On the origin of the methyl radical loss from deprotonated ferulic and isoferulic acids: Electronic excitation of a transient structure

Article abstract of DOI:10.1007/s13361-013-0604-2

Formation of radical fragments from even-electron ions is an exception to the "even-electron rule". In this work, ferulic acid (FA) and isoferulic acid (IFA) were used as the model compounds to probe the fragmentation mechanisms and the isomeric effects o

Full text of DOI:10.1007/s13361-013-0604-2

B American Society for Mass Spectrometry, 2013
J. Am. Soc. Mass Spectrom. (2013) 24:941Y948
DOI: 10.1007/s13361-013-0604-2
RESEARCH ARTICLE
On the Origin of the Methyl Radical Loss
from Deprotonated Ferulic and Isoferulic Acids:
Electronic Excitation of a Transient Structure
Xiaoping Zhang,1,2 Fei Li,1 Huiqing Lv,3 Yanqing Wu,1 Gaofeng Bian,1 Kezhi Jiang1
1Key Laboratory of Organosilicon Chemistry and Material Technology, Hangzhou Normal University, Hangzhou 310012, China
2College of Chemical Engineering and Material Science, Zhejiang University of Technology, Hangzhou 310014, China
3College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China
Abstract. Formation of radical fragments from even-electron ions is an exception to
the “even-electron rule”. In this work, ferulic acid (FA) and isoferulic acid (IFA) were
used as the model compounds to probe the fragmentation mechanisms and the
isomeric effects on homolytic cleavage. Elimination of methyl radical and CO2 are the
two competing reactions observed in the CID-MS of [FA – H]− and [IFA – H]−, of which
losing methyl radical violates the “even-electron rule”. The relative intensity of their
product ions is significantly different, and thereby the two isomeric compounds can be
differentiated by tandem MS. Theoretical calculations indicate that both the singlet-
triplet gap and the excitation energy decrease in the transient structures, as the
breaking C–O bond is lengthened. The methyl radical elimination has been
rationalized as the intramolecular electronic excitation of a transient structure with an elongating C–O bond.
The potential energy diagrams, completed by the addition of the energy barrier of the radical elimination, have
provided a reasonable explanation of the different CID-MS behaviors of [FA – H]− and [IFA – H]−.
Key words: Methyl radical elimination, Ferulic acid, Isoferulic acid, Electronic excitation, Transient structure,
Isomeric differentiation
Received: 21 December 2012/Revised: 28 January 2013/Accepted: 5 February 2013/Published online: 12 April 2013
Over the years, several reports have appeared in the literature
describing violations of the “even-electron rule”, in which an
Introduction
ESI-generated closed-shell ion dissociates to give an odd-
he combination of electrospray ionization (ESI) mass
spectrometry with collision-induced dissociation (CID)
electronic (OE) ion [7–19]. It is desirable and significant to
investigate the underlying mechanism on the formation of OE
ions from even-electronic (EE) ions because such consider-
ations are important for better structural elucidation by ESI
tandem mass spectrometry.
Most of the violations occur in cases that involve
aromatic, resonance-stabilized radicals as fragmentation
products [9–13], indicating that they also obey thermody-
namic rules [4]. Thus, the resonance stabilization renders the
spin forbidden decomposition channel accessible. It was also
proposed that aromatic stabilization leads to lower energy
barriers, which might kinetically favor the generation of the
radical fragments [4]. Sometimes, when the competing
decomposition channels are not available, radical species
appear as the fragment products [12].
T
technique is an indispensable analytical tool, which has been
widely used to analyze a large variety of organic and
pharmacologic compounds [1–3]. Successful structure eluci-
dation based on tandem mass spectrometry (MS/MS) data is
dependent on correct interpretation of the fragmentation
pathways. The ions generated in the electrospray process are
predominantly stable ionic species with closed-shell electronic
structures, such as [M + H]+, [M + Na]+, and [M – H]−. Upon
low energy collisional activation, dissociation of the closed-
shell ions usually follows the “even-electron rule” [4], and
leads to closed-shell fragment ions and neutral fragments [5, 6].
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-013-0604-2) contains supplementary material, which
is available to authorized users.
