Mass spectral analysis of N-oxides of nitrogen mustards, and N,N-dialkylaminoethyl-2-chlorides under electrospray ionization conditions

Article abstract of DOI:10.1016/j.ijms.2012.08.005

Chemical weapons convention (CWC) includes verification of chemical warfare agents and their precursors/degradation products in various environmental matrices. Nitrogen mustards and N,N-dialkylaminoethyl-2-chlorides, the precursors for the synthesis of nerve agents (VX type of compounds), are prone to undergo oxidation in environmental matrices or during decontamination process. Thus, screening of the oxidized products of the CWC related compounds is also an important task in the verification process. Here we report the successful characterization of the N-oxides of nitrogen mustards and N,N-dialkylaminoethyl-2-chlorides under positive ion electrospray ionization tandem mass spectrometry conditions. The collision-induced dissociation spectra of [M+H]⁺ ions show distinct product ions that enable unambiguous identification of studied N-oxides, including those of isomeric compounds. The proposed fragmentation pattern of these compounds is based on isotopic abundance ratio, high-resolution mass spectrometry data and product/precursor ion spectra. The CID spectra of [M+H]⁺ ions of the studied compounds include a specific product ion, [MH-(?OH + ?CH₂Cl)] ⁺, and distinctive alkene losses to recognize the attached alkyl group to the nitrogen.

Full text of DOI:10.1016/j.ijms.2012.08.005

International Journal of Mass Spectrometry 333 (2013) 15–20
Contents lists available at SciVerse ScienceDirect
International Journal of Mass Spectrometry
journal homepage: www.elsevier.com/locate/ijms
Mass spectral analysis of N-oxides of nitrogen mustards, and
N,N-dialkylaminoethyl-2-chlorides under electrospray ionization conditions
L. Sridhara, R. Karthikraja, M.R.V.S. Murtya, N. Prasada Rajua, M. Vairamania,b, S. Prabhakara,∗
a National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad 500007, India
b School of Bio-Engineering, SRM University, Chennai 603203, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 April 2012
Received in revised form 31 July 2012
Accepted 2 August 2012
Available online 10 August 2012
Chemical weapons convention (CWC) includes verification of chemical warfare agents and their
precursors/degradation products in various environmental matrices. Nitrogen mustards and N,N-
dialkylaminoethyl-2-chlorides, the precursors for the synthesis of nerve agents (VX type of compounds),
are prone to undergo oxidation in environmental matrices or during decontamination process. Thus,
screening of the oxidized products of the CWC related compounds is also an important task in the verifi-
cation process. Here we report the successful characterization of the N-oxides of nitrogen mustards and
N,N-dialkylaminoethyl-2-chlorides under positive ion electrospray ionization tandem mass spectrome-
try conditions. The collision-induced dissociation spectra of [M+H]+ ions show distinct product ions that
enable unambiguous identification of studied N-oxides, including those of isomeric compounds. The pro-
posed fragmentation pattern of these compounds is based on isotopic abundance ratio, high-resolution
mass spectrometry data and product/precursor ion spectra. The CID spectra of [M+H]+ ions of the stud-
ied compounds include a specific product ion, [MH−(•OH + •CH2Cl)]+, and distinctive alkene losses to
recognize the attached alkyl group to the nitrogen.
Keywords:
Chemical warfare agents
N-oxides of nitrogen mustards
N-oxides of dialkylaminoethyl-2-chlorides
[M+H]+ ions
ESI-MS/MS
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
identification of CWAs and their degradation products from envi-
ronmental matrices [13–17].
Chemical weapons are toxic chemicals that have a great poten-
tial to inflict death, injury, temporary incapacitation or sensory
irritation through its chemical action. The Chemical Weapons Con-
vention (CWC) enters into force in April 1997 with the objective of
prohibition of proliferation of chemical weapons [1,2]. The goal of
CWC is being met by execution of its unique verification program.
