Gas-phase formation of protonated benzene during collision-induced dissociation of certain protonated mono-substituted aromatic molecules produced in electrospray ionization

Article abstract of DOI:10.1002/rcm.4569

Protonated benzene, C₆H⁺₇, has been studied extensively to understand the structure and energy of a protonated organic molecule in the gas phase. The formation of C₆H⁺₇ is either through direct protonation of benzene, i.e., chemical ionization, or through fragmentation of certain radical cations produced from electron ionization or photon ionization. We report a novel observation of C₆H⁺₇ as a product ion formed in the collision-induced dissociation (CID) of protonated benzamide and related molecules produced via electrospray ionization (ESI). The formation of C₆H⁺₇ from these even-electron precursor ions during the CID process, which has not been previously reported, is proposed to occur from the protonated molecules via a proton migration in a five-membered ring intermediate followed by the cleavage of the mono-substituent C-C bond and concurrent formation of an ion-molecule complex. This unique mechanism has been scrutinized by examining some deuterated molecules and a series of structurally related model compounds. This finding provides a convenient mean to generate C₆H⁺₇, a reactive intermediate of considerable interest, for further physical or chemical investigation. Further studies indicate that the occurrence of C₆H⁺₇ in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) appears to be a rather common phenomenon for many compounds that contain 'benzoyl-type' moieties. Hence, the observation of the C₆H⁺₇ ion in LC/ESI-MS/MS can be used as an informative fragmentation pathway which should facilitate the identification of a great number of compounds containing the 'benzoyl-type' and similar structural features. These compounds are frequently present in food and pharmaceutical products as leachable impurities that require strict control and rapid elucidation of their identities.

Full text of DOI:10.1002/rcm.4569

RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4569
Gas-phase formation of protonated benzene during
collision-induced dissociation of certain protonated
mono-substituted aromatic molecules produced in
electrospray ionization
Min Li*, Mingxiang Lin and Abu M. Rustum
Global Quality Services – Analytical Sciences, Merck & Co. Inc., 1011 Morris Ave, Union, NJ 07083, USA
Received 23 February 2010; Revised 3 April 2010; Accepted 5 April 2010
R
7
Protonated benzene, C H , has been studied extensively to understand the structure and energy of a
6
R
7
protonated organic molecule in the gas phase. The formation of C H is either through direct
6
protonation of benzene, i.e., chemical ionization, or through fragmentation of certain radical cations
R
produced from electron ionization or photon ionization. We report a novel observation of C H as a
6
7
product ion formed in the collision-induced dissociation (CID) of protonated benzamide and related
R
molecules produced via electrospray ionization (ESI). The formation of C H from these even-
6
7
electron precursor ions during the CID process, which has not been previously reported, is proposed
to occur from the protonated molecules via a proton migration in a five-membered ring intermediate
followed by the cleavage of the mono-substituent CꢀC bond and concurrent formation of an ion-
molecule complex. This unique mechanism has been scrutinized by examining some deuterated
molecules and a series of structurally related model compounds. This finding provides a convenient
R
mean to generate C H , a reactive intermediate of considerable interest, for further physical or
6
7
R
7
chemical investigation. Further studies indicate that the occurrence of C H in liquid chromatog-
6
raphy/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) appears to be a rather
common phenomenon for many compounds that contain ‘benzoyl-type’ moieties. Hence, the
R
observation of the C H ion in LC/ESI-MS/MS can be used as an informative fragmentation pathway
6
7
which should facilitate the identification of a great number of compounds containing the ‘benzoyl-
type’ and similar structural features. These compounds are frequently present in food and pharma-
ceutical products as leachable impurities that require strict control and rapid elucidation of their
identities. Copyright # 2010 John Wiley & Sons, Ltd.