A single electron transfer (SET) mechanism via the
electron excitation in an ion/neutral complex (INC), which
results from the heterolytic cleavage of the EE precursor ions
driven by the positive charge, was proposed to be the key
Correspondence to: Kezhi Jiang; e-mail: jiangkezhi@hznu.edu.cn

942
X. Zhang et al.: Mechanism of the Methyl Radical Loss
step in the OE ions formation from the dissociation of some
protonated molecules, rather than the simple homolytic
bond cleavage [14–16]. Formation of radicals from the
homolytic cleavage of the precursor ion may also occur
with the involvement of an electronically excited state. Hau
et al. described the radical elimination of closed shell
pyridinium cations with the involvement of a cation
diradical [17]. Although not specified by the authors, such
an intermediate may correspond to a triplet state. Theoret-
ical calculations show that the singlet-triplet gap for
transient structures with an elongated benzylic C–C bond
is very low for protonated and alkylated pyridinium cations
and radical formation may result from the mixing of these
states [18]. Recently, Cabrera et al. proposed that dissoci-
ation of the lowest triplet excited state would provide a
reasonable interpretation for the radical loss of the
protonated hydroxypyridine N-oxides [19]. However, the
fragmentation mechanisms for the homolytic cleavage
processes have not been well documented, especially for
the radical elimination of anions [11].
Many natural products, such as methoxyl phenolic com-
pounds [20], flavonoid O-glycosides [3, 21], and oxygenated
tetracyclic triterpenoids [22], have reported to form radical
anions in the negative tandem MS. Ferulic acid (FA) and
isoferulic acid (IFA) are phenolic plant metabolite compounds,
that exhibits antioxidative properties and a large variety of
therapeutic effects against various diseases [23–25]. In the
present work, we demonstrate a special case where the methyl
radical expulsion competes with the CO2 elimination upon
low-energy dissociation of the deprotonated FA and IFA
molecules in the negative ESI tandem mass spectrometry.
Experimental and theoretical approaches are employed to
investigate the mechanisms of this decomposition and the
isomeric effect.
and ethyl isoferulate (IFE) were synthesized by the reaction
of the corresponding acid with ethanol in the presence of
SOCl2, and identified by IR, MS, and NMR.
Methanol (HPLC grade) was obtained from Merck
(Darmstadt, Germany). Purified water was prepared by
using Milli-Q water purification system (Millipore, Bedford,
USA).
Mass Spectrometry
The MS/MS experiments were performed on a micrOTOF QII
(Q-TOF) mass spectrometer from Bruker Daltonics (Billerica,
MA, USA), equipped with an ESI ion source. The diluted
solvent was directly infused into the electrospray ionization
source of the mass spectrometer with a syringe pump at a flow
rate of 180 μL/h. The parameters of Q-TOF mass spectrometers
were set as follows: drying gas flow in the ion source was set up
to 4 L/h and nebulizer gas pressure to 0.4×105Pa (both were
N2), the temperature of ion source was 180 °C, and voltage was
−3.5 kV. In MS/MS experiments, mass width of the selected
ion was 6 Da, and the collision energy of CID for the selected
ions was set at the range of 3 to 15 eV with Argon as the
collision gas. The instrument was operated at a resolution
higher than 15,000 full width at half maximum using the
micrOTOF-Q control program ver. 2.3. The data were analyzed
using the Data Analysis ver. 4 software package delivered by
Bruker Daltonics.
Theoretical Calculation
The theoretical calculations were performed using the
Gaussian 03 program [26]. The equilibrium geometries of
the target species were optimized using the density func-
tional theory (DFT) method at the B3LYP/6-311+G(2d, p)
level. The optimized structures were identified as the true
minima in energy by the absence of imaginary frequencies.
Vibrational frequencies and zero-point energies (ZPE) for all
the key species were calculated at the same level of theory.
The electron excitation energy of a transient structure was
obtained by the Configuration Interaction approach (CI-
Singles). The optimized structures were displayed by the
Gauss View (Version 3.09) software. Hard data on geom-
etries of all structures involved are available as Supporting
Information. The energies discussed here are the sum of
electronic and thermal free energy.
Experimental
Chemicals
FA and IFA (purity 99.0 %, Scheme 1) were purchased from
Sigma-Aldrich (Milwaukee, WI, USA). Ethyl ferulate (FE)
10
11
OH
O
O
12
O
7
2
O
1
3
13
O
9
8
4
6
HO
HO
14
5 FA
FE
Results and Discussion
CID Behavior
10
11
9
O
12
HO
O
7
2
3
1
HO
O
OH
O
8
4
Fragmentation of the Deprotonated Ferulic Acid Figure 1a
gives the CID mass spectrum of the deprotonated FA ([FA –
H]−, m/z 193) obtained with a Q-TOF mass spectrometer,
and the accurate MS/MS results are summarized in Table 1S
in the Supporting Information. Decomposition of [FA – H]−
mainly generates three fragment ions at m/z 178, 149, and
14
6
O
5
13
IFE
IFA
Scheme 1. Structures of ferulic acid, isoferulic acid, and
their ethyl and benzyl esters

X. Zhang et al.: Mechanism of the Methyl Radical Loss
943
Figure 1. Tandem mass spectrometry of the deprotonated ferulic acid (a) and the deprotonated isoferulic acid (b) by ESI-Q/TOF
with the collision energy at 8 eV
178 and 134 are more attractive because they are radical
anions with the electron open-shell structure and their
formation involves the violation of the “even-electron rule”
[4–7]. The fragmentation mechanisms and the isomeric
effects for the radical elimination will be documented in
detail in the following work.
