Online Investigation of Aqueous-Phase Electrochemical Reactions by Desorption Electrospray Ionization Mass Spectrometry

Article abstract of DOI:10.1007/s13361-015-1210-2

Electrochemistry (EC) combined with mass spectrometry (MS) is a powerful tool for elucidation of electrochemical reaction mechanisms. However, direct online analysis of electrochemical reaction in aqueous phase was rarely explored. This paper presents the online investigation of several electrochemical reactions with biological relevance in the aqueous phase, such as nitrosothiol reduction, carbohydrate oxidation, and carbamazepine oxidation using desorption electrospray ionization mass spectrometry (DESI-MS). It was found that electroreduction of nitrosothiols [e.g.; nitrosylated insulin B (13-23)] leads to free thiols by loss of NO, as confirmed by online MS analysis for the first time. The characteristic mass shift of 29 Da and the reduced intensity provide a quick way to identify nitrosylated species. Equally importantly, upon collision-induced dissociation (CID), the reduced peptide ion produces more fragment ions than its nitrosylated precursor ion (presumably the backbone fragmentation cannot compete with the facile NO loss for the precursor ion), thus facilitating peptide sequencing. In the case of saccharide oxidation, it was found that glucose undergoes electro-oxidation to produce gluconic acid at alkaline pH, but not at neutral and acidic pHs. Such a pH-dependent electrochemical behavior was also observed for disaccharides such as maltose and cellobiose. Upon electrochemical oxidation, carbamazepine was found to undergo ring contraction and amide bond cleavage, which parallels the oxidative metabolism observed for this drug in leucocytes. The mechanistic information of these redox reactions revealed by EC/DESI-MS would be of value in nitroso-proteome research and carbohydrate/drug metabolic studies.

Full text of DOI:10.1007/s13361-015-1210-2

B American Society for Mass Spectrometry, 2015
J. Am. Soc. Mass Spectrom. (2015) 26:1676Y1685
DOI: 10.1007/s13361-015-1210-2
RESEARCH ARTICLE
Online Investigation of Aqueous-Phase Electrochemical
Reactions by Desorption Electrospray Ionization Mass
Spectrometry
Mei Lu,1 Yong Liu,2 Roy Helmy,2 Gary E. Martin,2 Howard D. Dewald,1 Hao Chen1
1Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Edison Biotechnology Institute,
Ohio University, Athens, OH 45701, USA
2Department of Process and Analytical Chemistry, Merck Research Laboratories, Merck and Co., Inc., Rahway, NJ 07065, USA
Abstract. Electrochemistry (EC) combined with mass spectrometry (MS) is a powerful
tool for elucidation of electrochemical reaction mechanisms. However, direct online
analysis of electrochemical reaction in aqueous phase was rarely explored. This paper
presents the online investigation of several electrochemical reactions with biological
relevance in the aqueous phase, such as nitrosothiol reduction, carbohydrate oxida-
tion, and carbamazepine oxidation using desorption electrospray ionization mass
spectrometry (DESI-MS). It was found that electroreduction of nitrosothiols [e.g.,
nitrosylated insulin B (13-23)] leads to free thiols by loss of NO, as confirmed by online
MS analysis for the first time. The characteristic mass shift of 29 Da and the reduced
intensity provide a quick way to identify nitrosylated species. Equally importantly, upon
collision-induced dissociation (CID), the reduced peptide ion produces more fragment ions than its nitrosylated
precursor ion (presumably the backbone fragmentation cannot compete with the facile NO loss for the precursor
ion), thus facilitating peptide sequencing. In the case of saccharide oxidation, it was found that glucose undergoes
electro-oxidation to produce gluconic acid at alkaline pH, but not at neutral and acidic pHs. Such a pH-dependent
electrochemical behavior was also observed for disaccharides such as maltose and cellobiose. Upon electro-
chemical oxidation, carbamazepine was found to undergo ring contraction and amide bond cleavage, which
parallels the oxidative metabolism observed for this drug in leucocytes. The mechanistic information of these redox
reactions revealed by EC/DESI-MS would be of value in nitroso-proteome research and carbohydrate/drug
metabolic studies.