þ
Protonated benzene, C H , is a prototype organic cation that
EI-generated phenylarsenium radical cation through mass-
analyzed ion kinetic energy (MIKE) spectrometry and
6
7
has been studied extensively as a model to understand the
structure and energy of a protonated organic molecule in
1
1
þ
H ion could
7
collision-induced dissociation (CID). The C
6
1
the gas phase. The formation and structure, as well as the
also be formed from the photon ionization of carboxymethyl
þ
5
þ
reactivity and thermodynamic properties, of C
6
H
have
cyclohexa-2,5-diene. Upon its formation, C
6
H
can be
7
7
2
–10
been investigated both experimentally and theoretically.
mass-selected and studied by various spectroscopic tech-
niques or allowed to react with different molecules in the gas
þ
The formation of C
6
H
is reported to occur either through
7
direct protonation of benzene via chemical ionization or
through fragmentation of certain radical cations produced
phase to probe its structure and reactivity. Nevertheless,
þ
C H has never been observed as a product ion in LC/ESI-
6
7
from electron ionization or photon ionization. For
MS/MS, despite the widespread use of the technique. We
þ
þ
instance, C
6
H
can be generated from gas-phase proton
report a novel observation of C
6
H
from the CID of
7
7
þ
þ
2
transfer from CH5 and C
2
H
to benzene, i.e., chemical
protonated benzamide (and related compounds), an even-
electron precursor ion produced via electrospray ionization
5
ionization of benzene. Protonated benzene can also be
produced as a fragment ion from the electron ionization (EI)
(ESI) in a typical LC/ESI-MS/MS experiment. The most
3
4
þ
of O-toluidine and N-methylaniline, or benzylamine.
plausible formation mechanism of C
6
H
is proposed; the
7
þ
Recently, C H7 was observed in the product ions of an
results obtained from various experiments, including
deuterium labeling of the benzamide molecule and exam-
ination of the fragmentation of a series of model compounds
that are structurally related to benzamide, are consistent
with the proposed mechanism.
6
*
Correspondence to: M. Li, Global Quality Services – Analytical
Sciences, Merck & Co. Inc., 1011 Morris Ave, Union, NJ 07083,
USA.
E-mail: minli88@yahoo.com
Copyright # 2010 John Wiley & Sons, Ltd.

1
708 M. Li, M. Lin and A. M. Rustum
EXPERIMENTAL
are given in Scheme 1. The formation of the ions at m/z 105
and 77 can be readily explained. The protonated benzamide
Materials
All chemicals except N-cyclohexylbenzamide were obtained
from Sigma-Aldrich (St. Louis, MO, USA). N-Cyclohexyl-
(
I) can easily lose NH
3
at elevated collision energy, resulting
þ
in the m/z 105 ion (C
6
H
5
CO ). The latter ion can further
undergo a decarbonylation process to form the phenyl
benzamide-d
5
was prepared by mixing equal molar amounts
chloride and triethylamine in
þ
cation C H , the m/z 77 ion (pathway a, Scheme 1). On the
6
5
of cyclohexylamine, benzoyl-d
5
other hand, the occurrence of the m/z 79 ion as a major
product ion appeared somewhat puzzling initially, as there
have been no reports of such an event in the CID of an even-
electron precursor ion. After carefully reviewing all the data,
it occurred to us that m/z 79 might be protonated
methylene chloride at room temperature; the reaction was
completed instantly.
Mass spectrometric analysis
þ
þ
cannot be explained
7
Mass spectrometric analysis was primarily performed on a
Waters (Milford, MA, USA) Q-Tof Premier quadrupole time-
of-flight mass spectrometer operating in electrospray
positive ion mode. Analyte solutions were prepared in
benzene, C
6
H . The formation of C
6
H
7
by a single bond cleavage from protonated benzamide (I). To
explain this unusual formation, a novel mechanism starting
from a five-membered ring intermediate (Ia) was proposed
(pathway b): upon activation by CID, one proton transfers
from the protonated amide nitrogen to the ortho-position of
the benzene ring. This is followed by cleavage of the CꢀC
bond between the carbonyl and the benzene ring, resulting in
5
0:50 methanol and water mixture, while deuterated
compounds were dissolved in 50:50 deuterated methanol
CH OD) and deuterated water (D O). Sample solutions
(
3
2
were infused via the embedded syringe pump at a flow rate
of 5 mL/min into the ESI source. The ESI source was operated
with the following parameters: electrospray voltage 3.5 kV,
cone voltage 35 V, source temperature 1008C, desolvation
temperature 2508C, cone gas flow rate 60 L/h and
desolvation gas flow rate 600 L/h. The TOF mass analyzer
was operated in V mode at a rate of 1 scan/s and 0.1 s inter-
scan time. Mass calibration was performed using a sodium
cesium iodide solution. For MS/MS experiments, the
quadrupole mass selection window width was set to 1 m/z
unit and the lowest mono-isotopoic peaks of appropriate
analyte ions were selected as precursor ions. Argon was used
as the collision gas and the collision energy was set to 10 eV
for most MS/MS experiments unless indicated otherwise.