134, and the reaction pathways are given in Scheme 2. The
basic fragment ion at m/z 178 is identified as the
deprotonated 3-(3,4-dihydroxy phenyl)acrylic acid radical
according to the “nitrogen rule”, which resulted from the
methyl radical elimination of the precursor ion m/z 193.
Methyl radical loss is a shared feature of methoxyl-
substituted aromatic compounds [20]. The fragment ion m/z
149 is attributed to the 2-methoxy-5-vinyl phenolate anion,
originating from the CO2 elimination of the precursor ion.
The fragment ion m/z 134 is also a radical anion, the
deprotonated 4-vinylbenzene-1,2-diol radical, resulting ei-
ther from the CO2 elimination of the fragment ion m/z 178 or
from the methyl radical elimination of the ion m/z 149. The
formulas of these proposed fragment ions are supported by
accurate mass measurements obtained by ESI-Q-TOF, in
which the relative errors between the measured and the
calculated masses for the assigned structures are less than
5 ppm for all the abundant ions in Table 1S in the
Supporting Information. Herein, the fragment ions at m/z
The Effect of the Substitution Pattern To probe the effect of
the substituent pattern on the formation of the radical anions,
fragmentation of the deprotonated IFA, [IFA – H]−, has also
been investigated to compare the fragmentation behavior of [FA
– H]− (Figure 1). As expected, [IFA – H]− displays similar
reaction channels to [FA – H]− in the CID process, which leads
to the corresponding product ions at m/z 178, 149, and 134
(Scheme 1S in the Supporting Information).
Interestingly, the relative intensity of these product ions is
significantly different between the two isomers under the
same CID conditions, indicating that the two isomeric
compounds can be differentiated by tandem MS. The
O
O
O
O
O
O
O
OH
O
OH
HO
O
FA-Ha 67.3 kJ/mol
m/z 193
FA-178
IFA-178
Path1
Path2
m/z 178
O
O
O
O
O
O
O
O
O
OH
FA-134
IFA-134
m/z 134
FA-149
m/z 149
FA-Hb 0.0 kJ/mol
m/z 193
Scheme 2. The proposed fragmentation channels for FA–Hb

944
X. Zhang et al.: Mechanism of the Methyl Radical Loss
precursor ion (m/z 193) of [FA – H]− is the base peak, and
its fragment ion m/z 178 has a relative intensity of 46.7 %
(Table 1S in the Supporting Information). In contrast, the
fragment ion m/z 178 rather than the precursor ion becomes
the base peak in the MS/MS spectrum for [IFA – H]−, and
the relative intensity of the precursor ion reduces to 71.2 %.
Furthermore, the fragment ion m/z 149 originating from the
CO2 elimination of [FA – H]− is significantly more abundant
than the corresponding fragment ion from [IFA – H]−
(24.6 % in FA versus 3.4 % in IFA).
ation channel of losing CO2. As expected, only the
radical species at m/z 206, resulting from the methyl
radical elimination of [FE – H]− or [IFE – H]−, has been
observed in the CID mass spectra (Figure 1S in the
Supporting Information). Identification of the radical
fragments has also been substantiated by the accurate
mass spectral data (Table 1S in the Supporting Informa-
tion). Similar to FA and IFA, [IFE – H]− is somewhat
more facile towards methyl radical elimination than
[FE – H]−.
To gain more insight into these different fragmentation
behaviors, CID product ion mass spectra of [FA – H]− and
[IFA – H]− have been acquired with various collision
energies ranging from 3 to 15 eV [11]. Breakdown curves
have been plotted in Figure 2, and show the relationship
between the relative abundance of selected ions and the
collision energy. It is apparent that both [FA – H]− and [IFA
– H]− undergo decomposition to the first product ions at m/z
178 and 149, and that the secondary product ion m/z 134 is
formed from the ion at m/z 178 or 149. Decomposition of
[IFA – H]− is more facile than that of [FA – H]− at various
collision energies, which mainly leads to the open-shell
product ion at m/z 178. For the competing reactions leading
to the first product ions, CO2 elimination of [FA – H]− is
significantly more facile than that of [IFA – H]−.
