Competitive homolytic and heterolytic decomposition pathways of gas-phase negative ions generated from aminobenzoate esters

Article abstract of DOI:10.1002/jms.3740

An alkyl-radical loss and an alkene loss are two competitive fragmentation pathways that deprotonated aminobenzoate esters undergo upon activation under mass spectrometric conditions. For the meta and para isomers, the alkyl-radical loss by a homolytic cleavage of the alkyl-oxygen bond of the ester moiety is the predominant fragmentation pathway, while the contribution from the alkene elimination by a heterolytic pathway is less significant. In contrast, owing to a pronounced charge-mediated ortho effect, the alkene loss becomes the predominant pathway for the ortho isomers of ethyl and higher esters. Results from isotope-labeled compounds confirmed that the alkene loss proceeds by a specific γ-hydrogen transfer mechanism that resembles the McLafferty rearrangement for radical cations. Even for the para compounds, if the alkoxide moiety bears structural motifs required for the elimination of a more stable alkene molecule, the heterolytic pathway becomes the predominant pathway. For example, in the spectrum of deprotonated 2-phenylethyl 4-aminobenzoate, m/z 136 peak is the base peak because the alkene eliminated is styrene. Owing to the fact that all deprotonated aminobenzoate esters, irrespective of the size of the alkoxy group, upon activation fragment to form an m/z 135 ion, aminobenzoate esters in mixtures can be quantified by precursor ion discovery mass spectrometric experiments.

Full text of DOI:10.1002/jms.3740

Journal of
MASS
SPECTROMETRY
Research article
Received: 13 September 2015
Revised: 11 November 2015
Accepted: 24 November 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3740
Competitive homolytic and heterolytic
decomposition pathways of gas-phase negative
ions generated from aminobenzoate esters
Hanxue Xia, Yong Zhang, Julius Pavlov, Freneil B. Jariwala
and Athula B. Attygalle*
An alkyl-radical loss and an alkene loss are two competitive fragmentation pathways that deprotonated aminobenzoate esters
undergo upon activation under mass spectrometric conditions. For the meta and para isomers, the alkyl-radical loss by a homo-
lytic cleavage of the alkyl-oxygen bond of the ester moiety is the predominant fragmentation pathway, while the contribution
from the alkene elimination by a heterolytic pathway is less significant. In contrast, owing to a pronounced charge-mediated ortho
effect, the alkene loss becomes the predominant pathway for the ortho isomers of ethyl and higher esters. Results from isotope-
labeled compounds confirmed that the alkene loss proceeds by a specific γ-hydrogen transfer mechanism that resembles the
McLafferty rearrangement for radical cations. Even for the para compounds, if the alkoxide moiety bears structural motifs re-
quired for the elimination of a more stable alkene molecule, the heterolytic pathway becomes the predominant pathway. For ex-
ample, in the spectrum of deprotonated 2-phenylethyl 4-aminobenzoate, m/z 136 peak is the base peak because the alkene
eliminated is styrene. Owing to the fact that all deprotonated aminobenzoate esters, irrespective of the size of the alkoxy group,
upon activation fragment to form an m/z 135 ion, aminobenzoate esters in mixtures can be quantified by precursor ion discovery
mass spectrometric experiments. Copyright © 2016 John Wiley & Sons, Ltd.
Additional Supporting Information may be found in the online version of this article at the publisher’s website
Keywords: procaine, benzocaine, collision-induced dissociation; even-electron negative ions; aminobenzoate esters; McLafferty
rearrangement; even-electron rule
acidity of hydroperoxy radical (H–O–O•) (ΔGacid = 346.7 kcal molꢀ1
)
Introduction
is lower than that reported for methyl 4-aminobenzoate
(ΔGacid = 345.2 kcal molꢀ1). [9]
Several para-aminobenzoic acid (PABA) derivatives are known as
drugs or drug candidates. For example, the PABA derivatives pro-
caine [N,N-diethylaminoethyl 4-aminobenzoate](1) and benzocaine
(2) are widely used as local anesthetics.[1,2]
Interestingly, the m/z 235 ion generated by deprotonation of
procaine (1), upon collisional activation, underwent fragmentation
with ease by a homolytic mechanism to generate a rather unex-
pected distonic radical-anion (Scheme 1). In this study, we carried
out a detailed investigation of fragmentation mechanisms of
aminobenzoate anions, because a better understanding of dissoci-
ation pathways assists the characterization of unknown metabolites
and aids the development of more pragmatic quantitative methods
by multiple reaction monitoring procedures.
