Identification of position isomers by energy-resolved mass spectrometry

Article abstract of DOI:10.1002/jms.3607

This study reports an energy-resolved mass spectrometric (ERMS) strategy for the characterization of position isomers derived from the reaction of hydroxyl radicals (^-OH) with diphenhydramine (DPH) that are usually hard to differentiate by other methods. The isomer analogues formed by ^?OH attack on the side chain of DPH are identified with the help of a specific fragment ion peak (m/z 88) in the collision-induced dissociation (CID) spectrum of the protonated molecule. In the negative ion mode, the breakdown curves of the deprotonated molecules show an order of stability (supported by density functional theory (DFT) calculations) ortho > meta > para of the positional isomers formed by the hydroxylation of the aromatic ring. The gas phase stability of the deprotonated molecules [M - H]^- towards the benzylic cleavage depends mainly on the formation of intramolecular hydrogen bonds and of the mesomeric effect of the phenol hydroxyl. The [M - H]^- molecules of ortho and meta isomers result a peak at m/z 183 with notably different intensities because of the presence/absence of an intramolecular hydrogen bonding between the OH group and C9 protons. The ERMS approach discussed in this report might be an effective replacement for the conventional methods that requires very costly and time-consuming separation/purification methods along with the use of multi-spectroscopic methods.

Full text of DOI:10.1002/jms.3607

Journal of
MASS
SPECTROMETRY
Research article
Received: 14 December 2014
Revised: 14 March 2015
Accepted: 22 April 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3607
Identification of position isomers by
energy-resolved mass spectrometry
a
b
c
Sunil Paul M. Menachery, Olivier Laprévote, Thao P. Nguyen,
d
e
a,f
Usha K. Aravind, Pramod Gopinathan and Charuvila T. Aravindakumar *
This study reports an energy-resolved mass spectrometric (ERMS) strategy for the characterization of position isomers derived
●
from the reaction of hydroxyl radicals ( OH) with diphenhydramine (DPH) that are usually hard to differentiate by other methods.
●
The isomer analogues formed by OH attack on the side chain of DPH are identified with the help of a specific fragment ion peak
(m/z 88) in the collision-induced dissociation (CID) spectrum of the protonated molecule. In the negative ion mode, the breakdown
curves of the deprotonated molecules show an order of stability (supported by density functional theory (DFT) calculations)
ortho> meta> para of the positional isomers formed by the hydroxylation of the aromatic ring. The gas phase stability of the
ꢀ
deprotonated molecules [M ꢀ H] towards the benzylic cleavage depends mainly on the formation of intramolecular hydrogen
ꢀ
bonds and of the mesomeric effect of the phenol hydroxyl. The [Mꢀ H] molecules of ortho and meta isomers result a peak at
m/z 183 with notably different intensities because of the presence/absence of an intramolecular hydrogen bonding between
the OH group and C9 protons. The ERMS approach discussed in this report might be an effective replacement for the conventional
methods that requires very costly and time-consuming separation/purification methods along with the use of multi-spectroscopic
methods. Copyright © 2015 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: position isomerism; energy-resolved mass spectrometry; hydroxyl radicals; collision-induced dissociation; mesomeric effect
though the liquid chromatography (LC) system can separate each
of them.
Introduction
[15]
●
The crucial role of hydroxyl radicals ( OH) in the oxidative destruc-
tion of environmental pollutants in Advanced Oxidation Processes
A possible alternative to gain the structural characteristics of
these isomers is the investigation of the fragmentation of their
gas phase ions using energy-resolved mass spectrometry (ERMS).
The technique involves the collisional activation of the gas phase
ions either at the interface of an electrospray ionization source or
(AOPs) and in many biological processes including DNA damage,
[1–8]
mutation and ageing is well established.
The non position-
selective reaction of ●OH with aromatic compounds generates
isomeric hydroxylated compounds in various chemical pro-
by means of an inert collision gas in tandem mass spectro-
[6,9,10]
[16,17]
cesses.
absorption spectra obtained by pulse radiolysis experiments
a widely used technique to probe the spectroscopic details of
The exact structural assignments of the transient
metry.