The Organisation for Prohibition of Chemical Weapons (OPCW),
whose headquarters is in The Netherlands, is responsible for imple-
mentation of the CWC through its verification mechanism and it has
188 signatory states-parties [1,2]. Verification of the CWC requires
unequivocal identification of CWAs as well as their precursors,
degradation and reaction products in the samples collected from
suspected sites [3–7]. The analysis is performed either on-site by
the inspectors or off-site by at least two designated laboratories
appointed by the OPCW [8,9]. The OPCW maintains a network of
designated laboratories by periodically evaluating their analytical
capabilities through official proficiency tests (PTs) [10–12]. Only
the designated laboratories are authorized to undertake the task
of off-site analysis of CWAs and related compounds. A number of
methods have been developed for the extraction, detection and
The CWC related chemicals are enlisted in three scheduled cate-
gories, [1,2] among them the Schedule 1 includes highly toxic nerve
agents and blister agents (the sulphur mustards, lewisites, and
nitrogen mustards). The N,N-dialkylaminoethyl-2-chlorides and
N,N-dialkylaminoethanols are the precursors and the hydrolyzed
products of nerve agents (VX type of compounds, 1.A.03) [18].
Generally, the nitrogen containing CWAs like VX type compounds,
nitrogen mustards, N,N-dialkylaminoethyl-2-chlorides and N,N-
dialkylaminoethanols are prone to undergo N-oxidation during the
decontamination process [18–20]. Though these N-oxides are non-
scheduled chemicals, they are relevant to the verification of CWC,
because presence of these degradation products reveals alleged
use of nitrogen mustards or precursors of VX compounds. In fact,
OPCW included these N-oxides in the non-scheduled reportable
chemicals (NSRC), which are relevant to the CWC. N-oxidation of
tertiary amines is also known to occur in mammalian metabolism
[21,22]. Thus, detection of these compounds in suspected matrices
such as organic, oil, soil, and sand indicates the alleged usage and
production of the corresponding parent CWAs.
The N-oxides are known to be thermally labile [23,24]
and, hence, they cannot be analyzed by GC/MS where sam-
ple vaporization is a prerequisite [25]. Literature reports reveal
that the characterization of N-oxide using LC–MS/MS, especially
under atmospheric pressure chemical ionization conditions, is
∗
Corresponding author. Tel.: +91 40 27191343; fax: +91 40 27193156.
E-mail address: prabhakar@iict.res.in (S. Prabhakar).
1387-3806/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijms.2012.08.005

16
L. Sridhar et al. / International Journal of Mass Spectrometry 333 (2013) 15–20
were introduced into the source by flow injection (10 ␮L loop) using
O
N
R1
R2
Cl
methanol as the mobile phase at a flow rate of 30 ␮L/min. The typ-
ical source conditions were: capillary voltage, 5 kV; declustering
potentials (DP1), 60 V; DP2, 10 V; focusing potential, 250 V; mass
resolution 10,000 (FWHM). Ultra high purity nitrogen was used as
the curtain gas and the collision gas, whereas zero air was used as
the nebulizer. For the CID experiments, the precursor ion of interest
was selected using the quadrupole analyzer and the product ions
were analyzed using the TOF analyzer. The collision energies used
were between 15 and 25 eV. All the spectra reported were averages
of 25–30 scans. The spectra of isomers were recorded under identi-
cal experimental conditions. Elemental composition for the parent
as well as product ions was obtained from the measured accurate
mass values by using the software.
R
1, R2 = CH2CH2Cl (1)
R1, R2 = C2H5 (8)
R1 = CH3, R2 = CH2CH2Cl (2)
R1 = C2H5, R2 = n-C3H7 (9)
R1 = C2H5, R2 = CH2CH2Cl (3)
R1, R2 = CH3 (4)
R1 = C2H5, R2 = i-C3H7 (10)
R1 , R2 = n-C3H7 (11)
R1 , R2 = i-C3H7 (12)
R1 = CH3, R2 = C2H5 (5)
R1 = CH3, R2 = n-C3H7 (6)
R1 = CH3, R2 = i-C3H7 (7)
R1 = n-C3H7, R2 = i-C3H7 (13)
R1 = CH3, R2 = CH2CH2OH (14)
Precursor ion and some additional CID experiments were per-
formed using a triple quadrupole mass spectrometer (Quattro LC,
Micromass, Manchester, UK) using Masslynx software. Capillary
and cone voltages were kept at 3.50 kV and 30 V, respectively. Argon
was used as the collision gas, and the collision gas cell kept at pres-
sure of 4.0 × 10−4 mbar. In this case, the samples were introduced
into the source using a direct infusion pump (Harvard Apparatus)
at a flow rate of 10 ␮L/min.
