of the reaction was monitored by GC/MS.
2.4 Partial hydrolysis of the NM saltes: The general experimental procedure used to synthesis
partial hydrolysis products of NMs (pHpNMs) was as follows. 1000 µg of the NM salt was
taken in a 4-mL screw cap vial and, subsequently 1 mL of acetone-water (2:1 v/v) was added.
To obtain partial the hydrolysis product of the NM (pHpNM), the mixture was incubated
overnight at room temperature with continuous stirring. The solvents were evaporated under
gentle nitrogen stream and the solid residue was collected (≥ 90% yields). The obtained solid
residue was reconstituted in dry DCM to obtain a final pHpNM concentration of 1000 µg mL-
1. These standard solutions were stored at -20 °C. The schematic representation of the synthesis
of the pHpNMs is given in Scheme 1
2.5 Derivatization of pHpNMs: The two derivatizing agents (BSTFA and MTBSTFA) were
used to make the pHpNMs GC amenable. The reaction of pHpNMs with BSTFA and
MTBSTFA produces their trimethylsilyl (TMS) and tertiary butyl dimethylsilyl (TBDMS )
derivatives, respectively.
2.5.1 Trimethylsilyl (TMS) Derivatives of pHpNMs: 20 µL of each pHpNM was taken in a
screw cap vial from its standard stock solution (1000 µg mL-1). To this solution100 µL of
BSTFA was added and the sealed vial was kept at 70˚C for 30 min. The vial was then cooled
to room temperature and 100 µL of DCM was added. 1 µL of the aliquot was used for mass
spectral analysis.
2.5.2 tertiary-Butyldimethylsilyl (TBDMS ) Derivatives of pHpNMs: 20 µL of each pHpNM
was taken in a screw cap vial from its standard stock solution (1000 µg mL-1). To this solution,
100 µL of MTBSTFA was added and the sealed vial was kept at 100˚C for 60 min. The vial
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was then cooled to room temperature and 100 µL of DCM was added. 1 µL of the aliquot was
used for mass spectral analysis.
3. Results and Discussion:
3.1 Synthesis of pHpNMs: As the hydrolysis of NMs often yields their fully hydrolyzed
products,12 the selective synthesis of pHpNMs is quite challenging. In order to synthesize
pHpNMs, the hydrolysis kinetics of NMs salts was studied in a controlled way using an
acetone: water (2:1) system. By using this approach, the pHpNMs shown in Table 1, 2-[(2-
chloroethyl)(ethyl)amino]ethanol
(CEEAE),
2-[(2-chloroethyl)(methyl)amino]ethanol
(BCEAE) and 2,2′-[(2-
(CEMAE), 2-[bis-(2-chloroethyl)amino]ethanol
chloroethyl)amino]diethanol (CEADE), were synthesized. The obtained pHpNMs were
derivatized using BSTFA and MTBSTFA and subjected to mass spectral characterization and
elucidation of fragmentation pathways using GC/MS and GC/MS/MS analysis.
3.2 Mass spectral fragmentation of Trimethylsilyl (TMS) Derivatives:
3.2.1 EI-MS interpretation: Silylation by BSTFA converts hydroxyl group / groups of
pHpNMs into their corresponding trimethylsilyl (TMS) ethers (Scheme 2), and the TMS
derivatives of pHpNMs were represented as halfHN1TMS, halfHN2TMS, 1halfHN3TMS and
2halfHN3TMS (Table 2). The derivatized pHpNMs were analyzed by GC/MS in full scan EI
mode to obtain fragmentation patterns that allowed the unambiguous identification of each
compound.
The EI-MS spectra of halfHNxTMS are shown in Figure 1 and the major fragment ions
obtained are listed in Table 3. It can be seen from Figure 1 that the molecular ions (M+•) of
halfHNxTMS was formed in very low abundance. Most of the fragment ions were formed by
α-cleavage (α) and inductive cleavage (i) of M+•. The obtained fragment ions of halfHNxTMS
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and their postulated pathways from the molecular ion are depicted in Scheme 3. Depending
upon the site of generation of the initial radical cation, there are three possible α-cleavages in
halfHNxTMS. A fragment ion (1) at m/z [M-103]+ can be formed by α-cleavage of the C-C
bond eliminating a methoxy-trimethylsilane radical, initiated from a radical cation formed at
the nitrogen atom. Cleavage of the C-C bond in the chloroethyl group results in a m/z [M-49]+
fragment ion (4). Formation of a radical cation on the oxygen atom initiates cleavage of the Si-
C bond and loss of a methyl radical to form the m/z [M-15]+ ion (5). These proposed
fragmentation pathways of halfHNxTMS were supported by precursor ion scans for fragment
ion types (1), (4) and (5) (entries A, B and C in Table 4).