By using the low-energy collisional activation and tan-
dem mass spectrometry, one can control the amount of energy
transferred in to an ion by varying the collision energy (CE) and
(
the transient intermediate species) are consequently very difficult,
and hence many authors interpret the experimental spectra in to
[
6,10–13]
a cumulative effect of undistinguishable OH adducts.
An
*
Correspondence to: Charuvila T. Aravindakumar, Inter University Instrumentation
Centre (IUIC), Mahatma Gandhi University, Priyadarsini Hills, Kottayam, Kerala.
India. E-mail: cta@mgu.ac.in
important challenge in analytical chemistry over the past few de-
cades was therefore the development of suitable techniques for
the rapid characterization of these isomers. Nuclear magnetic reso-
nance (NMR) spectroscopy is an undisputed tool for the chemical
characterization of any kind of organic molecules. However, re-
quirement of relatively large sample amounts, high cost of deuter-
ated solvents and complicated (and costly) pre-separation/
a School of Environmental Sciences, Mahatma Gandhi University, Priyadarsini Hills,
Kottayam, Kerala, India
b Laboratory of Analytical Chemistry and Experimental Toxicology, University Paris
Descartes, Paris, France
[
14]
purification steps are the major drawback of this technique.
c Department of Chemistry, Pohang University of Science and Technology
Another possibility is the use of authentic standards that are
relatively costly and unavailable in many cases. The utilization of
tandem mass spectrometry alone is exceedingly difficult in this
context because of the close structural similarities of hydroxylated
isomers. In a previous report, Fu et al. suggested that the exact
structural assignment of individual isomers is very challenging
because of the close resemblances in the collision-induced dissoci-
ation (CID) spectra (commonly referred as MS/MS spectra) even
(
POSTECH), Pohang 790-784, Republic of Korea
d Advanced Centre of Environmental Studies and Sustainable Development
(ACESSD), Mahatma Gandhi University, Priyadarsini Hills, Kottayam, Kerala, India
e Department of Chemistry, N.S.S. Hindu College, Changanachery, Kerala, India
f
Inter University Instrumentation Centre (IUIC), Mahatma Gandhi University,
Priyadarsini Hills, Kottayam, Kerala, India
J. Mass Spectrom. 2015, 50, 944–950
Copyright © 2015 John Wiley & Sons, Ltd.

Journal of
MASS
SPECTROMETRY
Position isomers by ERMS
accordingly the fragmentation rate. The compound specific ERMS
curves are therefore exploited by many authors for the characteri-
zation of position and stereo isomers and even for conformational
Gaussian 09 W in order to predict the relative stability of individual
isomers.
[
24]
[16–19]
analysis.
Results and discussion
In this report, we present an ERMS-based strategy for the rapid
characterization of position isomers derived from the reaction of
Photo-irradiation experiments of the aqueous solution of DPH in
●
OH with diphenhydramine (DPH), a first-generation antihistamine
2 2
the presence of H O were performed on a medium pressure
and a potential candidate from the group of so-called ‘emerging
organic contaminants’. Destruction of this compound by various
mercury lamp (254nm)-based photo-reactor. The interaction of
●
UV radiation with H
according to reaction 1.
2
O
2
is reported to produce OH in the medium
[
9,20]
AOPs is thoroughly experimented.
Very recently, Feng et al.
[11]
reported that the reaction of ●OH on DPH yields different iso-
[
9]
•
meric hydroxylated compounds. The authors were however
unable to characterize the individual isomers because of the
lack of significant differences in the MS/MS spectra. Testa
et al. suggested that the pharmacological activities and the
affinity for specific targets were retained in the case of hydrox-
H O þ hν→2 OH
(1)
2
2
Analysis of hydroxylated products by mass spectrometry
[
21]
ylated metabolites.