Liquid chromatography (LC)–MS and LC–MS/MS experiments
were carried out for the compounds 3 and 6 using LCQ ion trap
mass spectrometer (Thermo Fisher, San Jose, CA, USA) coupled with
Surveyor HPLC system. The data acquisition was under the con-
trol of Xcalibur software. The data was acquired in the positive
mode and the typical source conditions (ESI) were: spray volt-
age, 4.8 kV, capillary voltage 20 V, capillary temperature 300 ◦C, and
tube lens offset voltage 10 V. Nitrogen (40 psi) was used as sheath
gas and helium (1.8 mTorr) was used as damping gas and collision
gas. Chromatographic analysis was achieved using a ZORBAX SB-
C18 (4.6 mm × 250 mm, 5 ␮m) analytical column. The mobile phase
contains methanol (A) and acetonitrile (B) in ratio of 90:10, at a con-
stant flow rate of 1 mL/min. The collision energy used for LC–MS/MS
experiments was 25 eV (6) and 30 eV (3).
Scheme 1. Molecular structures of the N-oxides studied (1–14).
challenging because of thermally induced deoxygenation or ther-
mally induced ‘Meisenheimer rearrangement’ [26–28]. Recently,
we reported the successful characterization of the N-oxides of N,N-
dialkylaminoethanols, alkyl diethanolamines, and triethanolamine
using positive ion electrospray ionization (ESI) tandem mass spec-
trometry [18]. The CID spectra of [M+H]+ ions of these compounds
showed a characteristic product ion corresponding to the loss of
CH4O2 that could be used for their unambiguous identification.
Therefore, ESI-MS, using direct flow injection or LC separation, is
a facile technique for the identification and characterization of the
N-oxides. Here, we extend our study to the ESI-MS/MS analysis of
the N-oxides of nitrogen mustards and N,N-dialkylaminoethyl-2-
chlorides (compounds 1–14, Scheme 1).
2. Experimental
2.1. Materials
The compounds 1–14 were synthesized from corresponding
aminoethyl-2-chloride by oxidation as per the known procedure
[29]. Briefly, the nitrogen mustard/dialkyl aminoethyl-2-chloride
was oxidized with urea hydrogen peroxide (UHP)/phthalic anhy-
dride system (Scheme 2). The precursors (nitrogen mustard/dialkyl
aminoethyl-2-chlorides) used in the present study were either
commercially available or synthesized from suitable precursors
by known procedure [30]. The compound 14 was the by product
in the synthesis of compound 2. The progress of oxidation reac-
tion was monitored by recording ESI mass spectra time to time by
inspecting the [M+H]+ ions of parent aminoethyl-2-chloride and
oxidized product that are separated by +16 mass units. UHP was
purchased from Sigma Aldrich (St. Louis, USA). Thionyl chloride and
the solvents used for the synthesis were purchased from Sd-Fine
chemicals (Mumbai, India). Analytical grade solvents were used for
mass spectrometric analysis, and they were obtained from E-Merck
(Mumbai, India).
3. Results and discussion
Initially, we attempted the synthesis of N-oxides of
the aminoethyl-2-chlorides by chlorination of N-oxides of
aminoethanols using thionyl chloride (SOCl2) and phosphorus
pentachloride (PCl5). But these experiments failed to yield the
intended products; instead the corresponding aminoethyl-2-
chlorides were formed due to expulsion of the oxygen on nitrogen.
Hence, we changed the strategy to oxidize aminoethyl-2-chlorides
by the H2O2, but in this case the aminoethyl-2-chlorides were
hydrolysed to the corresponding aminoethanols due to the pres-
ence of water in H2O2 and consequently resulted in the formation
of N-oxides of aminoethanols. Finally, oxidation with UHP/phthalic
anhydride system was successful for the synthesis of N-oxides
used in the current study.
The studied compounds include the N-oxides of three nitrogen
mustards (1–3) and ten N,N-dialkylaminoethyl-2-chlorides (4–13),
where the alkyl groups attached to nitrogen are methyl, ethyl,
propyl or isopropyl. All the parent compounds are listed sched-
ule compounds of the CWC, i.e., the nitrogen mustards are listed as
schedule 1.A.06 and N,N-dialkylaminoethyl-2-chlorides are listed
as schedule 2.B.10, respectively, of the CWC. We have also added the
N-oxide of N-(2-chloroethyl)-N-(2-hydroxylethyl)methylamine
(14), the partially hydrolyzed product of 2 in the list of studied com-
pounds to reveal the effects of the functional groups in the study.
