gas-phase fragmentation of protonated benzodiazepines
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RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2007; 21: 2273–2281
) DOI: 10.1002/rcm.3084
Published online in Wiley InterScience (www.interscience.wiley.comGas-phase fragmentation of protonated benzodiazepines
Antonella Risoli1,2, Joey B. Y. Cheng1, Udo H. Verkerk1, Junfang Zhao1, Gaetano Ragno2,
Alan C. Hopkinson1 and K. W. Michael Siu1*1Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario,
Canada M3J 1P32Department of Pharmaceutical Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy
Received 5 April 2007; Revised 7 May 2007; Accepted 8 May 2007
*CorrespoCentre foKeele StrE-mail: kContract/Research
Protonated 1,4-benzodiazepines dissociate in the gas phase by the common pathway of CO eli-
mination and by unique pathways dictated by the substituents; the latter typically differentiate
one benzodiazepine from another. Protonated 3-dihydro-5-phenyl-1,4-benzodiazepin-2-one, the
base diazepam devoid of substituents, dissociates by eliminating CO, HNCO, benzene, and ben-
zonitrile. Mechanisms of these reactions are proposed with ionic products being resonance stabil-
ized. The abundant [MHSCO]R ion dissociates to secondary products via elimination of benzene,
benzonitrile, the NH2 radical, and ammonia, yielding again ionic products that are stabilized by
resonance. Copyright # 2007 John Wiley & Sons, Ltd.
Benzodiazepines were discovered serendipitously in 1957
in the Roche laboratories.1 The benzodiazepine class of
drugs consists of approximately 50 commercially available
compounds with anxiolytic, muscle-relaxant and sedative
effects.2,3 In clinical practice these drugs are widely pres-
cribed for the treatment of insomnia and anxiety disorders.
Benzodiazepines are central nervous system (CNS) depress-
ants4,5 and are characterized by a strong and specific inter-
action with local g-aminobutyric acid chloride channel
receptor (GABAA) sites.6–10 Various benzodiazepine deriva-
tives were originally marketed as an improvement over the
then available barbiturates with regard to overdose effects,
drug interactions, dependence development, and illicit
abuse. Over-prescription in the 1970s and 1980s revealed
side effects and led to illicit use; this eventually resulted in a
decline in benzodiazepine prescriptions.3 Use of benzo-
diazepines can lead to physiological and psychological
withdrawal symptoms upon discontinuation;11,12 when
taken in combination with other CNS depressants, includ-
ing alcohol, benzodiazepines can induce severe, even fatal,
respiratory failure.13–15
The determination of benzodiazepines is often required
in forensic and clinical analyses. The large number of
benzodiazepines, together with the multiple metabolites of
varying biological activity that each of these will form,2,3,12,16
poses a significant analytical challenge.12,13,17 Currently,
gas chromatography (GC)18 and liquid chromatography
(LC)17,19–22 coupled with mass spectrometry (MS) are the
most commonly employed methods. MS has superior selec-
tivity and sensitivity than other detection methods; MS
evidence is considered to be the most reliable and less likely
ndence to: K. W. M. Siu, Department of Chemistry andr Research inMass Spectrometry, York University, 4700eet, Toronto, Ontario, Canada M3J 1P3.wmsiu@yorku.cagrant sponsor: Natural Sciences and EngineeringCouncil (NSERC) of Canada and MDS SCIEX.
to be challenged in court.12,23 While electron ionization is the
major ionization technique in GC/MS,24–27 softer ionization
techniques, including electrospray ionization (ESI), have
been widely used in LC/MS in recent years. For these softer
ionization techniques, structural information on the benzo-
diazepines can be derived from tandem mass spectrometry
(MS/MS). Qualitative analysis has been demonstrated using
an MS/MS library acquired under specific experimental
conditions.28,29
In general, the collision-induced dissociation (CID) of
benzodiazepines results in rich MS/MS spectra.11,17,28–34
Typically, the cleavage of characteristic substituents, includ-
ing Cl, NO2 and OH, coupled with GC or LC retention times
and molecular weights discernible from ESI, can result in the
(near) unambiguous identification of existing benzo-
diazepines. The present study was driven by a desire to
better understand the CID fragmentation chemistries in
order to enhance the ability to assign structural components,
when a new benzodiazepine is encountered. Towards this
end, we first examine the intrinsic dissociation reactions
of benzodiazepines by analyzing the CID of a skeletal
benzodiazepine, 3-dihydro-5-phenyl-1,4-benzodiazepin-2-
one (see Scheme 1 for the structure), that is devoid of any
functional group which typically differentiates the various
benzodiazepine products. For simplicity, this benzo-
diazepine will be referred hereafter as the base diazepam.