Characterizations of these isomeric com-
The samples resulting from the photo-irradiation experiments were
subjected to HRMS analysis to elucidate the identity of the major
transformation products formed during the reaction. Total ion chro-
matogram (TIC) in the positive ionization mode as shown in Fig. S1
shows four reasonably resolved peaks at RT 4.6 (A), 4.7 (B), 4.9 (C)
and 5.2min (D) corresponding to a mass-to-charge ratio (m/z) of
pounds are thus very interesting and highly important for the
evaluation of the pharmacological (and hence toxicological)
status of these reactions. Further, the minute structural details
of these kinds are very necessary to obtain an in-depth under-
standing of the transformation mechanism of DPH which is utmost
important in biological and environmental systems. To the best
of our knowledge, this is the first report on the application of
ERMS for the characterization of position isomers derived from
2
72 (obs. 272.1646; calcd. for C H NO 272.1645). The comparison
17 22 2
of the accurate mass and the elemental composition of these peaks
with that of the parent molecule (obs. 256.1697; calcd. for C H NO
17 22
●
the reaction of OH with any organic molecule of biological
256.1701) suggested that the difference in the m/z value is likely
and environmental importance, though a number of ERMS-based
isomeric characterizations were experimented with authentic
because of the hydroxylation of the parent compound.
[10,16,17,19,22]
standards.
CID experiments in positive ionization mode
Further, structural features are gained by performing CID experi-
+
ments of the respective precursor ions [M + H] . The corresponding
MS/MS spectra are shown in Fig. 1. The protonated molecules of A,
B and C at m/z 272 led to an abundant fragment at m/z 183 (obs.
Materials and methods
Diphenhydramine (CAS No. 58-73-1) was purchased from Sigma
Aldrich and was used without further purification. All other re-
agents used in the present study were in the highest available
purities. The various hydroxylated analogues of DPH were analysed
by ultra performance liquid chromatography (UPLC) coupled with a
quadrupole-time-of-flight (Q-TOF) high resolution mass spectrome-
ter (Xevo G2, Waters, USA). The samples were ionized by elec-
trospray ionization (ESI) after separating on a BEH C18 column
183.0814; calcd. for C13H11O 183.0810). This m/z value is around
16 Da higher than 167 (obs. 167.0857; calcd. for C13H11 167.0861),
which is the major fragment observed in the MS/MS spectrum of
protonated DPH. On the basis of the above observations, it is sug-
gested that the mass difference of 16Da is because of the hydrox-
ylation of the aromatic part of DPH.
Conversely, the MS/MS spectrum of D exhibits an intense frag-
ment at m/z 167. Because of the presence of a similar fragment in
the MS/MS spectrum of the parent molecule (Fig. S2), it seems
highly logical that the aromatic part of this hydroxylated derivative
remained intact, the hydroxyl group being located on the side
(100 mm × 2.1mm×1.7 μm, Waters). An elution gradient of metha-
nol and water (both having 1 mM ammonium acetate) at a flow rate
ꢀ
1
of 0.25 ml min was used as the mobile phase. The mass spectra
were recorded in both positive and negative ionization modes in
m/z ranges of 50–600 Th. The CID spectra were recorded by
fragmenting the selected precursor ions by collision with high
purity argon gas. For obtaining mass accuracy in high resolution
●
chain. The hydrogen abstraction reaction of OH from one of the
methyl hydrogen atoms of DPH followed by further hydroxylation
is a probable explanation for the formation of this product. A similar
product was also observed by Feng et al. during their end-product
+
ꢀ
mass spectra, leucine enkephalin ([M+ H] : 556.2771; [M ꢀ H] :
[
23]
[9]
554.2615) was used as a reference standard.
The stability of
studies. To explain the identity of this compound, the authors
different isomeric hydroxylated DPH analogues, identified by the
high resolution mass spectrometry, was further evaluated by ERMS
analysis. The ERMS curves were determined by recording the CID
spectrum of individual compounds using a series of collision ener-
gies (CE) starting from 6 eV and increased until the precursor ions
no longer exist. The structures of tested subjects were fully relaxed
without geometry constraint using density functional theory (DFT)
calculations with hybrid B3LYP functional using basis set 6-31G,
propose a C10-hydroxyl substituted DPH analogue with the help
of limited MS/MS studies. However, the assignment of an exact
structure for this spectrum in the case of compounds like DPH
seems to be very difficult because of the availability of more than
one hydrogen atoms (one at C , two at C and C and three at
7
9
10
●
C12 and C , Scheme 1) which could be the target of OH attack.