Subsequent cleavage of C-N bond of the [M-103]+ ion yields the +CH2CH2Cl fragment ion (3)
at m/z 63/65 with a relative abundance ratio of 3:1, thus confirming the presence of the
chloroethyl group. Inductive cleavage of the Si-O bond (when the charge is on the O atom) of
M+• give the trimethylsilyl cation at m/z 73 (8), which is further confirmed by a precursor ion
scan of halfHNxTMS (entry D in Table 4). 1,3 C-N hydrogen migration followed by inductive
cleavage at the C-N bond of ions 1, 4 and 5 (when R' is CH2CH2Cl) gives the characteristic
odd electron species (6). This proposed fragmentation route was based on a precursor ion scan
of m/z 93 and a product ion scan of m/z 208 (entry A in Table 4). Ion (7) is produced after
neutral loss of HCl through 1, 3 C-Cl hydrogen rearrangement from the molecular ion.
3.2.2 Retention indices: The retention indices of each derivatized compound were calculated
for halfHNxTMS (Table 3) and halfHNxTBDMS (Table 7) according to the Van Den Dool
and Kartz formula13 to provide an additional degree of specificity during the detection and
identification of targeted metabolites by EI-MS.
3.2.3 CI-MS interpretation: During EI-MS analysis it was found that the relative
abundances of the molecular ions of all the halfHNxTMS were very low (below 1%). In order
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to obtain information about the molecular weight of each halfHNxTMS, the samples were
analysed by full scan CI-MS and CI-MS/MS. The CI-MS spectra of halfHNxTMS are given
in Figure 2 and Table 5. All the compounds (entries A2-D2, Table 5) formed abundant
[M+H]+ ions, as shown in Figure 2, formed by protonation on the tertiary amine group. The
product ion spectra of the [M+H]+ ions were recorded at the collision energies (CEs) shown in
Figure 2.
MS/MS at 10 eV provides only one product ion at m/z [M-90]+ (1), but at CEs above 20 eV
some more product ions were observed, a combination of sequential fragmentation of the [M-
90]+ product ion and directly from the [M+H]+ ion at higher energy. The optimum CE, where
the spectrum contained the most abundant product ions, was found to be 20 eV for A2-C2 and
25 eV for D2 (Table 5). From Figure 2, it can be seen that the halfHNxTMSs showed some
common product ions viz. [M-90]+ (1), m/z 92 (when R' = CH3) (2)a. or 106 (when R' =
CH2CH3) (2)b., m/z 84 (3), m/z 70 (4), m/z 117 (5) and m/z 63(6), as illustrated in Scheme 4.
Product ion (1) was formed after elimination of trimethylsilanol, and ion (2) formed after α-
cleavage followed by inductive cleavage at the C-N bond. α-Cleavage of [M+H]+ followed by
cyclization gave a five membered cation at m/z 84 (3). β-Cleavage followed by inductive
cleavage then cyclization via elimination of a. chloride radical provides the product ion at m/z
70 (4). Inductive cleavage at the C-N bond from sites containing the TMS group yielded the
ion at m/z 117 (5). The appearance of the chloroethyl cation at m/z 63 (6) confirms the presence
of at least one chloroethyl group on the nitrogen atom. In addition to these common product
ions, an additional peak at m/z 140 (7) was observed for 1halfHN3TMS (where R =
CH2CH2Cl), while for 2halfHN3TMS (where R= CH2CH2OsiMe3) additional peaks were
observed at m/z 132 (8), m/z 178 (9), m/z 148 (10) and m/z 90 (11).
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3.3. Mass spectral fragmentation of tertiary-butyldimethylsilyl Derivatives:
To authenticate the retrospective detection of alleged exposure to any toxicant, at least two
techniques or two derivatives are required. Hence, the mass spectral characterization of
pHpNMs was performed using another derivatizing agent, MTBSTFA. Because of their
analytical response and chromatographic separation TBDMS derivatives are actually generally
more suitable than TMS derivatives14 because of their higher stability, good chromatographic
properties and greater separation from the solvent front. Silylation by MTBSTFA converts the
hydroxyl group(s) of pHpNMs into their corresponding tertiary-butyldimethylsilyl (TBDMS)
ethers (b 5); the TBDMS derivatives are represented as halfHN1TBDMS, halfHN2TBDMS,
1halfHN3TBDMS and 2halfHN3TBDMS (Table 6). The derivatized pHpNMs were analyzed
by GC/MS.