Characteristic fragmentations of benzodiazepine products
will then be introduced and discussed with respect to
features that will aid identification of these products by
recognizing the functional groups or substituents that they
carry.
Copyright # 2007 John Wiley & Sons, Ltd.
Scheme 1.
2274 A. Risoli et al.
EXPERIMENTAL
MaterialsNitrazepam, norfludiazepam, lorazepam (see Scheme 1 for
structures and molecular weights) were obtained from
Cambridge Isotope Laboratories (Quebec, Canada) as
solutions at a certified concentration of 1mg/mL in
methanol. All other benzodiazepines, including base diaze-
pam, nordiazepam, clonazepam, diazepam, oxazepam,
temazepam, and flunitrazepam (Scheme 1), at 10 ng/mL in
water/methanol, were a gift from MDS SCIEX (Concord,
ON, Canada). Sample solutions for MS analysis were at a
concentration of 4mM in 50:50 water/methanol containing
1% acetic acid. Methanol and acetic acid were from
Sigma (St. Louis, MO, USA); water was purified using a
Milli-Q system (Millipore, Billerica, MA, USA). All diazepam
materials were used as received and stored at 48C except for
analysis.
Mass spectrometryExperiments were conducted on a pre-commercial version of
the QTRAP linear ion-trap mass spectrometer (Applied
Biosystem/MDS SCIEX). Sample solutions were infused
into the pneumatically assisted ESI source at a flow rate
Copyright # 2007 John Wiley & Sons, Ltd.
of 2–3mL/min using a syringe pump. The typical ESI voltage
was between 5 and 5.5 kV. Dry air was used as the nebuli-
zer gas. The orifice and lens voltages were optimized
for maximum precursor ion signal intensity and between
100 and 200 scans were summed to produce a mass
spectrum. In a typical MS/MS experiment, the precursor
ion was mass-selected using the first quadrupole (Q1),
allowed to collide with nitrogen at a pressure of approxi-
mately 7.5mTorr (CAD gas setting of 4) in the second
quadrupole (q2), and mass-analyzed using the third
quadrupole (Q3). For chlorine-containing benzodiazepines,
unless otherwise stated, the reported results are based on
analyses of the 35Cl-containing ions, although the 35Cl- and
the 37Cl- containing ions were both examined to verify ion
assignments. For CID, the laboratory collision energy (Elab)
typically varied between 5 and 50 eV (100 eV for the base
diazepam). The ion lineage of specific product ions was
further investigated using the enhanced product ion (EPI)
scan mode, which traps and accumulates product ions in Q3
for better sensitivity.
Computational methodsGeometry optimizations and energy calculations were
performed with Gaussian 0335 using the non-local hybrid
Rapid Commun. Mass Spectrom. 2007; 21: 2273–2281
DOI: 10.1002/rcm
Figure 1. Product ion spectrum of protonated base diaze-
pam, m/z 237, at Elab¼ 30 eV.
Gas-phase fragmentation of protonated benzodiazepines 2275
three parameter B3LYP density functional theory (DFT)
approach36,37 with the 6-31þG(d) split-valence basis sets. All
stationary points were characterized by harmonic frequen-
cies as local minima (all real frequencies) and saddle points
(one imaginary frequency). Connections between transition
states and corresponding minima were verified using the
intrinsic reaction coordinate method.38,39
RESULTS AND DISCUSSION
A benzodiazepine has three heteroatoms on the seven-
membered ring and each potentially can accommodate
the ‘ionizing’ proton to give a protonated benzodiazepine
(MHþ). These are the imine nitrogen at N4, the amide oxy-
gen attached to C2, and the amide nitrogen at N1 (Scheme 1),
Figure 2. (a) Energy-resolved CID results of MHþ, m/z 237 and (b) details of secondary
products of the [MH�CO]þ ion, m/z 209.
Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 2273–2281
DOI: 10.1002/rcm
Figure 3. Energy profile for the elimination of CO from protonated base diazepam at B3LYP/6-31þG(d).
2276 A. Risoli et al.
in decreasing order of basicity. DFT calculations show that
protonation on the imine nitrogen produces ion I that is 23.1
and 36.9 kcal/mol lower in enthalpy (DH80) than the ions
produced by protonation on the amide oxygen, II, and on the
amide nitrogen, III, respectively (Scheme 2).
Figure 1 shows the CID spectrum (product ion scan) of
protonated base diazepam at a laboratory collision energy
(Elab) of 30 eV. Energy-resolved CID results are shown in
Fig. 2 for the MHþ (m/z 237) as well as details of secondary
products from a prominent product ion at m/z 209, formed
by elimination of CO. The loss of CO is a feature shared
by all benzodiazepines.11,17,28–34 Figure 3 gives the energy
profiles for the loss of CO from ion I directly and indi-
rectly via ion III. The former has a higher enthalpy barrier
(DH80¼ 73.3 kcal/mol) than the latter (56.3 kcal/mol). Pro-
tonation on the amide nitrogen weakens the amide bond
Scheme
Copyright # 2007 John Wiley & Sons, Ltd.
(a fact well known in the CID of protonated peptides).40 It is,
therefore, not surprising that the reaction pathway involving
ion III has a lower energy barrier. The barrier against
conversion of ion I into ion III, involving a 1,4-proton shift, is
54.2 kcal/mol – slightly lower than the barrier against the loss
of CO from III.
In addition to the loss of CO, protonated base diazepam
can also lose neutrals of 43, 78, and 103Da to give relatively
minor product ions at m/z 194, 159, and 134, respectively
(Figs. 1 and 2(a)). Plausible mechanisms are shown in
Scheme 3. In pathway B, ring contraction leads to the loss of
HNCO, 43Da. The resulting ion at m/z 194 delocalizes its
positive charge onto the original N4 (IV), the benzylic C5,
and both benzene rings. In pathwayC, a 1,3-proton shift from
N4 to C10 on the phenyl ring induces elimination of the
phenyl ring as benzene, 78Da, giving a benzodiazepine ion at
2.
Rapid Commun. Mass Spectrom. 2007; 21: 2273–2281
DOI: 10.1002/rcm
Scheme 3.
Gas-phase fragmentation of protonated benzodiazepines 2277
m/z 159 (V). By contrast, pathway D involves a 1,4-proton
shift that results in protonation of the amide oxygen to give
ion II. Contraction of the diazepine ring leads to elimination
of benzonitrile, 103Da, and formation of protonated oxindole
(m/z 134), ion VI. Alternatively, elimination of oxindole from
I by ring contraction would produce protonated benzonitrile
and a subsequent proton transfer would yield VI.
The majority of product ions seen in Fig. 1 are second-
ary product ions (Fig. 2(b)) as a result of dissociation of
the primary product ion, amino-protonated 1,2-dihydro-4-
phenylquinazoline, ion P atm/z 209. Ion P(, imino-protonated
1,2-dihydro-4-phenylquinazoline, is at the global minimum,
23.7 kcal/mol below P, but the barrier against interconver-
sion between these tautomers is 33.8 kcal/mol above P. From
Copyright # 2007 John Wiley & Sons, Ltd.