13
The small, however potentially important, fragment at m/z 88
(obs. 88.0767; calcd. for C H NO 88.0762) in the MS/MS spectrum
4
10
6-31G(d,p) and polarized basis set 6-31G+, 6-31G+(d). In addition
of D was not observed in the case of other precursor ions of A–C
B3PW91 method with basis set 6-31G+ was used, and a compara-
tive study on the effect of basis sets on the calculated structures
was performed. All the calculations were carried out using
(Fig. 2). This fragment ion allows the assignment of compound D
[9]
as either the C10 (an identical structure proposed by Feng et al.)
or C /C hydroxylated derivative of DPH. This assignment is based
12
13
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S.P.M. Menachery et al.
Figure 2. CID spectra of the deprotonated molecules of A–C (m/z 270).
Collision energy: 10 eV.
molecule of water from the complementary fragment ion at m/z
106 is very likely at the origin of the m/z 88 ion signal (Scheme 1).
Reports on the benzylic dissociation in the case of diaryl-
pyrimidines, benzyl esters and thioethers are available in the
Figure 1. The CID spectra of the protonated ions of A–D. Collision energy:
[
25]
[9]
literature. The MS/MS spectrum reported by Feng et al. how-
ever lacks the fragment ion peaks at m/z 88, 90 and 167. It is thus
reasonable to rule out the C10 hydroxylated analogue of DPH from
10 eV.
●
the present case. The possibility of OH attack on the nitrogen atom
of DPH seems very unlikely because of the lack of stabilization of
resulting nitrogen-centred radical cation. The above stated reasons
allow us to attribute a C12/C13 hydroxylated derivative of DPH for
isomer D. Relatively less intense fragments at m/z 90 (obs.
9
0.0919; calcd. for C
for C13 11O 183.0811) were also observed in this spectrum (Fig. 1D).
The identity of these fragment ions in the case of [D + H] is ratio-
nalized by the dissociation of oxygen (O )–carbon (C ) bond as
shown in Scheme 1.
4
H12NO 90.0919) and 183 (obs. 183.0808; calcd.
H
+
8
9
On the other hand, three distinguished sites namely ortho (2, 2′,
●
6
, 6′), meta (3, 3′, 5, 5′) and para (4, 4′) are available for OH attack
on the aromatic part of DPH (Scheme 1). The exact assignments
of the individual isomers (A–C) are very difficult at this stage be-
cause of the close structural features and significant similarities in
the MS/MS spectra. However, identification of the corresponding
isomeric hydroxylated products of DPH, which might include ortho,
meta and para derivatives, is utmost important for evaluating the
pharmacological activities of the metabolites/transformation prod-
ucts of compounds like DPH.
Scheme 1. Proposed fragmentation pathways of the (M + H) + ion (m/z
72) of D leading to the formation of fragment ions at m/z 167, 88 (1), 183
2
and 90 (2).
CID experiments in negative ionization mode
In order to simplify the interpretation, the CID spectra correspond-
ing to deprotonated A–C were recorded in the negative ionization
mode. It is noteworthy to mention the absence of peak D in the
case of the negative ionization mode (Fig. S1). A likely reason for
on the observed instability of the N-hydroxymethyl intermediate
which leads to a diphenylmethylium ion (m/z 167) by benzylic dis-
sociation of the precursor ion [D + H] (Scheme 1). The loss of a
+
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Position isomers by ERMS
the absence of this peak is the lack of ionization of the correspond-
ing product. It can be attributed to a lower gas-phase acidity of the
alcohol function with regards to the phenol group. The influence of
the hydroxylation site (such as ortho, meta and para) on the gas-
phase stability of the protonated molecules of A–C is however
much less significant because the OH group is far away from the
protonation site (very likely the tertiary amino group). By contrast,
the stability of the deprotonated molecules of A–C (obs.