3.3.1 EI-MS interpretation: The fragmentation pathways of TBDMS derivatives of all
pHpNMs are similar, and moreover their EI mass spectra are similar to those of the TMS
derivatives described above. The EI-MS spectra of the halfHNxTBDMSs are given in Figure
3 and the major fragment ions obtained are listed in Table 7. The obtained fragment ions of
halfHNxTBDMS and their possible pathways from M+• are depicted in Scheme 6.
The most abundance fragment ion [M-145]+ (1) was formed by charge localization at the
nitrogen atom and loss of a TBDMS-OCH2 radical (145 u) by α-cleavage (Scheme 6) while
ion (2) was obtained by α-cleavage followed by loss of a chloromethyl radical (49 u) from the
molecular ion (M+•). Ion (6) is the most common fragment ion for TBDMS derivatives15,
formed by the elimination of the stable tertiary-butyl radical. Similarly, fragment ion (4) was
formed by loss of a methyl radical. An odd electron species at m/z 93 (7) was formed from ions
(4) and (6) after a hydrogen rearrangement reaction followed by inductive cleavage, which was
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further confirmed by a precursor ion scan (entry C3, Table 8). The characteristic fragment ion
at m/z 73 (5) was formed from (3) and (4), with the pathways supported by precursor ion scans
(entries A3 and D3, Table 8).
3.3.2 CI-MS interpretation: The abundance of molecular ion peaks in EI-MS was very low.
Thus, in order to obtain molecular weight information and as an alternative technique, the
samples were subjected to CI-MS/MS analysis. The CI-MS spectra of halfHNxTBDMS are
given in Figure 4 and tabulated in Table 9. The mass spectral fragmentation pathway for
halfHNxTBDMS is given in Scheme 7. Abundant [M+H]+ ions were obtained for all the
halfHNxTBDMSs in CI mode. The CI-MS and CI-MS/MS spectra of the halfHNxTBDMSs
were similar to those of the TMS derivatives. The optimized CE values used to generate the
MS/MS spectra and their corresponding product ions are summarized in Table 9. At the
optimized CE, all the [M+H]+ ions dissociated into common product ions such as [M-132]+
(1), m/z 92 (when R' = CH3) (2)a or 106 (when R' = CH2CH3) (2)b, m/z 70 (3), m/z 63 (4), and
m/z 73 (5). Ion (1) is formed after elimination of t-butyldimethylsilanol, while ion (2) is formed
after α-cleavage followed by inductive cleavage at the C-N bond. β-cleavage of [M+H]+
followed by C-N hydrogen rearrangement and subsequent inductive cleavage followed by
expulsion of chloride radical provides the azeridinium cation at m/z 70 (3). The ion at m/z 63
(4) corresponds to the chloroethyl cation, whose appearance in the spectra confirms the
presence of at least one chloroethyl group on the nitrogen atom. In addition to these common
ions, peaks at m/z 78 (6) (when R = CH2CH3) and m/z 140 (7) were obtained in the CI-MS/MS
spectra of 1halfHN3TBDMS (when R = CH2CH2Cl), while for 2halfHN3TMS (when R=
CH2CH2OSi t-BuMe2) peaks at m/z 132 (8) and m/z 103 (9) were obtained. These
fragmentation pathways were further confirmed by product ion scans of [M+H]+ (shown in
entries A4-D4, Table 8).
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4. Conclusion:
The characterization of pHpNMs by GC/MS after derivatization by BSTFA and
MTBSTFA yielded valuable MS data for the retrospective detection of nitrogen mustards.
Derivatization by two different silylating agents provides authenticity to the identified results.
Furthermore, mass spectral interpretation by two different ionization technique (EI and CI)
operating in product and precursor ion scan mode reveals their probable fragmentation
pathways. These results provide a basis for the development of an accurate and precise GC/MS-
based detection method for the assessment of NMs exposure in both environmental and
biomedical samples. These proposed techniques can be used for the detection of metabolites of
NMs in environmental and biomedical samples (such as plasma and urine).
ACKNOWLEDGEMENTS
Buddhadeb Chandra is thankful to DRDE, Gwalior and DRDO, New Delhi for funding
and fellowship. We are thankful to Dr D K Dubey, Director, DRDE, Gwalior for his keen
interest and encouragement, to Dr G Rama Rao, Dr A. Majumdar, Mr R. Goud, Mr M. K.
Sahoo and Dr.A. K. Purohit for their scientific suggestions, and to all my colleagues and lab
mates for their unconditional support. This article is assigned DRDE accession no.