Computational Study
According to the above experimental results, two reaction
channels have been proposed for the dissociation of [FA –
H]− and [IFA – H]−. Path-1 involves methyl radical
elimination prior to CO2 decomposition, whereas CO2
decomposition is followed by methyl radical elimination in
Path-2 (Scheme 2 and Scheme 1S). The fragmentation
channels of [FA – H]− and [IFA – H]− have been
investigated by DFT theoretical calculations at the
uB3LYP/6-311+G(2d,p) level to probe the fragmentation
reactions.
Deprotonation Site FA has two active protons in its
structure, the carboxylic proton H11 and the phenolic
hydroxyl proton H14 (Scheme 1), and each has the potential
to undergo deprotonation during the negative ESI process,
and give FA–Ha and FA–Hb, respectively. The deproton-
ation site was reported to be affected largely by the solvent
used in the ESI process under the conditions that trace (mM)
KOH or LiOH was added in to the ESI solvent [27, 28]. In
the absence of KOH or LiOH, however, the MS/MS spectra
The Effect of Esterification To gain more insight into the
effect of the chemical structure on the homolytic
cleavage of the EE ions, ethyl ferulate (FE) and ethyl
isoferulate (IFE) have been synthesized for the negative
tandem MS investigation. For the occurrence of FE and
IFE, the carboxyl group has been protected by ethyl
esterification, which will prevent the competing dissoci-
Figure 2. The energy-resolved plot for [FA – H]− (a) and [IFA – H]− (b)

X. Zhang et al.: Mechanism of the Methyl Radical Loss
945
of [FA – H]− are practically identical irrespective whether
methanol or acetonitrile is used in the electrospray process,
while the MS/MS spectrum varies distinctively in the
presence of KOH (Figure 2S in the Supporting Information).
Therefore, deprotonation of the neutral molecule of FA in
the ESI process is hardly affected by the ESI solvent.
The calculated free energy of FA–Ha is 67.3 kJ/mol
higher than that of FA–Hb, indicating that the phenolic
hydroxyl proton is more likely to undergo dissociation in the
ESI process than the carboxylic proton. The stability of FA–
Hb results from the resonance effect, which disperses the
negative charge onto the phenyl group and the allyl acetic
moiety (Scheme 3); the negative charge in FA–Ha is mainly
localized on the carboxylic group. Thus, deprotonation of
FA primarily generates the gas-phase FA–Hb in the negative
ESI process due to the remarkable energetic difference
between FA–Ha and FA–Hb.
Figure 3. Calculated potential energy curves for the radical
fragmentation of the singlet (dots) and the triplet (triangles)
FA–Hb
Similar to FA, the phenolic hydroxyl group of IFA is
also thermodynamically more favorable to undergo
deprotonation than the carboxylic group does in the gas
phase because of the resonance stabilization (Scheme 3).
However, the negative charge in IFA-b cannot be
dispersed onto the allyl acetic moiety, because of the
meta position of the phenolic hydroxyl anion. As a
result, IFA-b lies 32.9 kJ/mol above FA-b in free energy,
indicating that fragmentation of the deprotonated IFA is
more favorable than FA, which is in agreement with the
CID MS results.
calculated transition state (TS) may represent a plateau in the
fragmentation channel rather than a saddle point. DFT methods
often fail to locate a transition state along the reaction
coordinates in the homolytic reactions, due to difficulties in
treating the self-interaction of electrons [12, 18, 19, 29]. This
leads to inaccurate estimation of TS energies for the radical loss
process. The calculated considerable TS value (247.8 kJ/mol)
obviously disagrees with the highly efficient homolytic
cleavage observed in the CID-MS experiments, and distinc-
tively overestimates the actual energy barrier in the radical
elimination of FA–Hb.