When in-source fragmentation was needed, the cone voltage
was increased to 50 V to induce additional fragmentation in
the source region. To evaluate the impact of the collision gas
and to compare the fragmentation patterns between different
types of instruments, a few selected MS/MS experiments
were also carried out on a ThermoScientific (San Jose, CA,
USA) LTQ linear ion trap mass spectrometer. The ESI source
of the LTQ was operated with the ESI voltage set to 4.0 kV
and the capillary temperature set to 3008C. The MS/MS data
were collected with an isolation width of 1 m/z unit. Helium
was used as the collision gas and the normalized collision
energy (collision energy normalized across the mass range
scanned) was set to 35%. The data presented in the following
Results and Discussion section were acquired from the Q-Tof
mass spectrometer unless indicated otherwise.
b
the formation of an ion-molecule complex (I ) consisting of a
þ
neutral benzene molecule and a O¼C¼NH ion (Scheme 1).
2
þ
This [benzeneꢁO¼C¼NH ] complex can be regarded as a
2
proton-bound dimer of benzene and isocyanic acid
(O¼C¼NH), which could have three outcomes: (1) it can
revert back to Ia; (2) it can dissociate with a proton transfer
þ
from O¼C¼NH to benzene, producing protonated benzene
2
(m/z 79) and neutral isocyanic acid; and (3) it can dissociate
without proton transfer, leading to the formation of
protonated isocyanic acid O¼C¼NHþ2 (m/z 44) and neutral
benzene.
Design of various experiments to verify the
proposed mechanism for the formation of
protonated benzene
In order for the proposed mechanism to be operative, two
requirements must be met: (1) there should be a hydrogen
atom available on the amide nitrogen to migrate as a proton
to the ortho-position of the benzene ring, and (2) the benzene
formed in the complex I should be able to abstract a proton
b
þ
from O¼C¼NH . Thus, the end result is that two protons are
2
transferred from the amide nitrogen to form the
þ
resulting C
6
H . To verify this hypothesis, deuterium labels
7
were introduced on the benzene ring and the amide nitrogen,
respectively, in order to examine if proton transfer in the five-
membered ring intermediate did indeed occur during the
CID of protonated benzamide. In addition, the fragmentation
behavior of three groups of structural analogs of benzamide
was examined to probe the impact of different functional
groups in benzamide on the formation of protonated
benzene. First, N,N-dimethylbenzamide that contains no
amide hydrogen was examined; in this case, no hydrogen is
available for the initial proton migration. Hence, formation of
protonated benzene would not be expected from N,N-
dimethylbenzamide. Second, the oxygen in the benzamide
carbonyl group was replaced with other heteroatoms (X) to
assess the impact of the electron-withdrawing capability of
the C¼X bond on the formation of the critical five-membered
ring intermediate (Ia). Third, different substituents were
introduced into the benzene ring to modify the proton
affinity of the benzene moiety and thus to evaluate its impact
RESULTS AND DISCUSSION
R
7
Formation of protonated benzene, C H (m/z 79),
6
from benzamide and a proposed formation
mechanism
CID of protonated benzamide (m/z 122) yielded three major
product ions: m/z 105, 79 and 77 (Fig. 1(a)). The structures of
benzamide and all other molecules examined in this study
are summarized in Table 1, along with the assignments of the
product ions observed from the CID of each precursor ion.