DRDE/VTX/10/2019.
CONFLICT OF INTEREST
The authors have declared that there is no conflict of interest.
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Table 1: NMs and their corresponding partial hydrolyzed product (pHps) as well
as complete hydrolyzed product (fHps).
Nitrogen
mustards
(NMs)
Partial hydrolyzed
product of NMs
(pHpNMs)
Fully hydrolyzed
product of NMs
(fHpNMs)
S.
No.
Structure
1
2
HN1
HN2
3
HN3
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Table 2: Abbreviation of TMS derivatives of HN depending upon different
substituent R′.
Entry
R′
CH3
Compound
Abbreviation
halfHN2TMS
halfHN1TMS
1halfHN3TMS
2halfHN3TMS
1
2
3
4
TMS derivative of halfHN2
TMS derivative of halfHN1
TMS derivative of 1halfHN3
TMS derivative of 2halfHN3
C2H5
CH2CH2Cl
CH2CH2OSiMe3
Table 3: EI-MS data of TMS derivatives of pHpNMs (halfHNxTMS)
m/z values of fragment ions (% relative abundance)
RI
*
R'
Compound
M•+
(1)
(2)
(3)
194
(2.6)
208
(2.8)
242
(4)
(5)
(6)
(7)
56
(1.9)
56
(8.5)
56
(8)
173
(1.6)
halfHN2
TMS
120
1
106
160
93
73
63
Me
Et
209
223
257
(100) (5.4)
120 174
(100) (3.7)
154 208
(100) (7.3)
208 262
(100) (2.0)
(7.9) (12.9) (4.7)
93 73 63
(6.2) (14.5) (4.1)
93 73 63
halfHN1
TMS
125
4
-
1halfHN3
TMS
151
9
221
CH2CH2Cl
(2.4) (14.0) (27.5) (16.8) (13.9) (2.0)
CH2CH2O
SiMe3
2halfHN3
TMS
158
4
296
93
73
63
56
311
-
(2.7)
(3.5) (65.5) (2.8)
(3.8)
*RI stands for retention indices.
Table 4: Product and precursor ions of representative ions.
m/z value
Entry
Compound
Precursor ions
Product ions
311, (M+•)
296, [M-15]+
262, [M-49]+
73
257, (M+•)
154, [M-103]+
296, 262, 208
147, 137, 130, 93, 73
137, 130, 117, 93, 73
58, 45, 43, 29
A
B
2halfHN3TMS
242, 208, 154
1halfHN3TMS
halfHN1TMS
halfHN2TMS
63, 56
208 [M-15]+, 187 [M-36]+,
174 [M-49]+
C
D
73
209, (M+•)
106, [M-103]+
106, 194
78, 70, 63, 57
Ions in BOLD represent those ions which are selected for and confirmed by precursor ion
scanning.
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Table 5: CI-MS data of TMS derivatives of pHpNMs
CE
(eV)
Entry
A2
R'
Me
Et
Compound
HalfHN2TMS 209
halfHN1TMS 223
M
[M+H]+
CI-MS/MS product ions
120 [M-90]+, 117 (5), 92 (2),
84 (3), 63 (6)
134 [M-90]+, 106 (2), 117(5),
70 (4), 63 (6)
168 [M-90]+, 140 (7), 117 (7),
106, 73, 63 (6)
210
20
20
20
B2
224
C2
CH2CH2Cl 1halfHN3TMS 257
258
222 [M-90]+, 178 (9), 148,
132 (9), 117, 106, 90 (11), 73,
63 (6)
CH2CH2OSi
2halfHN3TMS 311
Me3
D2
312
25
Table 6: Abbreviation of TBDMS derivatives of HN depending upon the different
substituent R′.
Entry
R′
Compound
Abbreviation
1
CH3
TBDMS derivative of halfHN2
halfHN2TBDMS
halfHN1TBDMS
1halfHN3TBDMS
2halfHN3TBDMS
2
3
4
C2H5
TBDMS derivative of halfHN1
TBDMS derivative of 1halfHN3
TBDMS derivative of 2halfHN3
CH2CH2Cl
CH2CH2OsitBuMe2
Table 7: EI-MS data of TBDMS derivative of pHpNMs
Fragment Ions (% relative abundance)
R’
Compounds
RI*
1343
1363
1648
1718
M•+
251
265
299
395
(1)
(2)
(3)
(4)
(5)
93
(6)
(7)
HalfHN2
TBDMS
halfHN1
TBDMS
1halfHN3
TBDMS
2halfHN3
TBDMS
106
202
194
236
73
63
Me
Et
(99.9) (5.1) (9.1) (1.7)
120 216 208 250
(99.9) (4.5) (3.9) (2.2)
154 250 242 284
(7.9)
93
(10.0) (3.0)
63
-
(6.2)
93
(1.9)
73
63
CH2CH2Cl
(99.9) (7.8) (7.5) (1.5) (14.0) (13.9) (8.4)
CH2CH2O
SitBuMe2
250
(99.9) (1.7) (2.9) (2.6)
* RI stands for retention indices
246
338
380
93
73
-
(3.5)
(26.5)
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Table 8: Product and precursor scans of representative ions.