Radical formation from closed-shell ions has been reported
to occur via the involvement of an electronically excited state,
such as the triplet state [17–19]. A full geometry optimization
and frequency analysis have been carried out for the singlet and
the triplet states of FA-Hb. Both have near planar geometries
(Figure 3S in the Supporting Information), but the triplet one
(FA–Hb–t) has longer ring bond lengths than the singlet state
(FA–Hb), indicating that FA–Hb–t is formed by excitation of a
π electron of the phenyl ring into the anti π orbital. Moreover,
natural bond orbital (NBO) analysis of FA-Hb shows that the
HOMO (51) and LUMO (52) of FA–Hb are all mainly located
at the phenyl ring (Figure 4). The radical center at the phenyl
Mechanistic Investigation of the Radical Elimination The
radical elimination pathway of FA–Hb has been characterized
by stepwise increase in the length of the breaking methoxyl C–
O bond, while optimizing all other coordinates at each given
bond length. The curve with dots in Figure 3 describes the
potential energy curve for the disruption of the methoxyl O–C
bond in FA–Hb (the singlet state) calculated at the uB3LYP/6-
31g(d) level. For a typical reaction, it is straightforward to
optimize the transition state geometry from the point with the
highest energy along the path. However, the energy of FA–Hb
increases monotonically as the O–C bond is elongated. The
OH
O
OH
O
OH
O
O
FA-Hb
O
O
O
O
O
(c)
(a)
(f)
(b)
OH
H
OH
O
O
O
O
O
O
O
O
O
O
(d)
(e)
IFA-Hb
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
(d)
(b)
(c)
(a)
Scheme 3. The resonance of FA–Hb and IFA–Hb

946
X. Zhang et al.: Mechanism of the Methyl Radical Loss
Figure 4. HOMO and LUMO of FA–Hb
ring will initiate the disruption (α cleavage) of the neighboring
C–O bond in the methyl radical elimination of the FA–Hb–t.
As shown in Figure 3, there is a maximum, FA–TS–t, in
the potential energy curve (with triangles) for scanning the
methoxyl C–O bond distance in FA–Hb–t. DFT calculations
indicate that FA–TS–t lies 105.2 kJ/mol above FA–Hb–t in
free energy. Therefore, the overall energy barrier via FA–
TS–t is 270.3 kJ/mol relative to the initial FA–Hb geometry,
since FA–Hb–t is located at 164.9 kJ/mol above the singlet
FA–Hb. Such a considerable energy barrier seriously in-
hibits the occurrence of the reaction channel, in which
excitation occurs prior to homolytic cleavage.
Nevertheless, the calculated vertical singlet-triplet transition
energies decrease as the O–C bond is lengthened, as shown in
Figure 3. The calculated PES scans of the singlet ground state
and the triplet excited state cross at a certain point, at which the
electronic excitation is a thermodynamically favorable process.
The crossing point corresponds to a transient structure of FA–
Hb with the C–O bond length of approximately 2.343 Å.
Because electron excitation occurs on the femtosecond (10−15s)
times scale, while the vibrational motion occurs on the 100
femtosecond (10−13s) time scale, electronic excitation is well
likely to perform for a transient structure of FA–Hb. It is
noteworthy that the first excitation energy of the transient
structures of FA–Hb decreases gradually as the O–C bond
length increases, and that it decreases rapidly when the O–C
bond extends beyond 2.0 Å (Figure 5). The first excitation
energy drops to 0 eV at a C–O bond length of approximately
2.25 Å, and it is −0.78 eV for the transient structure at the
crossing point, indicating a spontaneous electronic excitation
process. In addition, there is no significant difference in the
structural geometry between FA–Hb and FA–Hb–t at the
crossing point. Therefore, the radical elimination channel of
FA–Hb can be considered to proceed in the following process:
(1) elongation of the O–C bond to approximately 2.343 Å, (2)
spontaneous electronic excitation, and (3) subsequent disruption
of the O–C bond. The transient structure of FA–Hb at the
crossing point can be regarded as the transition state in the
methyl radical loss process, with a calculated energy barrier of
203.3 kJ/mol.
Similarly, the PES scans of the singlet and the triplet state
of IFA–Hb also cross at a certain point, corresponding to a
C–O bond length of approximately 2.263 Å (Figure 4S in
the Supporting Information). Frequency analysis of the
transient structure of IFA–Hb at the crossing point indicates
the transient structure is located at 180.7 kJ/mol above the
singlet IFA–Hb in free energy. Similar results have been
obtained in investigation of the radical elimination of the
other closed shell anions. Therefore, the radical elimination
can be rationalized as the electronic excitation of a transient
structure, and thus the energy barrier of the radical
elimination can be estimated coarsely.
Dissociation Pathways In Figure 6, we present a schematic
potential energy diagram for the fragmentation reactions of
the deprotonated FA. The energy barrier has been estimated
to be 203.3 kJ/mol for the methyl radical loss of FA–Hb in
Path-1, which is much more favorable than the direct
Figure 5. Plot of the first excitation energy and the O–C
bond length for the transient structures of FA–Hb
Figure 6. The energy profile of fragmentation of FA–Hb

X. Zhang et al.: Mechanism of the Methyl Radical Loss
947
homolytic cleavage of the triplet state via FA–TS1–t (270.3 kJ/
mol). As for the resulting fragment ion FA–178, the radical
center is stabilized by aromatic resonance, in which the
unpaired electron is delocalized over the whole molecular
structure (Table 2S in the Supporting Information). The sum
free energy of FA–178 and methyl radical is 121.5 kJ/mol
more than that of the precursor ion. However, the subsequent
dissociation of FA–178 needs to surmount a considerable
energy barrier (304.9 kJ/mol), which greatly restrains the
occurrence of the reaction.
energy barrier of 143.8 kJ/mol to undergo the methyl radical
elimination, according to the frequency analysis of the transient
structure at the crossing point of the PES scans of the singlet
and triplet states (Figure 7S in the Supporting Information). As
a result, the fragment ion IFA–149 has a low intensity, because
it can readily undergo further fragmentation when it is formed.