Proposed fragmentation pathways leading to the formation
of the three major product ions from protonated benzamide
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
DOI: 10.1002/rcm

Gas-phase formation of protonated benzene 1709
Figure 1. Collision-induced dissociation product ion spectra of (a) protonated benzamide (m/z 122),
(
b) protonated benzamide-d (m/z 127), which was generated as an in-source fragment ion of N-cyclohex-
5
5 3 2
ylbenzamide-d , and (c) the benzamide-N-d cation (m/z 125) generated from ESI of benzamide-N-d in
deuterated solvent. (d) CID product ion spectrum of protonated benzamide obtained from the LTQ.
on the proton transfer step in forming the corresponding
protonated benzene derivatives from I -like intermediates.
the case of benzamide-d
was generated as a product ion of N-cyclohexylbenzamide-
. In the proposed fragmentation pathway of benzamide-d
(Scheme 2), protonated benzamide-d was produced as an in-
5 5
, protonated benzamide-d (m/z 127)
b
d
5
5
Deuterium-labeling experiments with
benzamide-d and benzamide-N-d3
5
source fragment ion of N-cyclohexylbenzamide-d5 after
eliminating cyclohexene. The same fragmentation pathways
as had been observed in protonated benzamide were
5
Two complementary deuterium-labeled benzamide mol-
ecules, benzamide-d5 with all five aromatic hydrogens
replaced by deuterium atoms and benzamide-N-d
3
with
observed in protonated benzamide-d
5
(Fig. 1(b)). A direct
three amide hydrogens (including one hydrogen gained in
solution during ESI) labeled by deuterium atoms, were
neutral loss of NH
3
from protonated benzamide-d resulted
5
þ
in C
6
D
5
CO (m/z 110), which further lost CO to produce the
þ
examined to probe the proposed initial proton migration and
benzene-d
5
cation C
6
D
(m/z 82). The protonated benzene
5
þ
subsequent proton transfer during the formation of C
6
H . In
observed from benzamide-d was observed at m/z 84
5
7
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
DOI: 10.1002/rcm

1
710 M. Li, M. Lin and A. M. Rustum
Table 1. Summary of product ions observed from collision-induced dissociation of protonated benzamide and its related
molecules
Product ions observed from
protonated molecules: m/z
Compound
Benzamide
Structure
(assignment)
Comment
Figure
1a
þ
þ
7
105 (C H CO ), 79 (C H ),
6
5
6
Protonated benzene
was observed.
þ
þ
7
7 (C
6
H ), 44 (O¼C¼NH )
5
2
þ
þ
Benzamide-d
5
110 (C
6
D
5
CO ), 109 (C
6
D
4
þ
HCO ),
Protonated benzene
1b
þ
8
4 (C
2 (C
6
D
5
H ), 83 (C
6
D
4
þ
H ),
was observed as
2
3
þ
þ
þ
þ
3
8
6
D ), 81 (C
6
D
4
H ), 44 (O¼C¼NH )
C
6
5
D H
and C
6
H
4
H .
5
2
2
þ
þ
Benzamide-N-d
2
106 (C
6
H
4
DCO ), 105 (C
6
þ
H
5
CO ),
Deuterated benzene
1c
þ
þ
þ
8
1 (C
6
H
5
D ), 78 (C
H
6 4
D ), 77 (C
6
H ),
C
6
H
5
D
was observed.
2
5
2
þ
4
6 (O¼C¼ND )
2
þ
þ
þ
N,N-Dimethylbenzamide
105 (C H CO ), 77 (C H ), 72 [(CH ) NCO ] Protonated benzene
2a
2b
6
5
6
5
3 2
was not observed.
þ
þ
Thiobenzamide
121 (C H CS ), 79 (C H ),
Protonated benzene (m/z 79)
6
5
þ
6
7
þ
þ
7
7 (C
6
H ), 60 (S¼C¼NH )
and S¼C¼NH (m/z 60)
5
2
2
were observed.
þ
þ
5
Benzamidine
Benzylamine
104 (C H C¼NH ), 77 (C H )
Protonated benzene
was not observed.
2c
6
5
6
þ
þ
5
91 (C H ), 65 (C H )
7
5
Protonated benzene
was not observed.
2d
7
þ
þ
2
4-Nitrobenzamide
150 (NO C H CO ), 137 (HOC H CONH ),
Protonated nitrobenzene
was observed at m/z 124.