Mass spectrometry is one of the most commonly deployed
methods, not only for identification and quality control but also
for toxicological, pharmacokinetic, and metabolic investigations of
drugs and drug candidates. Generally, the positive-ion-generating
ionization mode is preferred for mass spectrometric investigations
of aminobenzoate esters because the free amino group is expected
to undergo facile protonation. Although it is not customary to re-
cord negative ion mass spectra of amines or esters, during our ex-
plorations on atmospheric pressure helium–plasma ionization
(HePI) for mass spectrometry,[3,4] we observed that many aromatic
amines, including procaine, are sufficiently acidic to generate gas-
eous anions (Supporting Information Figure S1a). Presumably, the
superoxide radical anions (Oꢀ2 •) present in the HePI source act as
the base to abstract a proton from the analytes.5-8 At least for
aromatic amines, this observation is not a surprise because the
Although it is uncommon for closed-shell even-electron ions to
lose a radical to form an odd-electron ion,10-12 several observations
of radical losses under electrospray-ionization (ESI) collision-
induced dissociation (CID) conditions have been reported.13-16
Herein, we report a diagnostically useful homolytic cleavage path-
way that takes place with minimal ion activation. In addition, we re-
port that deprotonated aminobenzoate esters also undergo an
*
Correspondence to: Athula Attygalle, Center for Mass Spectrometry, Department of
Chemistry, Chemical Biology, and Biomedical Engineering, Stevens Institute of
Technology, Hoboken, New Jersey 07030, USA. E-mail: athula.attygalle@stevens.edu
Center for Mass Spectrometry, Department of Chemistry, Chemical Biology, and
Biomedical Engineering, Stevens Institute of Technology, Hoboken, NJ, 07030, USA
J. Mass Spectrom. (2016), 51, 245–253
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Synthesis of aminobenzoates
One molar equivalent of each alcohol (methanol, ethanol, n-
propanol, n-butanol, trans-3-hexene-1-ol, phenethyl alcohol, benzyl
alcohol, methanol-d4, [1-2H2]ethanol or [2-2H3]ethanol) was mixed
with aminobenzoic acid (10 mg), N,N-dicyclohexylcarbodiimide
(1eq.), and a catalytic amount of 4-dimethylaminopyridine in di-
chloromethane (1 ml) at room temperature. The mixture was stirred
overnight, and the product was isolated by flash chromatographic
elution through a silica gel (32–63μm) column, with a mixture of
hexane: ethyl acetate (1 : 1). Methyl 4-aminobenzoate, EIMS m/z
(%) 151(42), 120(38), 92(35), 65(33).; n-Propyl 4-aminobenzoate,
EIMS m/z (%) 179(20), 137(100), 120(64), 92(29), 65(28).; n-Butyl 4-
aminobenzoate, EIMS m/z (%) 193(20), 137(91), 120(100), 92(29),
65(29).; [2H3]Methyl 4-aminobenzoate, EIMS m/z (%) 154(43), 120
(100), 92(35), 65(31), 39(11).; [1-2H2]Ethyl 4-aminobenzoate, EIMS
m/z (%) 167(31), 137(26), 120(100), 92(27), 65(28), 39(13).; [2-2H3]
Ethyl 4-aminobenzoate, EIMS m/z (%) 168(32), 138(18), 120(100),
92(29), 65(29), 39(9).; n-Hexyl 4-aminobenzoate, EIMS m/z (%) 221
(14), 138(11), 137(100), 121(6), 120(71), 92(22), 65(18), 41(9), 39(7).;
trans-3-Hexenyl 4-aminobenzoate, EIMS m/z (%) 219(5), 138(10),
137(99), 121(8), 120(100), 92(27), 65(25), 41(14), 39(13).; 2-
Phenylethyl 4-aminobenzoate, EIMS m/z (%) 241(7), 137(100), 120
(79), 104(12), 92(30), 65(36).; Benzyl 4-aminobenzoate, EIMS m/z
(%) 227(29), 182(13), 120(100), 91(63), 65(47).; Methyl 3-
aminobenzoate, EIMS m/z (%) 151(49), 120(69), 119(100), 65(57).;
Ethyl 3-aminobenzoate, EIMS m/z (%) 165(82), 137(66), 120(100),
93(60), 92(86), 65(66), 39(23).; n-Propyl 3-aminobenzoate, EIMS m/z
(%) 179(60), 137(100), 120(88), 92(68), 65(52), 39(22).; n-Butyl 3-
ptaminobenzoate, EIMS m/z (%) 193(42), 137(100), 120(58), 92(49),
65(37), 39(15).; Methyl 2-aminobenzoate, EIMS m/z (%) 151(62), 120
(38), 119(100), 92(77), 65(45), 52(13), 39(19).; Ethyl 2-aminobenzoate,
EIMS m/z (%) 165(46), 120(32), 119(100), 92(58), 65(37), 39
(13).; [1-2H2]Ethyl 2-aminobenzoate, EIMS m/z (%) 167(43),
120(31), 119(100), 92(46), 65(26), 39(9).; [2-2H3]Ethyl 2-
aminobenzoate, EIMS m/z (%) 168(64), 120(44), 119(100), 92(48),
65(30), 39(12).; n-Propyl 2-aminobenzoate, EIMS m/z (%) 179(40),
137(17), 120(34), 119(100), 92(47), 65(32), 39(13).; n-Butyl 2-
aminobenzoate, EIMS m/z (%) 193(39), 137(29), 120(35), 119(100),
92(40), 65(29), 39(13).