In conclusion, these potential energy diagrams, completed by
the addition of the energic barrier for the radical elimination,
have helped to provide a reasonable explanation of the different
CID-MS behaviors for these isomeric species.
For Path-2, FA–Hb first undergoes CO2 elimination via a
four-membered ring transition state FA–TS2, which results in
the formation of the fragment ion FA–149 (m/z 149). To Conclusion
accomplish this decomposition, rotation of the carboxyl group
The fragmentation of deprotonated ferulic and isoferulic acids
is essential for migration of the carboxyl hydrogen to the allyl
C8, as supported by the dihedral angle Ф C7-C8-C9-O11 of
106.4° in FA–TS2. This process is exoergic by 15.6 kJ/mol,
and the energy barrier is 178.1 kJ/mol relative to FA–Hb,
which is 25.2 kJ/mol lower than the barrier in Path-1. The
generated FA-149 then undergoes the methyl radical elimina-
tion to yield the open-shell anion FA–134 at m/z 134. The
energy barrier was assessed to be 172.6 kJ/mol by frequency
analysis of the transient structure of FA–134 at the crossing
point, originating from the PES scans of its singlet and triplet
states (Figure 5S in the Supporting Information).
Comparison of the potential energy diagram of the two
reaction channels (Figure 6) indicates that the methyl radical
expulsion in Path-1 is kinetically and thermodynamically
less favorable than the CO2 elimination in Path-2. Never-
theless, the formed fragment ion at m/z 149 in Path-2 is
much facile to undergo further dissociation, because the
energy barrier in the second step is less than that in the first
one. As a result, abundant fragment ion at m/z 134 can be
obtained in the CID-MS spectrum, and the fragment ion m/z
178 becomes more abundant than the ion m/z 149, which is
in a good agreement with the tandem MS results (Figure 1a),
and is further validated by the breakdown curve in Figure 2.
As for IFA–Hb, the calculated energy barrier for the
methyl radical elimination via IFA-TS1 is 180.7 kJ/mol
relative to IFA–Hb, which is kinetically more favorable than
that of FA–Hb by 19.6 kJ/mol (Figure 6S in the Supporting
Information). In addition, the calculated energy of IFA–Hb
is 32.9 kJ/mol higher than that of FA–Hb, while their
fragment ions, IFA–178 and FA–178, are different reso-
nance forms of the same radical anion. As a result, it is also
thermodynamically more favorable for IFA–Hb to undergo
the methyl radical elimination than FA–Hb, which also
agrees with the tandem MS results.
has been investigated experimentally and theoretically. Losses
of methyl radical and CO2 are the two main fragmentation
pathways for [FA–H]− and [IFA–H]−, in which the methyl
radical elimination violates the “even-electron rule”. The
mechanism for the methyl radical elimination was proposed
to involve the electronic excitation of a transient structure.
Thus, the energy barriers can be estimated coarsely by frequent
analysis of the transient structure, obtained from the PES scan
of the singlet ground state and triplet excited states of the
precursor ion. The calculated potential energy diagrams for the
fragmentation of [FA – H]− and [IFA – H]− have helped us
rationalize their different CID-MS behaviors of the two
isomers. The results presented in this work will provide a
better understanding of the radical elimination in CID MS
spectra. Further work will be carried out to test this mechanism.
Acknowledgments
The authors gratefully acknowledge financial support from
the National Science Foundation of China (No.21205025)
and the National Science Foundation of Zhejiang Province
(Y4100020 and Y4110243). Computer time was made
available on the SGI Aktix 450 sever at Computational
Center for Molecular Design of Organosilicon Compounds,
Hangzhou Normal University.
References
1. Hopfgartner, G., Bourgogne, E.: Quantitative high-throughput analysis
of drugs in biological matrices by mass spectrometry. Mass Spectrom.