Key words: Electrochemistry, Mass spectrometry, Desorption electrospray ionization, Nitrosothiol reduction,
Carbohydrate oxidation
Received: 13 April 2015/Revised: 16 May 2015/Accepted: 29 May 2015/Published Online: 5 August 2015
thermospray (TS) [2, 3], fast atom bombardment (FAB) [4],
and electrospray ionization (ESI) [5–8]. However, the online
Introduction
nline coupling of electrochemistry (EC) with mass spec-
trometry (MS) is powerful for probing redox reaction
monitoring of electrolysis in aqueous solution has been very
limited [1, 2, 9, 10] and organic solvent was often doped into
sample solution for electrolysis in order to enhance ionization
efficiency in the previous EC/MS studies. As electrochemical
reactions are traditionally carried out in aqueous solution (for
the reason that water is an ideal medium for dissolving elec-
trolyte salts and maintaining solution conductivity) [11], and
biomolecules occur in aqueous media, it is necessary to extend
the EC/MS investigation to aqueous electrochemical reactions.
Ambient ionization methods [12–14] such as desorption
electrospray ionization (DESI) [15] introduced by Cooks
et al. are among a family of the latest ionization methods that
can be used to directly ionize samples with no or little
O
mechanisms. This is because MS can serve as a highly sensitive
detector providing molecular weight and structural information
for intermediates and stable products of electrochemical reac-
tions. Various EC/MS techniques have been designed, using
different ionization methods such as electron impact (EI) [1],
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-015-1210-2) contains supplementary material, which is
available to authorized users.
Correspondence to: Yong Liu; e-mail: yong_liu2@merck.com, Hao Chen;
e-mail: chenh2@ohio.edu

M. Lu et al.: Electrochemical Mass Spectrometry
1677
preparation under ambient conditions. With the advent of am-
bient ionization methods, EC/MS coupling has been diversi-
fied, using liquid sample DESI [10, 16–22], nanospray DESI
[23], or flow atmospheric pressure afterglow (FAPA) ioniza-
tion [24]. In particular, liquid sample DESI was developed for
directly analyzing liquid samples in our and other laboratories
[16, 25–28]. The ionization occurs via the interaction of liquid
sample with charged droplets generated by the DESI spray and
the resulting ions are collected and analyzed by a mass spec-
trometer. It is useful in directly analyzing samples including
large proteins/protein complexes from their native environ-
ments [16, 29–34]. Furthermore, it is possible to use liquid
sample DESI-MS for studying fast reaction kinetics in
submillisecond time resolution [35] or coupling with liquid
chromatography (LC) [18, 36–38], microfluidics [26], and
microextraction [39]. In terms of combination with EC [10], it
was shown that liquid sample DESI-MS can be used to capture
transient intermediates [20]. The combined EC/DESI-MS (i.e.,
the combination of EC with liquid sample DESI-MS) is also
useful for the structural analysis of disulfide bond-containing
proteins in either top-down [19] or bottom-up approaches [17,
21] and for probing protein 3D-structures and protein–protein
interactions in combination with cross-linking chemistry [22].
In this study, we investigated several electrochemical reac-
tions in the aqueous phase using EC/DESI-MS, including
nitrosothiol reduction as well as carbohydrate and carbamazepine
oxidation. These redox reactions were selected because of their
biological importance (see detailed discussion below) and the
fact that their reaction mechanisms have not been examined using
online MS techniques yet. For the first time, it is shown that
nitrosothiols such as S-nitroso-N-acetyl-penicillamine (SNAP),
S-nitrosoglutathione (GSNO), and nitrosylated insulin B (13-23)
can be electrochemically reduced into free thiols via NO loss and
detected online by MS. Also, pH-dependent electrochemical
reactivities are observed for saccharide oxidation. In addition,
carbamazepine exhibits an electrochemical oxidation pathway
that is similar to the metabolic process reported in leukocytes
[40]. In addition to providing these mechanistic information, the
study also uncovers the related analytical applications (e.g., for
quickly identifying and sequencing nitrosylated peptides) and
shows the applicability of DESI-MS for online analysis of elec-
trochemical reactions in aqueous solution, at different pHs.
water used for sample preparation was obtained using a
Nanopure Diamond Barnstead purification system (Barnstead
International, Dubuque, IA, USA).