Scheme 1. Proposed homolytic fragmentation mechanism of
deprotonated procaine (1a).
alkene loss owing to a specific γ-hydrogen transfer that resembles
the well-known McLafferty rearrangement.
Experimental
Materials and methods
Most chemicals, including benzocaine (2), procaine (1), anthranilic
acid, 3-aminobenzoic acid, 4-aminobenzoic acid, methanol-d4,
dicyclohexylcarbodiimide, trans-3-hexene-1-ol, phenethyl alcohol,
4-dimethylaminopyridine were obtained from Sigma–Aldrich
Chemical Co (St Louis, MO, USA) and used as purchased. The Struc-
tures of all synthetic products were confirmed by GC-MS (70 eV).
Mass spectrometry
A Micromass Quattro Ultima triple-quadrupole tandem mass spec-
trometer (Manchester, UK) was used to obtain all collision-induced
dissociation mass spectra. Gaseous ions from samples were gener-
ated by ESI or HePI procedures.[3,4] For HePI, high-purity helium
(99.999%) was passed through the sample introduction capillary
needle, held typically at ꢀ3.5 kV, at a flow rate of about
30 mL minꢀ1 [3,4]
Typically, the hot (300°C) desolvation gas (N2) at
.
a flow of 90 L hꢀ1 was used to heat and vaporize samples. The
source temperature was maintained at 100–150 °C, and the cone
voltage was set to 5–10 V and hexapole I at 10V. For HePI, sample
deposits on glass slides obtained by evaporating sample solutions
in dichloromethane were placed individually about 1.0 cm away
from the helium plasma region in an enclosed ion source. To facil-
itate negative ion formation, a cotton wad soaked in aqueous am-
monia (28% NH3 in H2O) was placed in the ion source.[17,18]
Quantification of procaine using benzocaine as the internal
standard
Two 1000ppm stock solutions of procaine and benzocaine were
made in acetonitrile : water : ammonia (50 : 50 : 1 v/v/v). For the
stock solutions, five standard 25.00 mL solutions containing
100 ppm benzocaine and 100, 200, 300, 400, and 500 ppm procaine
were made in acetonitrile : water : NH4OH (50 : 50 : 1 v/v/v)
(Supporting Information Table S1). Each solution was infused at a
flow rate of 20 μL minꢀ1 to the ESI source, and spectra for precursor
ions of m/z 135 were recorded on a triple quadrupole instrument at
a laboratory-frame collision energy settings of 20 eV, using collision
gas argon at a pressure of 4.8× 10ꢀ2 Pa.
CID mass spectra were recorded on a triple-quadrupole tandem
mass spectrometer. For CID experiments, the pressure of the argon
in the collision cell was held at 116 Pa, and laboratory-frame colli-
sion energy was kept at about 20eV (unless otherwise stated). On
occasion, samples were infused into the ESI source as
acetonitrile–water–NH4OH (50 : 50 : 1 v/v/v) solutions at a flow rate
of 15 μL minꢀ1
.
For O2 addition studies, mass spectra were acquired on a Sciex
API 3000 QqQ mass spectrometer equipped with an electrospray
ionization source. The ion spray voltage was set to 5.5 kV, and a
declustering potential of 50 V, or 125 V, was used to achieve low-
energy or high-energy in-source fragmentation, respectively. CID
mass spectra were acquired using oxygen (99.993%) as the collision
gas. The gas pressure in the collision cell was typically maintained at
2.8 × 10ꢀ3 Pa Samples (1μg mLꢀ1) were infused into the ESI source
as acetonitrile–water–NH4OH (50 : 50 : 0.01) solutions at a flow rate
Computational methods
All calculations were performed using Gaussian 09 (Gaussian, Inc,
Wallingford, CT).[19] The geometries of all species involved in the
several possible pathways were fully optimized by using the widely
used B3LYP[20,21] method with a large 6-311++G(2d,2p) basis set.