The chemical structures of the studied N-oxides (1–14) are shown
in Scheme 1. Detection of these N-oxides in the suspected matrices
confirms the prior presence of the corresponding toxic chemicals or
2.2. Mass spectrometry
The experiments were performed using a quadrupole time-
of-flight mass spectrometer (QSTAR XL, Applied Biosystems/MDS
Sciex, Foster City, CA, USA), equipped with an ESI source. The data
acquisition was under the control of Analyst QS software (Applied
Biosystems). The collision energy used was 10 eV. All the samples
O
N
R1
R2
Urea Hydrogen Peroxide/Phthalicanhydride
Stirring 30 min, RT
R1
R2
Cl
Cl
N
Scheme 2. Synthetic scheme for the studied N-oxides (1–14).

L. Sridhar et al. / International Journal of Mass Spectrometry 333 (2013) 15–20
17
precursors/degradation products of the toxic chemicals. As N-
oxides are expected to be thermally unstable, we have not
attempted GC/MS and APCI analyses of 1–14. The positive ion ESI-
MS analysis of these compounds yielded good [M+H]+ ion signals.
Therefore, the compounds 1–14 were analyzed by direct ESI-MS,
LC–MS and MS/MS techniques under positive ion mode.
The direct ESI mass spectra (flow-injection) of the studied N-
oxides (1–14) show dominant protonated molecule, [M+H]+. The
spectra do not show either adduct ions (e.g., sodiated species) or
thermally induced deoxygenation/Meisenheimer rearrangement
products under the used experimental conditions, typically in the
absence of source heating. The isotopic pattern of all the [M+H]+
ions clearly reflected the number of chlorine atoms present in the
molecule, particularly the abundances of [MH+2]+, [MH+4]+ and
[MH+6]+ ions with respected [M+H]+ ions match with that of sim-
ulated values. The m/z values of the [M+H]+ ions of all the N-oxides
are shifted by +16 units compared with the [M+H]+ ions of the cor-
responding parent aminoethyl-2-chloride, and the accurate mass
values obtained from high resolution data confirmed that this shift
of +16 m/z units is due to the addition of an oxygen (N-oxidation).
The molecular weight and the elemental composition information
of the detected ion are not sufficient for the unambiguous iden-
tification of the compounds present in the given environmental
sample, particularly for the verification of CWC related chemicals.
Thus, we have further studied the dissociation of the [M+H]+ ions
of the N-oxides to obtain additional structural information.
3.1. CID of [M+H]+ ions
All the compounds (1–14) formed abundant [M+H]+ ions under
positive ion ESI conditions. In general, amine oxides are weak bases,
but highly polar like quaternary ammonium salts, and upon proto-
nation they form the corresponding hydroxylamine cations [18].
We presume the same in the case for the N-oxides of the present
investigation. The CID spectra of [M+H]+ ions were recorded at dif-
ferent collision energy (CE) values. The optimum CE value, at which
the spectrum contained more abundant product ions, is found to
be 25 eV for 1 and 3; 20 eV for 4–8; 23 eV for 2 and 9–14. The spec-
tra recorded at the optimum CE value were summarized in Table 1
and considered for further discussion. However, for the identifica-
tion of isomeric compounds only the spectra recorded at a fixed CE
value are considered. The CID spectra of the protonated N-oxides
(4–13) were compared with those of the protonated parent N,N-
dialkylaminoethyl-2-chlorides [17], and, as expected, they were
found to be different. Papouskova et al. reported the ESI-MS/MS
spectra of [M+H]+ ions of N,N-dialkylaminoethyl-2-chlorides [17].
From this report, it can be understood that [M+H]+ ion of these
+
compounds mainly show [MH−HCl]+, [MH−CnH2n
]
(n = 2 or 3),
and an ion at m/z 63 (+CH2CH2Cl). In the current study, the CID
spectra of [M+H]+ ions of the N-oxides (1–13) show a distinctive
fragmentation pattern different from that of the corresponding par-
ent aminoethyl-2-chloride and this enables easy identification of
the oxidation products. The general fragmentation pathway of the
[M+H]+ ions of compounds 1–13 is summarized in Scheme 3.