Rev. 22, 195–214 (2003)
2. Fang, A.S., Miao, X., Tidswell, P.W., Towle, M.H., Goetzinger, W.K.,
Kyranos, J.N.: Mass spectrometry analysis of new chemical entities for
pharmaceutical discovery. Mass Spectrom. Rev. 27, 20–34 (2008)
3. Vukics, V., Guttman, A.: Structural characterization of flavonoid
glycosides by multi-stage mass spectrometry. Mass Spectrom. Rev. 29,
1–16 (2010)
4. Karni, M., Mandelbaum, A.: The even-electron rule. Org. Mass
Spectrom. 15, 53–64 (1980)
5. McLafferty, F.W., Turecek, F.: Interpretation of Mass Spectra, 4th edn.
University Science Books, Mill Valley, CA (1993)
6. Gross, J.H.: Mass Spectrometry: A Textbook. Springer-Verlag, Heidelberg,
Germany (2004)
As shown in Scheme 3, the C8 atom in FA–Hb possesses
more negative charge than IFA–Hb because of the resonance
effects, and thus FA–Hb is more facile to accept the
transferring proton from the carboxylic group during CO2
elimination. As a result, the CO2 elimination of IFA–Hb
needs to surmount an energy barrier of 202.4 kJ/mol, which is
kinetically less favorable than that of FA–Hb by 24.3 kJ/mol.
In contrast, the resulting IFA–149 needs only to surmount an

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X. Zhang et al.: Mechanism of the Methyl Radical Loss
7. Thurman, E.M., Ferrer, I., Pozo, O.J., Sancho, J.V., Hernandez, F.: The
even-electron rule in electrospray mass spectra of pesticides. Rapid
Commun. Mass Spectrom. 21, 3855–3868 (2007)
8. Williams, J.P., Nibbering, N.M.M., Green, B.N., Patel, V.J., Scrivens,
J.H.: Collision-induced fragmentation pathways including odd-electron
ion formation from desorption electrospray ionization generated
protonated and deprotonated drugs derived from tandem accurate mass
spectrometry. J. Mass Spectrom. 41, 1277–1286 (2006)
19. Butler, M., Mañez, P.A., Cabrera, G.M.: An experimental and
computational study on the dissociation behavior of hydroxypyridine
N-oxides in atmospheric pressure ionization mass spectrometry. J. Mass
Spectrom. 45, 536–544 (2010)
20. Vessecchi, R., Emery, F.S., Galembeck, S.E., Lopes, N.P.: Fragmenta-
tion studies and electrospray ionization mass spectrometry of lapachol:
Protonated, deprotonated and cationized species. Rapid Commun. Mass
Spectrom. 24, 2101–2108 (2010)
9. Levsen, K., Schiebel, H.-M., Terlouw, J.K., Jobst, K.J., Elend, M.,
Preiβ, A., Thiele, H., Ingendoh, A.: Even-electron ions: A systematic
study of the neutral species lost in the dissociation of quasimolecular
ions. J. Mass Spectrom. 42, 1024–1044 (2007)
10. Vessecchi, R., Carollo, C.A., Lopes, J.N.C., Crotti, A.E.M., Lopes,
N.P., Galembeck, S.E.: Gas-phase dissociation of 1,4-naphthoquinone
derivative anions by electrospray ionization tandem mass spectrometry.
J. Mass Spectrom. 44, 1224–1233 (2009)
11. Cai, Y., Mo, Z., Rannulu, N.S., Guan, B., Kannupal, S., Gibb, B.C., Cole,
R.B.: Characterization of an exception to the ‘even-electron rule’ upon low-
energy collision induced decomposition in negative ion electrospray
tandem mass spectrometry. J. Mass Spectrom. 45, 235–240 (2010)
12. Bajpai, L., Varshney, M., Seubert, C.N., Stevens, S.M., Johnson, J.V.,
Yost, R.A., Dennis, D.M.: Mass spectral fragmentation of the
intravenous anesthetic propofol and structurally related phenols. J.