Frequency calculations were performed at the same level to verify
the nature of each stationary state in the potential energy surface,
of 25 μL minꢀ1
.
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Fragmentations of deprotonated aminobenzoate esters
i.e. reactant/product are associated with all positive vibrational fre-
quencies, and transition state (TS) is associated with only one imag-
inary frequency, for which the normal vibrational mode
corresponds to the expected bond formation/breaking movements
in a specific reaction pathway. Gibbs free energies were calculated
for species at room temperature and ambient pressure.
A subsequent detailed study conducted on a series of
deprotonated 4-aminobenzoate esters proved that this type of rad-
ical elimination is in fact a diagnostically useful general phenome-
non. For example, the HePI spectra of deprotonated methyl (5),
ethyl (benzocaine; 2a), n-propyl (6), or n-butyl (7) esters of 4-
aminobenzoic acid showed prominent peaks at m/z 150, 164, 178,
and 192 for the respective deprotonated molecules (Supporting In-
formation Figure S2). All these precursor ions, upon mass isolation
and collisional activation, eliminated a radical corresponding to
the alkyl group to generate an ion of m/z 135 in a manner analo-
gous to the fragmentation proposed for procaine (Scheme 2).
Moreover, a fragment-ion spectrum recorded from the m/z 165
ion generated from benzocaine (2) under H/D-exchange source
conditions[17] (Fig. 1D) established the fact that the hydrogen
atom on the amino group was not eliminated during the radical
loss. Similar H/D-exchange results were obtained also from pro-
caine (Fig. 1B). Also, the product-ion spectrum recorded from
deprotonated [2H3]methyl 4-aminobenzoate proved that the
m/z 135 ion is generated by losing the methyl radical (Supporting
Information Figure S3).
Results and discussion
Procaine (1) is an aromatic ester bearing a free amino group. Under
positive-ion-generating mass spectrometric conditions, procaine un-
dergoes facile protonation, and a peak is observed at m/z 237 in its
ESI mass spectrum (Supporting Information Figure S1b). Procaine
also undergoes deprotonation to generate gaseous negative ions
under both electrospray ionization and HePI conditions (Supporting
Information Figure S1a). The m/z 235 negative ion (1a) generated in
this way undergoes facile fragmentation upon collisional activation
to generate a rather unanticipated product ion (m/z 135) (Fig. 1A).
For the formation of m/z 135 ion (3), a neutral radical must be elim-
inated from the precursor ion. Generally, a closed-shell even-electron
ion is predicted to fragment upon activation to generate an even-
electron ion and an even-electron neutral species.[11] Therefore, the
facile formation of the m/z 135 ion (3) is a deviation from this expec-
tation. Thus, the facile formation of m/z 135 ion adds to the repertoire
of noted exceptions16,22-24 and confirms that a radical loss from an
even-electron ion is in reality not a ‘forbidden’ process (Scheme 1).
The product-ion spectra of deprotonated methyl, ethyl (8), n-
propyl, and n-butyl esters of 3-amino benzoic acid (Supporting In-
formation Figure S4) are very similar to the spectra recorded from
the corresponding esters of 4-amino benzoic acid. For both series
of compounds, the m/z 135 ion is generated by the homolytic
cleavage of the alkyl–oxygen bond of the ester moiety. For the es-
ters of 2-aminobenzoic acid, only the spectrum of the methyl ester
Figure 1. Unit–mass resolution product-ion spectra of m/z 235 and 164 anions generated from procaine (A) and benzocaine (C), and those of m/z 236 and
165 anions generated from procaine (B) and benzocaine (D) by exposing samples to D2O vapor in a helium-plasma ionization source. The spectra were
recorded at a laboratory-frame collision energy settings of 20 eV on a tandem quadrupole mass spectrometer (all parameters, including collision gas
pressure and skimmer voltage, were identical).