Digestion of Ubiquitin
Digestion of ubiquitin were carried out using TPCK-treated
trypsin with a ratio of 1:50 (enzyme/protein) in 50 mM ammo-
nium bicarbonate aqueous solution overnight at 38°C incuba-
tion to finish digestion.
Online EC/DESI-MS Apparatus
A home-built apparatus for coupling a thin-layer electrochem-
ical flow cell with either a Waters Xevo QTof mass spectrom-
eter (Milford, MA, USA) or a Thermo Scientific LCQ DECA
mass spectrometer (San Jose, CA, USA) by liquid DESI was
used (apparatus shown in Scheme 1S, Supporting Information)
and described previously in detail [20]. The Reactor Cell
(Antec BV, Zoeterwoude, The Netherlands) equipped with a
gold disc working electrode (i.d. 8 mm) was used for
nitrosothiol reduction and saccharide oxidation. A HyREF
electrode was used as the reference electrode, and carbon-
loaded PTFE was used as a counter electrode. The μ-PrepCell
(Antec BV, Zoeterwoude, The Netherlands) equipped with a
glassy carbon working electrode (30 × 12 mm2) was employed
for carbamazepine oxidation. A HyREF electrode was used as
the reference electrode and titanium was used as the auxiliary
electrode. The electrochemically oxidized or reduced products
flowed out of the cell via a short piece of fused silica connection
capillary (i.d. 0.1 mm, length 4.0 cm) and underwent interactions
with the charged microdroplets from the DESI spray for ioniza-
tion. The capillary outlet was placed about 1 mm downstream
from the DESI spray probe tip and kept in line with the DESI
sprayer tip and the mass spectrometer’s inlet. For the positive ion
detection mode, the spray solvent for DESI was methanol/water
(1:1 by volume) containing 1% acetic acid, and a high voltage of
5 kV was applied to the spray probe. For the negative ion mode
used for saccharide oxidation monitoring, the spray solvent for
DESI was methanol/water (1:1 by volume) containing 1% am-
monia, and a high voltage of –5 kV was applied to the spray
probe. Injection flow rates for both the DESI spray solvent and
the sample solution for electrolysis were 10 μL/min.
In this study, all of the sample solutions except carbamaz-
epine were prepared using water as solvent with the pH adjust-
ed by HCOOH, NH4OH, or NaOH. In the case of carbamaz-
epine, because of its low solubilities in water, stock solutions
had to be prepared in methanol and diluted using water. How-
ever, the final content of methanol in the sample solutions used
for electrolysis was negligible (less than 2% by volume).
Experimental
Chemicals
S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-penicillamine
(SNAP), ubiquitin from bovine erythrocytes, trypsin from por-
cine pancreas, formic acid, 9-acridine carboxaldehyde, acridine,
acridone, glucose, maltose, and cellobiose were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Nitrosylated Results and Discussion
insulin B (13-23) was purchase from Protea (Morgantown,
Nitrosothiol Reduction
WV, USA). Acetic acid and HPLC-grade methanol were obtain-
ed from Fisher Scientific (Fair Lawn, NJ, USA) and GFS
Chemicals (Columbus, OH. USA), respectively. Deionized
Nitrosothiol compounds (RSNOs) are attracting increasing
attention because of their important biological activities [41].

1678
M. Lu et al.: Electrochemical Mass Spectrometry
Most of their biological functions are triggered via the cleavage
of the S–N bond to release nitric oxide, which plays a signif-
icant regulatory role in biological systems [42]. RSNOs are
also potentially implicated in the post-translational modifica-
tion of protein cysteine residues [41, 43]. Recently, the homo-
lytic cleavage of S–N bonds of nitrosothiol ions upon gas-
phase dissociation was used to generate sulfur-based radical
ions, the fragmentation behaviors and reactivities of which
were extensively investigated [44–46]. Among various
methods developed for analyses of RSNOs, electrochemical
detection based on reduction of RSNOs is sensitive and
selective [47–49]. However, it is difficult to identify the
reaction products using only an electrochemical detector. In
this regard, MS could provide more direct evidence for the
reaction mechanism elucidation. In this work, the electro-
chemical reduction of three nitrosothiols, including SNAP,
GSNO, and nitrosylated insulin B (13-23), followed by
online mass spectrometric detection were first investigated,
using our EC/DESI-MS method.