trend and indicated that there is a big gap in energy between meta
and ortho isomer predicting the stability in ortho structure. The re-
sults clearly showed that the gas phase ion of ortho analogue has
the lowest energy and is therefore the most stable. The energy dif-
ference (B3LYP/6-31G+(d)) between ortho to meta and meta to para
ꢀ
1
(least stable) were 3.16 and 1.93kcal mol , respectively. More spe-
cific results are shown in Table S1.
It is clearly visible from the optimized structure of ortho isomer
(Fig. 3) that the electronegative deprotonated phenol oxygen and
the alkyl hydrogen atoms are very close to each other. The stabiliza-
tion of gas phase ions by hydrogen bonding is reported in the case
2
270.1494; calcd. for C17H20NO 270.1494) is expected to be drasti-
cally affected by the position of the hydroxyl group because of
the involvement of the negative charge in the aromatic system
and of the expected mesomeric effects on the benzylic bond disso-
ciation. Consequently, the ease in fragmentation of these ions is
likely different for A–C. In this context, it was assumed that the dif-
ferences in the MS/MS spectra and ERMS curves of different ion
peaks could offer valuable information about the exact position of
the OH group.
[16,26,27]
of a range of compounds including biomolecules.
In the
case of ortho isomer, it is thus very logical to expect the formation
of an intramolecular hydrogen bonding between the above speci-
fied atoms. A similar stabilization in the case of meta and pare ana-
logues is however very unlikely because the phenol oxygen is
geometrically very far from the alkyl hydrogen atoms. On the basis
of theoretical calculations, it is thus expected that the gas phase
deprotonated ion of the para isomer induces higher fragmentation
rates.
The MS/MS spectra of the deprotonated molecules of A–C are
ꢀ
ꢀ
largely different (Fig. 2). The precursor ions [A ꢀ H] and [B ꢀ H]
led to an identical fragment at m/z 181 (obs. 181.0653; calcd. for
13 9
C H O 181.0653) but with very different intensities. Comparison
ꢀ
ꢀ
of the CID spectra of the [B ꢀ H] and [C ꢀ H] precursor ions at
m/z 270 showed an additional ion peak at m/z 183 (obs. 183.0804;
calcd. for C H O 183.0811) for the compound C with an intensity
ERMS analysis
To predict the stability of various deprotonated ions, ERMS studies
were performed on a range of CE. The results are shown in Fig. 4.
The relative ion current corresponding to the individual ions was
calculated using the following equation, where Ci and Cp stand
1
3 11
similar to the m/z 181 ion. A small, however potentially important,
peak at m/z 88 (obs. 88.0756; calcd. for C H NO 88.0762) was ob-
4
ꢀ
10
ꢀ
served in the case of [A ꢀ H] and [C ꢀ H] whereas this fragment
ꢀ
was not observed in the CID spectrum of [B ꢀ H] . Additionally, a
peak at m/z 153 (obs. 153.0700; calcd. for C H 153.0704) is visible
1
2 9
in the case of the C isomer. An extremely small peak at m/z 199
obs. 199.0755; calcd. for C H O 199.0759) is also observed in
(
13 11 2
the case of A and C. Further, the intensity of the precursor ion
ꢀ
[
A ꢀ H] was less than 30% at a CE of 10 eV whereas the other
two precursor ions formed from B and C showed a much higher
gas-phase stability under the same CE conditions.
Theoretical calculations
Theoretical calculations of gas phase deprotonated ions using DFT
method have been mainly carried out to find out the relative stabil-
ities of individual isomers. The two different DFT methods used in
this study showed very similar result in predicting the stability of
each ion. The optimized geometries of gas phase deprotonated
ions of ortho, meta and para are shown in Fig. 3. Application of
polarized basis sets showed a slight decrease in the molecular
energy difference among three isomers compared to 6-31G(d,p)
and 6-31G basis sets. However, all of the methods showed the same
Figure 4. Plot of relative ion current versus collision energy corresponding
to m/z 270 (A (•), B (▪) and (▲) C) and m/z 181 (A (ο), B (□) and (◊) C).