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atoms (each shares -0.77 e, Supporting Information Table S4) upon
homolytic fragmentation of the alkyl–oxygen bond. Moreover, the
unpaired electron in the product ion (3, m/z 135) is then delocalized
over the nitrogen atom (0.53 e, Supporting Information Table S4)
and the phenyl ring (0.49 e for six ring carbons, Supporting Informa-
tion Table S4). The data presented in Table 1 show that the energy
demand for the homolytic fragmentation of the anions from both
procaine (1) and benzocaine (2) is similar (about 30 kcalmolꢀ1). This
observation is congruent with the fact that essentially the same
type of chemical bond is broken in a similar manner for both com-
pounds. Therefore, we selected the relatively smaller ion, namely,
the deprotonated benzocaine ion (2a), for further computational
calculations in order to compare the energetics of other possible
fragmentation pathways that could be involved. It is known in or-
ganic chemistry that the carbonyl carbon atom of an ester moiety
is amenable to nucleophilic attacks, which promulgates a hetero-
lytic cleavage of the bond between the carbonyl carbon and the ox-
ygen atom of the alkoxide moiety. Despite the absence of any
external nucleophiles in the gas phase for a bimolecular reaction,
we theoretically evaluated this heterolytic bond-breaking pathway
in a unimolecular manner. Interestingly, the energy demand for an
elimination of an ethoxide ion (11) in this way (Reaction 3 in Table 1;
red line in Fig. 2A) was found to be more than the double that
which is required for the homolytic bond cleavage depicted in Re-
action 2. This result is congruent with the experimental observation
that a peak for the ethoxide ion (11, m/z 45), which is expected from
Reaction 3, was not detected. On the other hand, the CID mass
spectrum of the m/z 164 ion from benzocaine (2) showed an
intense peak at m/z 135 (3) confirming the active contribution of
Reaction 2 to the overall fragmentation process.
Scheme 2. Proposed homolytic fragmentation mechanism for
deprotonated methyl (5), ethyl (2a), n-propyl (6), and n-butyl (7) esters of
4-aminobenzoic acid.
shows a prominent peak at m/z 135 for the radical loss (Supporting
Information Figure S5a). In contrast, the spectra of deprotonated
ethyl (9) and higher esters of 2-aminobenzoic acid showed a more
pronounced peak at m/z 136 (10) owing to a charge-assisted alkene
loss (Supporting Information Figure S5b–d).
To comprehend the bond-breaking processes that take place
during the generation of fragment ions from anions derived from
procaine (1) and benzocaine (2) type of esters, a detailed quantum
chemical study was performed with B3LYP using a large 6-311++G
(2d,2p) basis set. The computational results showed that the nega-
tive charge on the anionic species m/z 235 and m/z 164, generated
by deprotonation of procaine (1) and benzocaine (2), respectively,
is accumulated primarily on the nitrogen atom because a formal
charge of ꢀ0.82 e was indicated on the nitrogen atom (Supporting
Information Tables S2 and S3). For both anions (m/z 235 and 164), a
homolytic cleavage of the bond between oxygen and carbon
atoms of the alkoxide moiety leads to the formation of an m/z
135 anion (3) and a corresponding neutral radical as depicted by
Reactions 1 and 2 in Table 1. Interestingly, the computational re-
sults showed the negative charge, initially located on the nitrogen
atom of the precursor ion, to shift primarily to carboxyl oxygen
Moreover, in the mass spectrum recorded from benzocaine (2), a
small peak was also observed at m/z 136 (12) (Fig. 1C). This peak
corresponds to an ethylene loss from the precursor ion by a rear-
rangement process. Product-ion spectra recorded from m/z 166
and 167 anions generated from [1-2H2]ethyl 4-aminobenzoate
and [2-2H3]ethyl 4-aminobenzoate confirmed that the fragmenta-
tion reaction follows a specific pathway because the spectrum of
the dideuterio compound showed a peak at m/z 136, whereas that
of the trideuterio congener showed a corresponding peak at m/z
137 (Fig. 3). Because both peaks (m/z 136 and m/z 137) repre-
sented a loss of H2C¼CD2 from the precursor ion, it was evident
that a proton transfer takes place specifically from the γ-position
(Fig. 3). To afford an ethylene loss, the ethyl group of the lowest
energy conformation of the m/z 164 ion (2a) should rotate to a
higher-energy conformer in which the γ-H in the ethyl moiety is
poised to transfer a proton to the carbonyl oxygen (Supporting In-
formation Figure S6). This transition state (TS 2a–12) then pro-
ceeds to generate the m/z 136 ion (12) by eliminating ethylene
(see Reactions 4 and 5 in Table 1). It is evident from the computed
TS 2a–12 structure that the γ-H is partially transferred to the car-
bonyl oxygen (RO…H = 1.145 Å), with a concomitant increase of
the C¼O bond length (ΔRC¼O = +0.079 Å), a decrease of the C–O
bond length (ΔRC–O = ꢀ0.090 Å), and a partial detachment of the
ethylene moiety (ΔRO–C = +0.327 Å), as envisaged by the proposed
pathway (Scheme 3). Although the overall process of formation of
m/z 136 ion and ethylene is only slightly thermodynamically favo-
rable (ꢀ1.30 kcal molꢀ1; Reaction 5 in Table 1), the energy require-
ment for this pathway (the activation energy of Reaction 4) is not
exceedingly high, 42.39 kcal molꢀ1, which is ~12 kcal molꢀ1 higher
than the demand of the homolytic pathway (Reaction 2), but still
much smaller than that needed for the heterolytic pathway
Table 1. Calculated Gibbs free energies of fragmentation reactions
Reaction no.