Figure 1a shows the DESI-MS spectrum acquired when a
solution of 100 μM SNAP in H2O containing 0.5% HCOOH
flowed through the thin-layer electrochemical cell with no
potential being applied. The protonated SNAP was detected
at m/z 221, along with its in-source CID fragment ion, the
sulfur radical ion of m/z 191, as a result of NO loss. This NO
loss is characteristic for the protonated nitrosothiol, which was
utilized to produce sulfur based radical ions [44–46]. As shown
in Figure 1b, when a negative potential (–1.4 V) was applied to
the gold working electrode, the intensity of m/z 221 decreased
significantly, while the abundance of the ion at m/z 192,
corresponding to the electrochemical reduction product of
SNAP, increased. The mass shift of 29 Da is due to NO loss
and subsequent hydrogen addition, which confirms the previ-
ously proposed electrochemical reduction mechanism for
nitrosothiol compounds as shown in Scheme 1 [50]. Upon
CID, m/z 221 mainly dissociates to the sulfur radical cation
at m/z 191 by loss of NO (structure shown in Figure 1S-a,
Supporting Information), which is typical for nitrosothiol
compounds because of the labile nature of S–N bond. CID
of m/z 191 generated from in-source CID of the protonated
SNAP shows the radical induced loss of (CH3)2C=S to
give rise to m/z 117 (Figure 1S-b, Supporting Information).
In contrast, CID of the reduced product ion at m/z 192
mainly leads to loss of small molecules such as H2O,
CH2=C=O, and HCOOH to form fragment ions at m/z
174, 150, and 146, respectively. The results suggest that
the reduced product ion has a different dissociation behav-
ior from both the intact precursor ion and the sulfur radical
cation resulting from the precursor ion by loss of NO.
Nitrosothiol peptide GSNO was also examined using EC/
DESI-MS. This compound is important for NO metabolism
and mediates many signaling pathways via protein post-
translational modification [49]. Figure 1c shows the DESI-
MS spectrum acquired when a solution of 100 μM GSNO in
H2O containing 0.5% HCOOH flowed through the thin-layer
electrochemical cell with no potential applied. Similar to
SNAP, the protonated GSNO is detected at m/z 337, along
with its in-source CID fragment ion m/z 307 by loss of NO
(structure shown as inset of Figure 1c). As shown in Figure 1d,
when a –1.4 V was applied to the gold working electrode, m/z
[SNAP+H]+
221.1
(c) Cell off
[GSNO+H]+
307.1
337.1
100
100
(a)Cell off
191.1
CH3
H2N
O
H3C
50
50
0
S
OH
O
CH3
0
195
215
185
205
340
300
320
(d) Cell on
308.1
192.1
CH3
O
100
OH
O
(b) Cell on
100
50
H3C
HS
NH3
O
O
OH
O
H2N
NH
H
N
50
0
SH
CH3
HO
[GSNO+H]+
337.1
[SNAP+H]+
221.1
O
0
195
205
185
215
340
300
320
m/z
Figure 1. DESI-MS spectra acquired when a solution of (a) 100 μM SNAP and (c) 100 μM GSNO in water containing 0.5% HCOOH
flowed through the thin-layer electrochemical cell with no potential applied. DESI-MS spectra acquired when a solution of (b) 100 μM
SNAP and (d) 100 μM GSNO in water containing 0.5% HCOOH flowed through the thin-layer electrochemical cell with an applied
potential of –1.4 V

M. Lu et al.: Electrochemical Mass Spectrometry
1679
R-SNO + e-
R-S- + H+
R-S- +NO
R-SH
HS loss (m/z 588.3), which is induced by the sulfur radical and
consistent with the previous literature report [52]. Besides,
additional fragment ions such as b4, b10 + H2O2+, y6, b6, y7,
and y9 are also detected (Figure 2e). The electrochemical
reduction of nitrosylated insulin B (13-23) generates the native
peptide, and the CID MS/MS spectrum of the reduced produc-
tion (m/z 605.3) is shown in Figure 2f. Obviously, there are
more observed fragment ions resulting from peptide backbone
dissociation in comparison to CID of either the nitrosylated
peptide ion or the sulfur radical peptide ion. The observed
fragment ions, including b3, b4, b5, b6, b7, b8, b9, b102+, y2,
Scheme 1. Electrochemical reduction mechanism for S-
nitrosothiol compounds
337 decreased considerably while the protonated glutathione
(GSH, m/z 308) was produced, as a result of electroreduction of
GSNO. The sulfur radical cation m/z 307 is detected as the
predominant fragment ion of the protonated GSNO (m/z 337)
by characteristic NO loss (Figure 2S-a, Supporting Informa-
tion). CID of m/z 307 shows abundant fragment ion [b2-H]+.