All the CID spectra show the [MH−OH]+• and [MH−H2O]+, but
these ions have low abundance in the compounds where an iso-
propyl group is attached to the nitrogen. There is only one source of
oxygen, i.e., N-oxide moiety, in the studied compounds, thus it must
be involved in the formation of the above two product ions. The
[MH−OH]+• ion formed by the loss of a hydroxy radical is known
from the earlier study of the protonated species of N-oxides [18,22].
The ion at m/z 63 corresponds to chloroethyl cation (+CH2CH2Cl), as
confirmed by HRMS data (Supplementary data, Table S1), is consis-
tently present in all the spectra, but it is a low abundant ion in some
cases. Appearance of this ion in the spectra concludes the presence
of at least a chloroethyl group on the nitrogen atom.

18
L. Sridhar et al. / International Journal of Mass Spectrometry 333 (2013) 15–20
OH
N
[MH-H2O]+
-H2O
R1
R2
Cl
Cl
Cl
Cl
-CH2-OH
-OH
Cl
CH2
m/ z 63
N
H3C
-C2H4O
N
H3C
H3C
N
H
OH
-CH2-Cl
-OH
-CH4O2
-CH2-Cl
N
OH
R1
N
[MH-H2O]+
R1
R2
[5,7-10,12,13]
-CH3OH
[MH-CH3OH]+
Cl
N
-H2O
R2
-CH3ClO
-C2H4
OH
Cl
H3C
[MH]+ ion of 1-13
H3C
N
-CH3ClO
OH
CH2
Cl
OH
OH
N
R1
H
Cl
-C2H5OH
Cl
[M+H]+ of 14
m/z 63
N
-C3H6
R1
Scheme 4. Fragmentation pattern of [M+H]+ ion of compound 14.
OH
R1
[11,13]
N
-C3H6
Cl
-H2O
-HCl
OH
N
N
H
[MH-C3H6-H2O-HCl]+
As expected, [MH−C3H6]+ is prominently observed in 6–7 and
9–13, in which a propyl/isopropyl group is attached to nitro-
gen. Furthermore, consecutive loss of two propene molecules to
form the [MH−2(C3H6)]+ ion is present where two such groups
(propyl/isopropyl) are present (11–13). Eliminations of HCl and
H2O (single or consecutive) from [MH−C3H6]+ and [MH−2(C3H6)]+
ions, wherever applicable, are also observed (Scheme 3). In the case
of N-ethyl group containing molecules, the expected [MH−C2H4]+
is found but in low abundance.
R1
H
[
Cl
6
,
7
]
,
9
3
-H2O
-
1
1
-
3
9
,
]
7
,
-HCl
6
[
-OH
-C3H6
-C2H4
]
[MH-C3H6-H2O]+
Cl
R1
N
Cl
10
[
H
OH
N
H
H
OH
N
H
H
Cl
-HCl
-H2O
[MH-2C3H6-H2O]+
OH
R1
N
In addition to the above, the CID spectra of the [M+H]+ ions
from N-ethyl and N-isopropyl containing N-oxides include loss of
CH3OH from [M+H]+ ions, and that from N-propyl containing N-
oxides show loss of C2H5OH. The ions by the loss of CH3OH from
[M+H]+ are all below 1% and the loss of C2H5OH creates more
intense ions, except for 13. These assignments are based on the
HRMS data. The observed loss of alcohol (methanol/ethanol) can
be explained involving a ␤-cleavage together with elimination of
–OH, through a concerted or a step-wise process as depicted in
Scheme 3. This proposed mechanism is similar to that envisaged to
explain the loss of (•OH + •CH2Cl) and H2O from [M+H]+ ions [18].
The compound 14 is a partially hydrolyzed product of 2, and
it has both 2-chloroethyl and 2-hydroxyethyl functional groups.
Thus, the CID spectrum of [M+H]+ ion from 14 include the ion
at m/z 106 and 110 corresponding to [MH−(•OH + •CH2OH)]+ and
[MH−C2H4O]+, respectively, the characteristic peaks for the N-
oxide of aminoethanol [18]. The spectrum also includes the ion at
m/z 88 corresponding to [MH−(•OH + •CH2Cl)]+ that is characteris-
tic of the N-oxide of aminoethyl-2-chloride (Scheme 4).
Scheme 3. General fragmentation pattern of [M+H]+ ion of compounds 1–13.
All the CID spectra (from 1 to 13) show a common product ion
due to the loss of 66 u. The [MH−66]+ is prominent for all the com-
pounds (70–100%), except for those having an N-isopropyl group
(7, 10, 12 and 13), where it is low abundant (<2%). High-resolution
data confirms that the loss of 66 u corresponds to the loss of CH3ClO.