3a, 3b
2
6
6
4
4
6
4
þ
þ
1
1
1
24 (NO
20 (OC
04 (C
2
C
H ), 121 (C
6
H
4
CONH ),
6
3
þ
þ
6
H
CO ), 109 (HOC
6
H
4
NH ),
2
þ
6
H
4
CO )
(
Continues)
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
DOI: 10.1002/rcm

Gas-phase formation of protonated benzene 1711
Table 1. (Continued)
Product ions observed from
protonated molecules: m/z
(assignment)
Compound
Structure
Comment
Figure
3c, 3d
þ
4-Methoxybenzamide
135 (CH
09 (CH
3
OC
OC
6
H
4
þ
CO ),
Protonated 4-methoxybenzene
was observed at m/z 109.
þ
1
3
6
H ), 94 (C
6
H
6
O )
6
þ
4
-Methoxybenzamide-N-d
2
136 (CH
3
3
3
OC
OC
OC
6
6
6
H
H
H
3
4
4
DCO ),
Deuterated 4-methoxybenzene Not shown
was observed at m/z 111.
þ
1
1
9
35 (CH
CO ),
þ
D ),
2
11 (CH
þ
6 4 2
6 (C H D O )
(
pathway A). In addition, another set of product ions was
completely deuterated solvent. Fragmentation of IV yielded
observed at m/z 109, 81 and 83, which were 1 m/z unit
the ions corresponding to those observed from protonated
less than the three expected ions, i.e., loss of NH
3
(m/z 110),
5
benzamide-d with the predicted mass shifts (Scheme 3). The
þ
loss of NH
3
and CO (m/z 82) and C
6
D
5
H
(m/z 84). These
neutral loss of ND
3
from benzamide-N-d
3
(IV) resulted
2
þ
ions can be explained as resulting from an intramolecular
hydrogen/deuterium (H/D) exchange process prior to
in C
6
H
5
CO (m/z 105), which further fragmented to the
þ
phenyl cation, C
6
H
(m/z 77) (Fig. 1(c)). Deuterated benzene
5
þ
or during the CID of protonated benzamide-d5.
A
was observed as C H D (m/z 81), with the two deuterium
6
5
2
deuterium in the benzene ring of II, probably the most
acidic deuterium on the ortho-position, can exchange with a
hydrogen atom on the amide nitrogen through pathways
B1/B2 to produce III (Scheme 2), which then fragments to
atoms that originated from the deuterated amide nitrogen in
IV. The proton-competing isocyanic acid cation was
observed as O¼C¼NDþ2 (m/z 46). An intramolecular H/D
5
exchange process similar to that for benzamide-d occurred
þ
þ
give C
nated benzene-d
In another set of experiments, the benzamide-N-d
IV) was generated from ESI of benzamide-N-d2 in a
6
D
4
HCO (m/z 109), C
6
D
4
H
(m/z 81), and proto-
3
for benzamide-N-d (IV), leading to the formation of V.
4
(m/z 83).
Subsequent fragmentation of V produced the ND
2
H neutral
þ
3
cation
loss product C
6
H
4
DCO at m/z 106 and the benzene-d
þ
(
cation C H D at m/z 78. The fragmentation behavior
6
4
þ
7
Scheme 1. Proposed mechanism for the formation of protonated benzene (C H ) and other product ions
6
during CID of electrospray-generated protonated benzamide.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
DOI: 10.1002/rcm

1
712 M. Li, M. Lin and A. M. Rustum
Scheme 2. Proposed fragmentation mechanism of protonated benzamide-d under CID.
5
þ
observed in the above studies of the two complementary
from the Q-Tof (Fig. 1(a)), the phenyl cation (C
6
H , m/z
5
2
deuterium-labeled benzamide molecules, benzamide-d
5
and
77) was not observed in the MS spectrum of protonated
benzamide obtained from the LTQ. This difference may be
due to the somewhat different collision modes used in the
two instruments. In the LTQ, only the targeted precursor
benzamide-N-d , provided conclusive evidence that the
3
amide hydrogen atoms are the source of the two hydrogens
required to form protonated benzene as proposed in
Scheme 1.