the energy-resolved results shown in Fig. 2(b), and ignoring
product ions with abundances <5%, it is apparent that the
following secondary product ions,m/z 131, 106, 193, and 192,
are formed directly from the [MH�CO]þ ion at m/z 209, and
with relatively low barriers. Mechanisms that can lead to
these ions are shown in Scheme 4. In pathway E, a 1,5-proton
shift in P fromN1 to C10 or a 1,3-proton shift in P( fromN4 to
C10 induces elimination of the phenyl ring as benzene and
gives the protonated 2-methaniminobenzonitrile ion (VII) at
m/z 131. Ring contraction in pathway F can occur from either
P or P( forming either protonated benzoazetine (ion VIII) at
m/z 106, or protonated benzonitrile at m/z 104. Ion VIII is
16.9 kcal/mol higher in enthalpy (DH80) than protonated
N-methylenebenzenamine, Ph�NHþ¼CH2; however, con-
version from the former into the latter is a high-energy
process, requiring a 1,3-proton shift and cleavage of a C–C
bond. The calculated proton affinities (PAs) of 1,2-dihydro-
4-phenylquinazoline (that protonates to give VIII) and
benzonitrile are 218.2 and 194.4 kcal/mol at B3LYP/6-31þG(d); the latter is in good agreement with the reference PA of
194 kcal/mol for benzonitrile.41 The observation of proto-
nated benzonitrile being formed at higher threshold energies
suggests that the dominant pathway is via ion P(. At low
collision energies, the lifetime of the initially formed
PhCNHþ/benzoazetine complex will be sufficiently long
to permit efficient proton transfer and protonated benzoa-
zetine will be the major product; at higher collision energies,
the lifetime of the complex will be shorter and direct
dissociation yields protonated benzonitrile. Pathways G and
H result in losses of the NH2 radical and NH3 leading to the
product ions atm/z 193 and 192, respectively. The initial step
is identical and involves cleavage of the N1–C2 bond in P,
giving IX, an ion in which there is extensive charge
delocalization. Elimination of NH3 (pathway G) yields a
benzyne ion X at m/z 192 that is similarly resonance
stabilized. Alternatively, elimination of �NH2 (pathway H)
results in a distonic phenyl radical XI at m/z 193 that is also
resonance stabilized.
Figure 4 shows the CID results of (a) protonated
nordiazepam and (b) protonated norfludiazepam, both
at Elab of 30 eV. Both benzodiazepines are simple derivatives
of the base diazepam: nordiazepam has a Cl substituent at
the C7 position, a rather common substitution pattern among
commercially available diazepams, while norfludiazepam
carries, in addition to the Cl substituent, a F substituent at the
C20 position, which is less common than the former
(Scheme 1). As observed for protonated base diazepam,
protonated nordiazepam (m/z 271) dissociates under CID
conditions by losing the following neutrals: CO, HNCO, and
benzonitrile. The product ion at m/z 193 is interpreted as
being formed via concomitant elimination of HNCO and Cl,
not benzene. This interpretation is a consequence of the
following observations: CID of the 37Cl-containing proto-
nated nordiazepam atm/z 273 yields a product ion atm/z 193;
and protonated norfludiazepam shows the same loss of 78Da
(HNCOþCl, Fig. 3(b)) and none at 96Da (the mass of
fluorobenzene). In addition, this interpretation is in agree-
ment with a previous one based on H/D-exchange.31
Protonated norfludiazepam (m/z 289) dissociates analo-
gously, via losses of CO (28Da), ClþHNCO (78Da), and
Rapid Commun. Mass Spectrom. 2007; 21: 2273–2281
DOI: 10.1002/rcm
Scheme 4.
2278 A. Risoli et al.
fluorobenzonitrile (121Da). The [MH�CO]þ ions of both
diazepam derivatives dissociate facilely to give secondary
product ions, as was observed for the [MH�CO]þ ion of the
base diazepam. For nordiazepam, the losses are Cl, benzene,
and benzonitrile; for norfludiazepam, they are Cl, fluoro-
Copyright # 2007 John Wiley & Sons, Ltd.
benzene, and fluorobenzonitrile. The resulting ions should
have structures analogous to those postulated in Scheme 4.
Substituting the N1 hydrogen of nordiazepam with a
methyl group results in diazepam; the CID spectrum of
protonated diazepam at Elab of 30 eV is shown in Fig. 5. It
Rapid Commun. Mass Spectrom. 2007; 21: 2273–2281
DOI: 10.1002/rcm
Figure 4. Product ion spectra of (a) protonated nordiaze-
pam, m/z 271 and (b) protonated norfludiazepam, m/z 289,
both at Elab¼ 30 eV.