Since this process is consistently observed in all the studied com-
pounds, irrespective of the size of the alkyl groups present on the
nitrogen, it can be presumed that the oxygen on nitrogen and the
chloroethyl group attached to nitrogen are involved in the expul-
sion of CH3ClO from the [M+H]+ ion. The precursor ion spectrum of
the [MH−CH3ClO]+ ion from 9 showed [M+H]+and [MH−OH]+• ions
as the precursors (spectrum not included). In fact the [MH−OH]+•
ion appeared in all the CID spectra. Thus, the loss of CH3ClO can
be explained by a step-wise process involving loss of two con-
secutive radicals (•OH followed by •CH2Cl) from the [M+H]+ ion.
The proposed step-wise mechanism is further supported from the
CID spectrum of the [MH−OH]+• ion, which exclusively consists
the peak due to the loss of •CH2Cl. The loss of 66 u from the
[M+H]+ ions in the current study is similar to the loss of 48 u
(•OH + •CH2OH) observed from the [M+H]+ ions of N-oxides of
dialkylaminoethanols. However, a contribution from direct loss of
HOCH2Cl from the [M+H]+ ion by a concerted process (Scheme 3)
cannot be ruled out for the formation of the [MH−CH3ClO]+ product
ion.
3.1.2. Differentiation of isomeric compounds
Among the studied N-oxides, some isomeric compounds are
present among the compounds 3–14. The isomerism is exclusively
due to size and structure of alkyl groups attached to the nitrogen
(structural isomers). One compound may be isomeric to another by
two different ways; (1) having difference in the structure of alkyl
group (e.g., propyl or isopropyl), and (2) having different size of
alkyl groups randomly on the nitrogen leading to same molecular
formulae (mixed alkyl groups). The N,N-dialkylaminoethane-2-
chlorides are listed in the CWC schedule list (2.B.10), and in this
case the alkyl groups can be methyl, ethyl, propyl or isopropyl.
Considering this information, three isomeric sets are possible for
the N-oxides of the N,N-dialkylaminoethyl-2-chlorides, i.e., com-
pounds 6–8, 9–10, and 11–13, possessing molecular weights of
151, 165, and 179, respectively. These isomeric compounds could
be easily differentiated based on the differences in the fragmenta-
tion of their [M+H]+ ions. The alkyl groups present on the nitrogen
atom are playing a major role in the differentiation of isomeric
compounds. Presence of N-propyl or N-isopropyl moiety is char-
acterized by the formation of abundant [MH−C3H6]+ ion, and this
ion is invariably more abundant in N-isopropyl compound than in
N-propyl compound. The [MH−C2H5OH]+ ion is observed for all
the compounds containing N-propyl group. These characteristic
The CID spectra of N-oxides of nitrogen mustards (1–3)
show a low abundant, but a conspicuous ion corresponding to
[MH−C2H4]+, as supported by HRMS data. Formation of this ion
in 1 and 2 can only be explained through a chlorine migration to
nitrogen. However, such a loss is not observed in the N-oxides of
N,N-dialkyl aminoethyl-2-chlorides.
3.1.1. Effect of alkyl substituents on nitrogen
In general, the [M+H]+ ions of alkyl substituted amines, where
the alkyl group is other than the methyl group, show an ion cor-
responding to the loss of an alkene after a hydrogen migration
from that alkyl group to nitrogen. In the present case, the N-
oxides of N,N-dialkylaminoethyl-2-chlorides (5–13) contain ethyl
and propyl/isopropyl groups, and hence, loss of an ethylene (C2H4)
and/or a propene (C3H6), is expected from their [M+H]+ ions.

L. Sridhar et al. / International Journal of Mass Spectrometry 333 (2013) 15–20
19
Fig. 2. CID mass spectra of [M+H]+ of (a) 9, and (b) 10.