Precursor
reactant
Reaction
ΔG
(kcal molꢀ1
)
1
Deprotonated
m/z 235 → m/z
135 + Et2N-CH2CH2•
m/z 164 → m/z
135 + Et•
31.11
30.14
64.61
42.39
ꢀ1.30
25.99
31.49
ꢀ20.35
21.66
44.38
ꢀ0.39
procaine
2
Deprotonated
benzocaine
3
Deprotonated
m/z 164 → 119
Da + m/z 45 (EtOꢀ)
m/z 164 → TS
benzocaine
4
Deprotonated
benzocaine
5
Deprotonated
m/z 164 → m/z
136 + H2C = CH2
m/z 164 → m/z
135 + Et•
benzocaine
6
Deprotonated ethyl
2-aminobenzoate
Deprotonated ethyl
2-aminobenzoate
Deprotonated ethyl
2-aminobenzoate
Deprotonated ethyl
3-aminobenzoate
Deprotonated ethyl
3-aminobenzoate
Deprotonated ethyl
3-aminobenzoate
7
m/z 164 → TS
8
m/z 164→m/z
136 + H2C = CH2
m/z 164→ m/z
135 + Et•
9
10
11
m/z 164→ TS
m/z 164→m/z
136 + H2C = CH2
(Reaction 3) by about 22 kcal molꢀ1
.
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Fragmentations of deprotonated aminobenzoate esters
Figure 2. Reaction pathways for fragmentation of m/z 164 ion generated from ethyl esters of 4-amino (A), 3-amino (B), 2-aminobenzoic acid (C), the
optimized molecular structures, and their relative energies. Color scheme for atoms: C = cyan, O = red, N = blue, and H = gray.
Figure 3. Unit–mass resolution product-ion spectra of m/z 166 and 167 anions generated from [1-2H2]ethyl 4-aminobenzoate (A) and [2-2H3]ethyl
4-aminobenzoate (B), recorded at a laboratory-frame collision energy settings of 20 eV on a tandem quadrupole mass spectrometer equipped with a
helium-plasma ionization source (all parameters, including collision-gas pressure and skimmer voltage, were identical).
Presumably, the charge on the NH group does not contribute
very significantly to the fragmentation mechanism because
computational results show that even for uncharged para-
H2NC6H4CO2CH2CH3 (benzocaine), the heterolytic fragmentation
pathway is an energetically feasible process (Supporting
Scheme 3. Proposed fragmentation mechanism for elimination of
Information Table S6). Furthermore, a charge-assisted mechanism
produces a non-aromatic quinone-like structure, which was not
supported by optimized computational calculations because the
charge on the nitrogen atom does not shift to the carbonyl oxy-
gen even after the alkene is eliminated.
Moreover, this alkene-loss mechanism is also supported by experi-
mental results displayed in Fig. 4. From plots of relative ion intensities
versus laboratory-frame collision energies (‘breakdown graphs’, Fig. 4),
it is evident that the ethylene loss (9→TS 9-10) is the preferred
ethylene from deprotonated ethyl ester of 4-aminobenzoic acid (2a).
The specific alkene elimination process discussed previously re-
sembles the well-known McLafferty rearrangement for radical
cations.[9] Our computational results suggest that the negative
charge (ꢀ0.82 e; Supporting Information Table S2) located primarily
on the nitrogen atom of deprotonated benzocaine (2a, m/z 164)
continues to remain on the nitrogen atom even after the m/z 136
product ion is formed (ꢀ0.81 e; 12, Supporting Information Table S5).
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Figure 4. Plots of relative intensities of ions versus laboratory-frame collision energy for m/z 164 ( ), 136( ), 135 ( ) ions derived from ethyl 2- (A1), 3- (B1), and 4-
aminobenzoate (C1), and product-ion spectrum of m/z 164 anions of each ester (A2, B2, and C2) recorded at a laboratory-frame collision energy settings of 20 eV
on a tandem quadrupole mass spectrometer equipped with a HePI source (all parameters, including collision gas pressure and skimmer voltage, were identical).
of an alkene from the ortho isomers of alkyloxy benzoic acids has
been noted previously.[22] Evidently, the alkene loss is a general
phenomenon because a significant peak at m/z 136 appears in all
spectra of 2-aminobenzoates synthesized from ethanol (9) and
higher n-alkanols (Supporting Information Figure S5). Results from
two deuterium-labeled compounds confirmed the role of the
ortho-isomer-specific fragmentation pathway (Scheme 4). The
spectrum recorded from the m/z 166 ion from [1-2H2]ethyl 2-
Scheme 4. Proposed fragmentation mechanism for deprotonated ethyl
ester of 2-aminobenzoic acid (9).