2
ion is activated by resonance excitation during an MS
experiment. The primary product ions formed in LTQ are
Protonated benzene obtained on different types
of mass spectrometers using different collision
gases
In addition to the MS/MS experiments performed on
the Q-Tof mass spectrometer where argon was used as
the collision gas, the fragmentation of protonated benza-
mide was also examined on a LTQ mass spectrometer
where helium was used as the collision gas, to evaluate
not activated again; thus no further fragmentation can be
þ
produced. This explains why C
6
H
was not observed in
5
2
the MS spectrum of protonated benzamide in the LTQ
þ
due to it being a secondary product ion from C
6
H
5
CO . An
3
þ
MS experiment on C
6
þ
H
5
CO performed in the LTQ did
indeed produce C H
6
(data not shown). On the other
5
hand, in a CID experiment carried out in a tandem-in-
space mass spectrometer (such as a triple-quadrupole type
instrument, or the Q-Tof in this case), primary product
ions can undergo additional collision activation resulting
in secondary and further product ions. The fact that
the impact of the collision gas and other instrumental
þ
factors on the formation of C H . The CID of protonated
6
7
benzamide in the LTQ yielded two major product
þ
þ
ions:
C
6
H
5
CO
at m/z 105 and
C
6
H
at m/z 79
protonated benzene was observed from protonated
7
þ
(
Fig. 1(d)). Therefore, it can be concluded that the type
benzamide in the LTQ not only confirms that C
6
H
(m/
7
of the inert collision gas used is not critical for the
z 79) is a primary product ion, which is consistent with the
þ
formation of C
6
H . Compared with the result obtained
proposed mechanism, but also indicates that the formation
7
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
DOI: 10.1002/rcm

Gas-phase formation of protonated benzene 1713
Scheme 3. Proposed fragmentation mechanism of protonated benzamide-N-d under CID.
3
þ
of C
6
H
is determined by the intrinsic chemical property
is one of the two weakest bonds in benzamide due to the
7
1
2
of the precursor ion, which is independent of the
instruments used.
presence of the electron-withdrawing carbonyl group.
Substituting the carbonyl oxygen with other heteroatoms (X)
possessing less electronegativity may have an effect on the
CꢀC bond cleavage, which would in turn affect the
Studies in the formation of protonated benzene
from structural analogs of benzamide
þ
formation of C
6
H from these benzamide analogs. Therefore,
7
thiobenzamide and benzamidine which contain C¼S and
C¼N in place of the C¼O, respectively, were chosen as the
model compounds to test this hypothesis. In addition,
benzylamine, which has a CH ꢀNH single bond instead of a
N,N-Dimethylbenzamide
Based on the proposed mechanism, N,N-dimethylbenza-
mide, which does not contain any exchangeable hydrogen on
2
2
þ
the amide nitrogen, should not be capable of forming C
6
H .
7
double C¼X bond, was also tested. The CID of thiobenza-
Indeed, the CID of protonated N,N-dimethylbenzamide
þ
mide led to direct neutral loss of NH to form C H CS (m/z
3
6
5
þ
yielded three major ions (Fig. 2(a)): C H CO (m/z 105), the
6
5
121), and a subsequent C¼S loss to yield the phenyl cation
þ
phenyl cation C H (m/z 77), and (CH ) NCO (m/z 72). As
þ
7
6
5
3 2
6
(m/z 77), as shown in Fig. 2(b). Although both C H (m/z 79)
þ
expected, no protonated benzene, C H , was observed.
þ
6
7
and protonated isothiocyanic acid (S¼C¼NH , m/z 60) were
2
þ
observed, the intensity ratio of C
6
H
(m/z 79) to the phenyl
7
Thiobenzamide, benzamidine and benzylamine
cation (m/z 77) was less than 1, while this ratio was greater
To further verify other aspects of the proposed mechanism,
than 1 in the case of benzamide (Fig. 1(a)). This result
þ
additional structural analogs of benzamide were examined
suggests that the pathway to form C H7 from protonated
6
to investigate the impact of modifying certain moieties of the
thiobenzamide is less favored than the competing pathway
þ
molecule on the formation of C H . Another critical step in
of NH
3
neutral loss. In the case of benzamidine, the
6
7
þ
the proposed mechanism is the cleavage of the carbonyl
benzene CꢀC bond, which should occur concertedly with the
proton migration but precede the formation of the presumed
formation of C
6
H
was completely suppressed. The neutral
7
þ
loss products, C
6
H
5
C¼NH (m/z 104) and the phenyl cation
(m/z 77), were the two dominant ions observed (Fig. 2(c));
þ
nor HN¼CNHþ3 was observed. Collectively,
proton-bound dimer of benzene and isocyanic acid (I
b
). The
neither C
6
H
7
CꢀC bond connecting the carbonyl group to the benzene ring
Copyright # 2010 John Wiley & Sons, Ltd.