3.1.2.3. Compounds 11, 12 and 13. As in the previous set of isomers
(9 and 10), this set of isomeric compounds (11–13) differ among
one another by the attachment of propyl and/or isopropyl to the
nitrogen. The dipropyl compound (11) shows the [MH−C2H5OH]+
ion (m/z 134) as the base peak, which is absent in the diisopropyl
compound (12). In contrast, the [MH−CH3OH]+ ion is specifically
observed in the compound 12, but it is a low abundant ion. In addi-
tion, the [MH−(•OH + •CH2Cl)]+ ion (m/z 114) is relatively more
abundant (70.5%) in 11 than in 12 (0.7%). The compound 12 shows
dominant product ions corresponding to [MH−C3H6]+ (m/z 138)
and [MH−2(C3H6)]+ (m/z 96), while these ions are relatively low
abundant in 11. The same trend is observed in the abundances of the
product ions that are formed by further losses of H2O and HCl from
the above two characteristic ions. With these specific/selective
characteristic ions, the isomers 11 and 12 can be easily recognized.
The compound 13 consists of both propyl and isopropyl group on
nitrogen, and hence show product ions due to mixed fragmentation.
The spectrum includes both [MH−CH3OH]+ and [MH−C2H5OH]+
ions. The [MH−C3H6]+ is more dominant (the base peak) in 13
when compared to that in 11 and 12. Therefore, the three isomeric
compounds in this group can be certainly identified without any
ambiguity.
Fig. 1. CID mass spectra of [M+H]+ of (a) 6, (b) 7, and (c) 8.
fragmentations that facilitate unambiguous differentiation of the
isomeric compounds are discussed below.
3.1.2.1. Compounds 6–8. The 6 and 7 have propyl or isopropyl
group on nitrogen, hence show the ions [MH−C3H6]+ (m/z 110)
[MH−C3H6−H2O]+ (m/z 92), and [MH−C3H6−HCl]+ (m/z 74), which
are specifically formed by the involvement of propyl/isopropyl
group (Fig. 1). These ions are absent in compound 8 because both
the alkyl groups are ethyl, thus the compound 8 can be easily dis-
tinguished from 6 and 7. The compounds 6 and 7 are isomeric
due to the attachment of propyl (6) or isopropyl (7) to the nitro-
gen. The [MH−C2H5OH]+ ion (m/z 106) specifically appear in 6
due to the presence of a propyl group, whereas it is absent in 7.
The [MH−C3H6]+ ion (m/z 110) is abundant in 7 (the base peak)
because of the facile expulsion of C3H6 from an isopropyl group,
whereas it is low abundant (21%) in 6 that having a propyl group.
The [MH−(•OH + •CH2Cl)]+ ion (m/z 86) is the base peak in 6, but in
contrast this ion is insignificant in 7 (1%).
3.2. LC–MS and LC–MS/MS analysis
LC–MS experiments were performed choosing the compounds
3 (N-alkyl) and 6 (N,N-dialkyl) as examples. The data (Fig. S1)
revealed that the compounds were successfully eluted from the col-
umn. The compounds 3 and 6 were eluted at 2.69 min and 3.60 min,
respectively, under the used LC conditions. Both the compounds
showed abundant [M+H]+ ions in their LC-ESI mass spectra. The
LC–MS/MS spectra of the [M+H]+ ions of 3 and 6 obtained from
the ion trap instrument were closely matching with that obtained
from Q-TOF mass spectrometer by direct ESI-MS/MS analyses. As
expected, there were some differences in the relative abundances
of the product ions in the spectra obtained from same compound
using two different instruments. However, all the structure indica-
tive fragments are present in both the spectra. This data suggests
that LC–MS/MS can also be applied for the verification of the studied
compounds in a sample mixture.
3.1.2.2. Compounds 9 and 10. The compounds 9 and 10 are isomeric
to each other because of the difference in the attached C3 alkyl
group (propyl or an isopropyl) to the nitrogen. Hence, the [M+H]+
ion fragmentation of 9 and 10 is solely controlled by the propyl and
isopropyl groups (Fig. 2). As expected, the [MH−C3H6]+ ion (m/z
124) is the base peak in 10 having an isopropyl group, whereas it
is low abundant (16.6%) in 9 having a propyl group. Likewise, the
[MH−C3H6−H2O]+, [MH−C3H6−OH]+• and [MH−C3H6−HCl]+ ions
are dominant only in 10. The [MH−C2H5OH]+ ion is specifically
observed in compound 9 that easily distinguishes the compound 9
from 10. The [MH−(•OH + •CH2Cl)]+ ion is the base peak in 9, but it
is low abundant in 10 (1.2%).

20
L. Sridhar et al. / International Journal of Mass Spectrometry 333 (2013) 15–20
4. Conclusion
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because they can be used as environmental and biological mark-
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