aminobenzoate showed a fragment-ion peak at m/z 136, analogous
to those observed from the non-labeled compound. This result con-
firmed that βꢀhydrogens do not directly participate in the proton
transfer process. On the other hand, the product-ion spectrum of
the m/z 167 ion from [2-2H3]ethyl 2-aminobenzoate showed a peak
at m/z 137, which indicated that a deuteron from the γꢀposition is
specifically transferred during the ethylene elimination process
(Supporting Information Figure S7; Scheme 4). The mechanism re-
sembles the well-known McLafferty rearrangement for radical cat-
ions. Grossert et al. previously reported a similar observation with
negative ions generated from 3-hydroxy or 3-aminocarboxylic acids.[27]
For the alkene loss to take place, the precursor ion must be acti-
vated to reach the corresponding transition state. However, com-
putational chemistry calculations showed that only the alkene
elimination reaction of the ortho isomer is exergonic by
ꢀ20.35 kcalmolꢀ1. In contrast, for both meta and para isomers,
the alkene loss process is neither exergonic nor endergonic (Fig. 2).
In other words, once the transition state is formed, only that of the
ortho isomer (TS 9-10) bears thermodynamic compulsion to pro-
ceed to an elimination product (10). In the absence of such a strong
thermodynamic driving force for an alkene loss, deprotonated meta
and para isomers upon activation proceed to fragment by the
charge-assisted pathway for the ortho isomer (Figure 4A1; Scheme 4),
although the ethylene loss is not the favored process for the meta and
para isomers, a weak loss is evidenced by the presence a m/z 136 peak
in spectra recorded at very high collision energies (Figure 4B2 and C2;
Supporting Information Figures S2 and S4). An analogous loss of
carbon monoxide from meta and para-hydroxyphenylketones[25]
,
or CO2 from meta- and para-hydroxybenzoic acids,[26] has been
reported previously.
A comparison of spectra recorded from deprotonated esters of
2-, 3-, and 4-aminobenzoic acids showed that the spectra of meta
and para isomers are not very different from each other. In contrast,
those of the ortho isomers were dramatically different. To start with,
the ortho isomers required less collisional activation to initiate frag-
mentation (Figure 4A1). Furthermore, the initial fragmentation that
meta and para isomers undergo is the radical loss (Figure 4B2 and
C2). On the other hand, the homolytic fragmentation pathway is to-
tally suppressed in the esters of 2-aminobenzoic acid because an
ortho-isomer-specific fragmentation mechanism is more favorable
for ortho isomers (Figure 4A1). A similar but less dramatic (because
several other fragmentations pathways predominate) elimination
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SPECTROMETRY
Fragmentations of deprotonated aminobenzoate esters
Figure 5. Unit–mass resolution product-ion spectra of m/z 220, 218, 240, and 226 anions generated by electrospray ionization from n-hexyl (A), trans-3-
hexenyl (B) 2-phenylethyl (C), and benzyl (D) esters of 4-aminobenzoate, recorded at a laboratory-frame collision energy settings of 20 eV on a tandem
quadrupole mass spectrometer (all parameters, including collision gas pressure and skimmer voltage, were identical).
Figure 6. Unit–mass resolution product-ion spectra acquired on a Sciex API 3000 instrument, equipped with an ESI source, from the m/z 164 ion (A) and that
of the in-source-generated m/z 91 ion (C) from deprotonated benzocaine, using N2 as collision gas. Analogously, product-ion spectra ‘B’ and ‘D’ were recorded
using O2 as the collision gas.
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SPECTROMETRY
homolytic pathway to generate an m/z 135 ion. Overall, our compu-
tational results agree well with the experimental observations that
the charge-assisted McLafferty type rearrangement is the more
favorable dissociation pathway, as opposed to the homolytic frag-
mentation pathway, for the ortho compounds.
To study the effect of the nature of the alkyl group on the
fragmentation pathways, spectra from n-hexyl, 3-hexenyl, 2-
phenylethyl, and benzyl esters of 4-aminobenzoic acid were re-
corded. The spectrum of n-hexyl 4-aminobenzoate (Fig. 5A) showed
a strong peak at m/z 135 (3) and a minute peak at m/z 136 (12),
which conforms to the trend observed for other n-alkyl 4-
aminobenzoates. However, the spectrum of trans-3-hexenyl 4-
aminobenzoate (Fig. 5B) showed a more intense peak at m/z 136
compared with that at m/z 135 owing to the fact that a conjugated
diene is now eliminated. Finally, in the spectrum of 2-phenylethyl 4-
aminobenzoate, the m/z 136 peak becomes the base peak because
the alkene eliminated is styrene (Fig. 5C). In addition, the proton
that is transferred in the fragmentation of trans-3-hexenyl and 2-
phenylethyl esters originate from an allylic position (Scheme 3).