these results appear to indicate that the CꢀC bonds between
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
DOI: 10.1002/rcm

1
714 M. Li, M. Lin and A. M. Rustum
Figure 2. Collision-induced dissociation product ion spectra of protonated (a) N,N-dimethylbenzamide (m/z
150), (b) thiobenzamide (m/z 138), (c) benzamidine (m/z 121), and (d) benzylamine (m/z 108).
the benzene ring and the C¼X group in both thiobenzamide
and benzamidine are increasingly stronger than the one in
benzamide, causing the two benzamide analogs to be less
likely to proceed via the five-membered ring mechanism
since cleavage of this CꢀC bond is a critical step, which will
probably occur concertedly with the proton migration in the
proposed mechanism (Scheme 1). On the other hand, CID of
protonated benzylamine did not yield protonated benzene,
which is not unexpected (Fig. 2(d)). This probably can be
attributed to the following two factors: (1) no electron-
withdrawing double bond is present to activate the CꢀC
bond connected to the benzene ring in benzylamine, and/or
91). The comparative study of the three model compounds
suggests that the electron-withdrawing C¼O group attached
to the benzene ring in benzamide decreases the carbonyl
1
2
benzene CꢀC bond order and thus renders the formation
þ
of C
6
H
quite efficient.
7
4-Nitrobenzamide and 4-methoxybenzamide
Another factor affecting the formation of C H7 in the
þ
6
proposed mechanism is the proton transfer step (pathway d,
Scheme 1) which takes place after cleavage of the carbonyl
benzene CꢀC bond and concomitant proton migration
leading to the formation of the proton-bound dimer (path-
(
2) neutral loss of NH
3
from protonated benzylamine should
way c). The proposed proton transfer step is essential to the
þ
þ
produce a very stable benzylium/tropylium ion (C H , m/z
formation of C H7 and the efficiency of this step should be
7
7
6
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
DOI: 10.1002/rcm

Gas-phase formation of protonated benzene 1715
determined by the relative proton affinities of the two entities
in the proton-bound dimer (I ). In the case of benzamide, the
benzamide derivatives, 4-nitrobenzamide and 4-methoxy-
benzamide, were examined. The proton affinity of nitroben-
zene is 191.3 kcal/mol, which indicates that proton transfer
from protonated nitrobenzene to O¼C¼NH is endothermic
b
two entities competing for the proton in the proton-bound
dimer are benzene and isocyanic acid (O¼C¼NH). Their
proton affinities are approximately the same: with benzene at
þ
by about 11 kcal/mol, i.e., the formation of O¼C¼NH from
2
1
3
1
79.3 kcal/mol and O¼C¼NH at 180 kcal/mol. As a result,
protonated 4-nitrobenzamide is less favored than from
protonated benzamide. The experimental results obtained
from 4-nitrobenzamide were indeed consistent with this
prediction. In the product ion spectrum of protonated
4-nitrobenzamide (Fig. 3(a)), protonated nitrobenzene was
observed at m/z 124 with 10 eV collision energy, while no
O¼C¼NHþ2 ion (m/z 44) was observed. Other major ions
observed from CID of protonated 4-nitrobenzamide resulted
from known fragmentation pathways and the assignments of
þ
both C
6
H
and protonated isocyanic acid were observed
7
þ
from benzamide, although the intensity of the C
6
H
ion was
7
greater than that of protonated isocyanic acid. This
observation is consistent with the proposed mechanism.