m/z value
Entry
Compound
Precursor ion
Product ion
395, (M+•)
250, 338, 346, 380
250 [M-145]+, 338 [M-
2halfHN3TBDMS 49]+, 359 [M-36]+, 380
159, 118, 115, 103, 100, 93,
73, 70, 59
A3
[M-15]+
380, 338
299, (M+•)
147
154, 242, 250, 264, 284
137
126, 118, 92, 63, 56
208, 134
134, 93, 84, 73, 58
92, 84, 63, 58, 56
106, 194, 202, 216, 236
B3
C3
1halfHN3TBDMS
halfHN1TBDMS
242 [M-57]+, 206
154 [M-145]+
265, (M+•)
208[M-57]+
120 [M-145]+
251, (M+•)
236 [M-15]+,
120, 93, 73
194 [M-57]+
D3
halfHN2TBDMS
236 [M-15]+, 194 [M-
57]+, 150, 137
236, 216, 202, 159
93
73
Ions in BOLD represent those ions which are selected for and confirmed by precursor
ion scanning.
Table 9: CI-MS data of TBDMS derivatives of pHpNMs
CI-MS/MS product CE
Entry
R'
Chemicals
M
[M+H]+
ions
(eV)
120 [M-132]+, 92 (2),
A4
B4
C4
D4
Me
Et
HalfHN2TBDMS
halfHN1TBDMS
1halfHN3TBDMS
2halfHN3TBDMS
251
265
299
395
252
266
300
396
20
73 (5), 63 (4)
134 [M-132]+, 106 (2),
78 (6), 73, 70, 63
168 [M-132]+, 140 (7),
70 (3)
25
20
30
CH2CH2Cl
CH2CH2OSitB uMe2
264 [M-132]+, 132 (8),
114, 103 (9), 90, 73
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Scheme 1: Equilibrium of HN salts with their free form in water and acetone - water
mixture.
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Scheme 2: Silylation of pHpNMs by BSTFA.
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Scheme 3: EI-MS fragmentation of TMS derivative of pHpNMs.
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Scheme 4: General fragmentation pattern of [M+H]+of TMS derivatives of pHpNMs in
CI mode.
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Scheme 5: Silylation of halfHNs by MTBSTFA.
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Scheme 6: EI-MS fragmentation of TBDMS derivatives of pHpNMs.
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Scheme 7: General fragmentation pattern of [M+H]+ ions of TBDMS derivatives of
pHpNMs in CI mode.
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Figure1: EI-MS spectra of (A) halfHN2TMS, (B) halfHN1TMS, (C) 1halfHN3TMS and (D)
2halfHN3TMS 1
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Figure 2: Full scan CI-MS spectra, and CI-MS/MS spectra of the respective [M+H]+ ions of
the TMS derivatives of pHpNMs, (A) CIFS-halfHN2TMS, (B) CI-MS/MS-halfHN2TMS,
(C) CIFS-halfHN1TMS, (D) CI-MS/MS-halfHN2TMS, (E) CIFS-1halfHN3TMS, (F) CI-
MS/MS-1halfHN3TMS, (G) CIFS-2halfHN3TMS and (H) CI-MS/MS-2halfHN3TMS
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Figure 3: EI-MS spectra of (A) halfHN2TBDMS, (B) halfHN1TBDMS, (C)
1halfHN3TBDMS and (D) 2halfHN3TBDMS
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Figure 4: Full scan CI-MS spectra followed by MSMS spectra of corresponding [M+H]+ ion
of TBDMS derivative of halfHNs, (A) CIFS-halfHN2TBDMS, (B) CI-MS/MS-
halfHN2TBDMS, (C) CIFS-halfHN1TBDMS, (D) CI-MS/MS-halfHN2TBDMS, (E) CIFS-
1halfHN3TBDMS , (F) CI-MS/MS-1halfHN3TBDMS, (G) CIFS-2halfHN3TBDMS and (H)
CI-MS/MS-2halfHN3TBDMS .
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