Am. Soc. Mass Spectrom. 16, 814–824 (2005)
13. Chen, K., Rannulu, N.S., Cai, Y., Lane, P., Liebl, A.L., Rees, B.B.,
Corre, C., Challis, G.L., Cole, R.B.: Unusual odd-electron fragments
from even-electron protonated prodiginine precursors using positive-ion
electrospray tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 19,
1856–1866 (2008)
14. Xu, G., Huang, T., Zhang, J., Huang, J.K., Carlson, T., Miao, S.:
Investigation of collision-induced dissociations involving odd-electron
ion formation under positive electrospray ionization conditions using
accurate mass. Rapid Commun. Mass Spectrom. 24, 321–327 (2010)
15. Hu, N., Tu, Y.-P., Jiang, K., Pan, Y.: Intramolecular charge transfer in
the gas phase: Fragmentation of protonated sulfonamides in mass
spectrometry. J. Org. Chem. 75, 4244–4250 (2010)
16. Chai, Y., Sun, H., Pan, Y., Sun, C.: N-centered odd-electron ions formation
from collision-induced dissociation of electrospray ionization generated
even-electron ions: Single electron transfer via ion/neutral complex in the
fragmentation of protonated N,N′-dibenzylpiperazines and protonated N-
benzylpiperazines. J. Am. Soc. Mass Spectrom. 22, 1526–1533 (2011)
17. Hau, J., Stadler, R., Jenny, T.A., Fay, L.B.: Tandem mass spectrometric
accurate mass performance of time-of-flight and Fourier transform ion
cyclotron resonance mass spectrometry: A case study with pyridine
derivatives. Rapid Commun. Mass Spectrom. 15, 1840–1848 (2001)
18. Denekamp, C., Tenetov, E., Horev, Y.: Homolytic cleavages in
pyridinium ions, an excited state process. J. Am. Soc. Mass. Spectrom.
14, 790–801 (2003)
21. Cuyckens, F., Claeys, M.: Mass spectrometry in the structural analysis
of flavonoids. J. Mass Spectrom. 39, 1–15 (2004)
22. Cheng, C.-R., Yang, M., Wu, Z.-Y., Wang, Y., Zeng, F., Wu, W.-Y.,
Guan, S.-H., Guo, D.-A.: Fragmentation pathways of oxygenated
tetracyclic triterpenoids and their application in the qualitative analysis
of Ganoderma lucidum by multistage tandem mass spectrometry. Rapid
Commun. Mass Spectrom. 25, 1323–1335 (2011)
23. Zhao, Z., Moghadasian, M.H.: Chemistry, natural sources, dietary
intake, and pharmacokinetic properties of ferulic acid: A review. Food
Chem. 109, 691–702 (2008)
24. Itagaki, S., Kurokawa, T., Nakata, C., Saito, Y., Oikawa, S., Kobayashi,
M., Hirano, T., Iseki, K.: In vitro and in vivo antioxidant properties of
ferulic acid: a comparative study with other natural oxidation inhibitors.
Food Chem. 114, 466–471 (2009)
25. Barone, E., Calabrese, V., Mancuso, C.: Ferulic acid and its therapeutic
potential as a hormetin for age-related diseases. Biogerontology 10, 97–
108 (2009)
26. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A.,
Cheeseman, J.R., Zakrzewski, V.G., Montgomery Jr., J.A., Stratmann,
R.E., Burant, J.C., Dapprich, S., Millam, J.M., Daniels, A.D., Kudin,
K.N., Strain, M.C., Farkas, O., Tomasi, J., Barone, V., Cossi, M.,
Cammi, R., Mennucci, B., Pomelli, C., Adamo, C., Clifford, S.,
Ochterski, J., Petersson, G.A., Ayala, P.Y., Cui, Q., Morokuma, K.,
Malick, D.K., Rabuck, A.D., Raghavachari, K., Foresman, J.B.,
Cioslowski, J., Ortiz, J.V., Stefanov, B.B., Liu, G., Liashenko, A.,
Piskorz, P., Komaromi, I., Gomperts, R., Martin, R.L., Fox, D.J., Keith,
T., Al-Laham, M.A., Peng, C.Y., Nanayakkara, A., Gonzalez, C.,
Challacombe, M., Gill, P.M.W., Johnson, B., Chen, W., Wong, M.W.,
Andres, J.L., Gonzalez, C., Head-Gordon, M.E., Replogle, S., Pople,
J.A.: Gaussian 03. Gaussian Inc., Pittsburgh, PA (2003)
27. Tain, Z., Wang, X.-B., Wang, L.-S., Kass, S.R.: Are carboxyl groups
the most acidic sites in amino acids? Gas-phase Acidities, photoelectron
spectra, and computations on tyrosine, p-hydroxybenzoic acid, and their
conjugate bases. J. Am. Chem. Soc. 131, 1174–1181 (2009)
28. Steill, J.D., Oomens, J.: Gas-phase deprotonation of p-hydroxybenzoic
acid investigated by IR spectroscopy: Solution-phase structure is
retained upon ESI. J. Am. Chem. Soc. 131, 13570–13571 (2009)
29. Nguyen, M.T., Creve, S., Ha, T.K.: On the formation of the
+
CH2CH2CH 0 NH2 distonic radical cation upon ionization of
cyclopropylamine and allylamine. Chem. Phys. Lett. 293, 90–96 (1998)