Therefore, it can be predicted that, if a substituted benzene
moiety possesses a higher proton affinity than isocyanic acid,
þ
the formation of O¼C¼NH (m/z 44) would be less favored
2
or even completely suppressed. To test this hypothesis, two
Figure 3. Collision-induced dissociation product ion spectra of protonated 4-nitrobenzamide at (a) 10 eV
collision energy and (b) 15 eV collision energy, and 4-methoxybenzamide at (c) 10 eV collision energy and (d)
15 eV collision energy.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
DOI: 10.1002/rcm

1
716 M. Li, M. Lin and A. M. Rustum
these ions are summarized in Table 1. When the collision
energy was increased from 10 to 15 eV, the intensity of
protonated nitrobenzene decreased and a trace amount of
m/z 44 was observed (Fig. 3(b)), suggesting that endothermic
proton transfer took place at the higher collision energy,
subsequent proton transfer – were examined and challenged
by the design of three sets of experiments. All the results
þ
unambiguously indicate that C
6
H
is formed through the
7
CID of the electrospray-generated protonated benzamide
(and the precursor ions of certain structurally related
þ
leading to the formation of O¼C¼NH . The proton affinity
compounds). This finding provides a convenient means to
2
þ
of 4-methoxybenzamide is 200.7 kcal/mol, which is about 21
kcal/mol greater than that of O¼C¼NH. Following the same
rationale outlined above, it can be predicted that the
formation of protonated methoxybenzene should be even
more favored than the formation of protonated nitrobenzene,
generate C
6
H , a reactive intermediate of much interest, for
7
further physical or chemical investigation. In addition, the
þ
observation of C
6
H
(m/z 79) can be used as an informative
7
diagnostic ion to facilitate the identification of unknown
species containing the ‘benzoyl-type’ structural feature. Our
þ
þ
which should result in further suppression of O¼C¼NH .
further studies indicate that the formation of C
6
H in the CID
2
7
Again, the experiment results agreed well with this
prediction: protonated methoxybenzene (m/z 109) was
observed as one of the major product ions (Fig. 3(c)), while
O¼C¼NHþ2 (m/z 44) was completely absent even at higher
collision energy (Fig. 3(d)). Furthermore, additional product
of electrospray-generated ions appears to be a common
phenomenon for a great number of compounds that contain
‘benzoyl-type’ or similar structural features. These com-
pounds are frequently present in food and pharmaceutical
products as leachable impurities that require strict control
and rapid elucidation of their identities.
3
ions of protonated 4-methoxybenzamide-N-d generated
from ESI of 4-methoxybenzamide in fully deuterated
solvents are consistent with the proposed mechanism with
the corresponding mass shifts due to the methoxyl group
and/or H/D exchange. These results suggest that the
proton transfer step, proposed in the protonated benzene
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1
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þ
(
C
6
H ) formation mechanism, is the final critical step
2. Jones W, Boissel P, Chiavarino B, Crestoni ME, Fornarini
7
þ
S, Lemaire J, Maitre P. Angew. Chem. Int. Ed. 2003; 42:
towards the formation of C
6
H . Although the ion energies
7
2
057.
in a CID process may not be controlled accurately, the
qualitative comparison results obtained in this study are
consistent with the intrinsic properties of the protonated
molecules in the gas phase.
3
4
5
6
. Selim ETM, Rabbih MA, Fahmey MA. Org. Mass Spectrom.
1992; 27: 919.
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CONCLUSIONS
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86/187: 241.
8. Freiser BS, Woodin RL, Beauchamp JL. J. Am. Chem. Soc.
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1
þ
6
7
Protonated benzene (C H ) has been observed in LC/ESI-
1
MS/MS during the CID of the protonated benzamides, an
event that has not previously been reported despite the
9
. Kuck VD, Ingeman S, Konig LJ, Grutzmacher HF, Nibbering
NMM. Angew. Chem. 1985; 97: 691.
10. Holman RW, Gross ML. J. Am. Chem. Soc. 1989; 111: 3560.
þ
widespread use of the technique. The formation of C
6
H
is
7
1
1
1
1. Letzel M, Kirchhoff D, Gr u¨ tzmacher HF, Stein D, Gr u¨ tzma-
cher H. Dalton Trans. 2006; 2008.
2. Al-Awadi NA. J. Chem. Soc., Perkin Trans. II 1990; 12:
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3. All proton affinities referred to in this study were taken from
the NIST Chemistry WebBook, NIST Standard Reference
Database Number 69. Available: http://webbook.nist.
gov/chemistry/.
proposed as a fragmentation product of the protonated
molecule via a proton migration and concomitant CꢀC bond
breakage in a five-membered ring intermediate followed by a
proton transfer process. These three important aspects of the
proposed mechanism – proton migration, concomitant
breakage of the carbonyl benzene CꢀC bond, and the
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1707–1716
DOI: 10.1002/rcm