Figure 6. Product ion spectra of (a) protonated nitrazepam,
m/z 282, at Elab¼ 30 eVand (b) protonated flunitrazepam,m/z
314, at Elab¼ 35 eV.
Gas-phase fragmentation of protonated benzodiazepines 2279
shows many similarities to the CID spectrum of protonated
nordiazepam in Fig. 2(a). The two new features are the
elimination of neutrals of 57Da from protonated diazepam
and of 29Da from the [MH�CO]þ ion. The 57Da neutral that
is lost is interpreted as CH3NCO; the mechanism for its
elimination is postulated to be analogous to that of HNCO
shown in Scheme 3. Protonated nordiazepam does not
appear to lose HNCO alone, but concomitantly with Cl. The
29Da neutral lost from the [MH�CO]þ ion corresponds to
methyl nitrene, CH3N (or the lower energy H2C¼NH),
forming protonated isoindole.
Substituting the C7 hydrogen on nordiazepamwith a nitro
group results in nitrazepam; the CID spectrum of protonated
nitrazepam at Elab of 30 eV is shown in Fig. 6(a). The most
prominent feature is the loss of NO2, 46Da. This same feature
Figure 5. Product ion spectrum of protonated diazepam,m/z
286, at Elab¼ 30 eV.
Copyright # 2007 John Wiley & Sons, Ltd.
is evident in the CID of protonated flunitrazepam (Fig. 6(b)),
which differs from nitrazepam in having a methyl sub-
stituent on N1 and a fluoro substituent on C20, and
clonazepam, a derivative of nitrazepam that contains a
chloro substituent at the C20 position.
Replacing the C3 hydrogen on nordiazepam with a
hydroxyl group produces oxazepam, an additional substi-
tution of Cl at the C20 position results in lorazepam.
Similarly, replacing the C3 hydrogen on diazepam with a
hydroxyl group produces temazepam. TheCID spectra of the
[MþH]þ ions of these OH-bearing derivatives are dominated
by two common neutral losses: that of water (18Da),
followed by CO (28Da). The spectrum for protonated
oxazepam is shown in Fig. 7. An additional prominent
loss involves the loss of a 56Da neutral. This loss is also
Figure 7. Product ion spectrum of protonated oxazepam,
m/z 287, at Elab¼ 35 eV.
Rapid Commun. Mass Spectrom. 2007; 21: 2273–2281
DOI: 10.1002/rcm
Scheme 5.
2280 A. Risoli et al.
evident in protonated lorazepam, albeit much less notably.
The proposed mechanism for the loss of water, shown in
Scheme 5, is in agreement with H/D exchange results.31
Nucleophilic attack by the hydroxyl oxygen on the N4
hydrogen, followed by ring contraction and elimination of
water, results in ion XIII at m/z 269. Ring contraction and
cleavage of CO produces ion XIV atm/z 241 that is resonance
stabilized. The 56Da neutral loss directly from protonated
oxazepam is postulated to be that of ethylenedione, C2O2, a
short-lived molecule that rapidly dissociates into two
molecules of CO.42 This hypothesis of C2O2 is consistent
with the absence of the stepwise elimination of the CO
molecules.
CONCLUSIONS
Substitution of chloro, hydroxyl, and nitro groups on the
base diazepam has a dramatic effect on gas-phase dis-
sociation chemistry. The losses of these groups are the most
facile and dominate the CID chemistry. The loss of CO is a
common, facile dissociation pathway of all benzodiazepines,
in evidence even in the aforementioned substituted benzo-
diazepines. Other common losses include the phenyl ring at
the C5 position, as either (substituted) benzene or benzoni-
trile. Ion structures and fragmentation mechanisms have
been postulated towards building a knowledge- and/or
intuition-based benzodiazepine information bank whose
contents will lead to eventual understanding of how
structure drives fragmentation chemistry and determines
products.
Copyright # 2007 John Wiley & Sons, Ltd.
AcknowledgementsThis work was supported by the Natural Sciences and
Engineering Research Council (NSERC) of Canada and
MDS SCIEX. A.R. acknowledges financial support from
the University of Calabria to allow her to carry out part of
her thesis research at York University.
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