Figure 3. Optimized structure of the deprotonated gas phase ions of A–C (basis set B3LYP/6-31G+(d)).
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S.P.M. Menachery et al.
for the observed ion current of a specific product ion as well as the
parent ion, respectively.
Further, the fragment at m/z 183 which was observed in the case
of C and which led to a very weak signal for B explicitly justifies the
assessment of ortho and meta isomers in to B and C, respectively
[
28]
ꢀ
ꢀ
X
ꢁꢁ
rel
(Fig. 2). Formation of this fragment is indeed easily rationalized with
C ¼ 100ꢁ Ci= Cp þ
Ci
(2)
the help of an intramolecular proton transfer followed by the re-
lease of a (dimethylamino)acetaldehyde moiety as depicted in
Scheme 3.
The ERMS curves of the deprotonated molecules displayed a
ꢀ
5
0% dissociation rate of the [A ꢀ H] ion at a CE of near 8.0eV
with a fast increase of the intensity of the fragment ion at m/z
The proposed mechanism corresponding to the formation of m/z
183 is very unlikely in the case of para analogue because of the
large distance between the phenoxide group and the alkyl hydro-
gen atoms. The absence (or very negligible intensity) of this frag-
ment in the MS/MS spectrum of the para isomer is hence
unambiguously anticipated. It is noteworthy to specify that the
small ion peaks at m/z 182 (obs. 182.0683) and 183 (obs.
183.0769) observed in the MS/MS spectrum of A (Fig. 2) are mainly
ꢀ
ꢀ
1
81 (Fig. 4). By comparison, [B ꢀ H] and [C ꢀ H] were much more
stable showing a 50% dissociation rate at 19.5 and 14.5 eV, respec-
tively. Further, the enhancement of the m/z 181 fragment intensi-
ties was relatively slow in the case of the deprotonated B and C
molecules suggesting a much higher stability of these ions toward
this fragmentation pathway.
The presence of hydroxyl groups at the ortho and para positions
is an important factor that enhances the benzylic dissociation
responsible for the formation of the dimethylamino ethanolate
anion (m/z 88) along with the complementary ortho/para quinone
methide ion as depicted in Scheme 2. The cleavage of the benzylic
carbon–oxygen bond leads directly to the m/z 88 fragment ion or,
via a proton transfer within an intermediate ion-neutral complex,
to the fragment at m/z 181. The involvement of phenol oxygen
atom in the intramolecular hydrogen bonding, that reduces elec-
tron density, however restricts this mesomeric enhancement in
the case of ortho isomer. Furthermore, the fragmentation of the
benzylic bond is expected to be less effective in the case of the
meta analogue because of the lack of any mesomeric enhance-
ment. These effects should reduce the fragmentation rate of the
deprotonated ortho and meta isomer and increase its gas-phase
stability. Because the ERMS analysis reveals that the stability of
the deprotonated molecules are in the order B > C > A (Fig. 4),
we assigned the ortho structure, which is predicted as the most
stable one by theoretical calculations, to the compound B. The
two other structures, i.e. meta and para, remained to be attributed
to the compounds A and C. The deprotonated ion of compound A
was clearly the less stable one and was then assigned the structure
para. Indeed the position of the phenoxy group does not allow any
intramolecular hydrogen bonding between the negative charge
and the rest of the structure. As a consequence, the compound C
was identified as the meta isomer.