The weaker allylic C–H bond renders the proton transfer more fac-
ile. Thus, we conclude that the McLafferty-type rearrangement
pathway described here becomes more favorable when the alkoxy
moieties bear specific structural motifs to eliminate a more stable
alkene molecule. For the homolytic pathway, the stability of leaving
radical also plays a major role. For example, the spectrum of benzyl
4-aminobenzoate is dominated by the peak at m/z 135 for the
distonic radical-anion because of the exceptional stability of the
leaving benzyl radical.
The spectra of procaine (1) and benzocaine (2) both showed
peaks at m/z 91 and 135, which represent distonic anions.[28] To
further investigate the radical nature of the m/z 135 and 91 ions,
an oxygen addition experiment[29] was conducted. With O2 as the
collision gas, product-ion spectra recorded from deprotonated
benzocaine (Fig. 6B) and from the m/z 91 ion (4) generated in-
source from a benzocaine sample (Fig. 6D) showed an additional
peak at m/z 107, which was absent in the spectra recorded with
N2 as the collision gas (Fig. 6A and C). The m/z 107 ion (14) is a
product-ion resulting from an ion-molecule reaction between
the m/z 91 ion (4) and molecular O2 in the collision cell
(Scheme 5).
Figure 7. A plot of intensity ratios of m/z 235 peak for procaine and m/z
164 peak for benzocaine against concentration ratios of procaine to
benzocaine from an m/z 135-precursor-ion experiment.
containing different concentrations of procaine and a constant con-
centration of benzocaine were prepared (Supporting Information
Table S1) and subjected to an m/z 135 ion precursor-ion scan exper-
iment (Supporting Information Figure S8). A plot of peak intensity
ratio values for (m/z 235)/(m/z 164) versus concentration ratios
gave a linear calibration curve (Fig. 7), demonstrating the efficacy
of the m/z-135 parent-ion scan experiment for quantification of
aminobenzoate esters.
Conclusions
We conclude that the ring position of the carboalkoxy group
and the nature of the alkoxy group determine the relative ex-
tent of the contribution of the homolytic (alkyl-radical loss) ver-
sus heterolytic cleavage (alkene loss) pathways in the gas-phase
fragmentation of deprotonated aminobenzoate esters. Generally,
the meta and para isomers favor the homolytic cleavage path-
way, whereas the ortho compounds prefer the charge-assisted
heterolytic fragmentation mechanism. However, when structural
motifs of the alkoxy substituent permit the elimination of more
conjugated alkene molecules, the alkene elimination pathway
then becomes the preferred mechanism even for para isomers.
The fact that all anions generated from deprotonation of
aminobenzoate esters upon collisional activation yield a frag-
ment to form an m/z 135 ion can be employed to quantify
components in mixtures of aminobenzoate esters made from
different alcohols by precursor-ion discovery mass spectrometric
experiments without the need of prior time-consuming chro-
matographic separations. Our generalizations on the dissocia-
tion of the carboxylate functionality of esters, based on
experimental observations and theoretical results, may be appli-
cable to a broad range of decomposition reactions such those
afforded by thermolysis and photolysis.
Because both deprotonated benzocaine and procaine fragment
to generate a common ion (m/z 135), we envisaged that one ester
can be deployed as an internal standard to quantify the other by a
precursor-ion discovery experiment. The precursor-ion spectrum of
the m/z 135 ion, recorded from a mixture of benzocaine and pro-
caine, is depicted in Supporting Information Figure S8. This result
shows that the peak at m/z 135 can be useful to identify
aminobenzoates in a mixture of congeners. To determine whether
precursor-ion spectra of m/z 135 will deliver accurate
aminobenzoate abundances in a mixture, a series of solutions
Acknowledgements
This research was supported by funds provided by Stevens Institute
of Technology (Hoboken, NJ). We thank Bristol-Myers Squibb
(New Brunswick, NJ) for the donation of the Quattro Ultima and
the API 3000 mass spectrometers.
Scheme 5. Identification of distonic radical anions by oxygen addition.
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Journal of
MASS
SPECTROMETRY
Fragmentations of deprotonated aminobenzoate esters
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Supporting Information
Additional Supporting Information may be found in the online
version of this article at the publisher’s website
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