13
because of the contribution from C isotope of fragment ion peak
at m/z 181, and then significantly differ from the m/z 183 fragment
ion (obs. 183.0804) formed from deprotonated B and C. The intra-
molecular hydrogen bonding involved in the case of ortho ana-
logue is expected to restrict the feasibility of this fragmentation
pathway though phenoxide group is very near to the alkyl hydro-
gen atoms. Consequently, the intensity of the corresponding frag-
ꢀ
ment at m/z 183 is very much weaker in the case of the [B ꢀ H]
ion. The reasonable distance between the phenoxide group and al-
kyl hydrogen atoms and the lack of intramolecular hydrogen bond-
ing result the most intense fragment peak corresponding to m/z
183, which is more or less equal to that of the fragment at m/z
181, in the case of meta isomer (Fig. 2).
Formation of remaining fragment ion such as m/z 153 and 199 is
explained in Scheme 4. The small fragment at m/z 153, which is
observed in the case of C (Fig. 2) as well as in the case of A and B
at higher CE values of 22 and 18, respectively (Figure S3 and
S4), is rationalized by a routine loss of CO (27.9949) from the
fragment at m/z 181 and corresponds to a cyclopentadienyl-type
Scheme 3. Proposed fragmentation pathway leading to the formation of
the fragment ion at m/z 183.
Scheme 2. Proposed fragmentation pathways of m/z 270 ion derived from
A and B.
Scheme 4. Proposed fragmentation pathway leading to the formation of
fragment ions at m/z 153 (1) and 199 (2).
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Position isomers by ERMS
[
10] R. Sreekanth, S. P. M. Menachery, U. K. Aravind, J.-L. Marignier, J. Belloni,
C. T. Aravindakumar. Oxidation reactions of hydroxy naphthoquinones:
Mechanistic investigation by LC-Q-TOF-MS analysis. Int. J. Radiat. Biol.
anion. The minute fragments observed at m/z 199 in the case of
para and meta analogues are likely due to a similar dissociation
of carbon–oxygen bond as explained in the case of protonated
molecule of D. The proposed structure of fragment ions at
m/z 153 and 199 is depicted in Scheme 4.
2
014, 90, 495.
[11] G. V. Buxton, C. L. Greenstock, W. P. Helman, A. B. Ross. Critical review of
rate constants for reactions of hydrated electrons, hydrogen atoms and
hydroxyl radicals ([center-dot]OH/[center-dot]O[sup - ] in aqueous
solution. J. Phys. Chem. Ref. Data 1988, 17, 513.
[
12] M. S. Kulkarni, P. Yadav, M. B. Shirdhonkar, B. S. M. Rao. Pulse radiolysis:
Pune University LINAC facility. Curr. Sci. 2007, 92, 599.
Conclusions
[
13] M. M. Sunil Paul, U. K. Aravind, G. Pramod, A. Saha, C. T. Aravindakumar.
Hydroxyl radical induced oxidation of theophylline in water: A kinetic
and mechanistic study. Org. Biomol. Chem. 2014, 12, 5611.
In conclusion, a very simple, rapid and cost-effective method, based
on energy-resolved mass spectrometry, has been developed for the
real-time characterization of position isomers (such as ortho, meta
and para) derived from the reaction of hydroxyl radical with
diphenhydramine. Because of the close structural similarities, the
characterization of position isomers of these kind otherwise re-
quires highly difficult and costly separation/purification procedures
and the use of multi spectroscopic techniques such as nuclear
magnetic resonance (NMR) and infrared (IR) spectroscopy. The
present strategy does not necessitate any time consuming purifica-
tion steps and use of any costly standards. It allows real-time
characterization of the position isomers within a chromatographic
run time of 6 min. In this context, the novel ERMS-based methodol-
ogy demonstrated in the present study is extremely relevant.
Further studies are however necessary for the development of a
more generalized procedure applicable for more number of mole-
cules which are currently in progress.
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Acknowledgements
This work was performed under collaborative research scheme
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(Ref No. UGC-DAE-CSR-KC/CRS/09/RC02/1455) of UGC-DAE Consor-
tium for Scientific Research, Kolkata Centre, Kolkata. The authors
acknowledge Dr. Ji Hoon Shim, Department of Chemistry, Pohang
University of Science and Technology (POSTECH), Republic of
Korea, for the computational facility. CTA is thankful to DST (under
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