(m/z 232) by loss of glycine and m/z 289 and 271 attributable to
water losses (Figure 2S-b, Supporting Information). Another
fragment ion m/z 202 arises from m/z 289 because of further
loss of CH2=C(NH2)COOH. The same fragmentation behavior
was reported [51]. Electroreduction of GSNO generates free
thiol species GSH and the protonated reduction product GSH
produces b2 and y2 ions upon CID (Figure 2S-c, Supporting
Information), which agrees with the MS/MS behavior of the
protonated GSH generated by ESI of authentic GSH compound
(data not shown), and confirms the formation of free thiol
species after EC reduction. In this case of the nitrosylated
tripeptide GSH, CID of the protonated GSNO does not give
much structure information as NO loss is the predominant
dissociation pathway, whereas the protonated reduction prod-
uct GSH gives rise to more structurally informative fragment
ions upon CID. This is also true for other peptides [e.g.,
nitrosylated insulin B (13-23) shown below], and shows the
potential application of the online EC/DESI-MS method in
structure analysis of nitrosylated peptides. Again, the charac-
teristic mass shift of 29 Da (attributable to NO loss and H
addition) between the protonated intact GSNO and the proton-
ated reduction product GSH upon electrolysis indicates a sim-
ple and fast way to identify nitrosylated species.
Another nitrosylated peptide, nitrosylated insulin B (13-23),
was examined using EC/DESI-MS. Insulin B (13-23) peptide
is a fragment of the insulin B chain, which contains 11 amino
acids, and its seventh amino acid cysteine is nitrosylated (se-
quence shown in Figure 2a). Figure 2b shows the DESI-MS
spectrum acquired when a solution of 10 μM nitrosylated
insulin B (13-23) in water containing 0.5% HCOOH flowed
through the electrochemical cell with no potential applied. Only
doubly charged peptide ion [M + 2H]2+ was detected at m/z
619.8 with high intensity. When a negative potential of −2 V
was applied to the working electrode to trigger electroreduction,
m/z 619.8 decreased and a new doubly charged product was
detected at m/z 605.3 (Figure 2c). Again, the mass difference of
the peptide before and after reduction is 29 Da, indicating NO
loss and H addition.
2+
y3, y4, y5, y6, y7, y8, y9, and y10 cover all of the backbone
cleavages from which the peptide full sequence can be deter-
mined. The enhanced peptide sequence coverage upon CID for
the electroreduced product ion is due to the absence of NO
compared with the nitrosylated peptide ion. Although CID of
the sulfur radical peptide ion also produces some fragment ions
from backbone cleavages (Figure 2e), the radical induced frag-
mentation via HS loss dominates the dissociation pathway so
that less fragment ions are observed in comparison to the
reduced product ion.
Our results show that EC/DESI-MS method is advanta-
geous in quickly identifying the nitrosylated species based
on their response to electroreduction (including the charac-
teristic mass decrease of 29 Da and intensity drop upon
electroreduction) and is helpful to peptide sequencing, which
suggests its potential application in nitroso-proteome re-
search. Indeed, the response to electrolysis can serve as a
simple and fast way to identify nitrosylated peptides from
other non-nitrosylated peptides in a mixture. As a demon-
stration for this application, a mixture of peptides from
trypsin-digested ubiquitin and the nitrosylated insulin B
(13-23) was subjected to EC/DESI-MS analysis (Figure 3S,
Supporting Information). Before electrolysis, the DESI-MS
spectrum (Figure 3S-a, Supporting Information) shows sin-
gly and doubly charged peptides from ubiquitin digestion as
well as the doubly charged nitrosylated insulin B (13-23) at
m/z 619.8. With a reduction potential applied to the cell
(Figure 3S-b, Supporting Information), only the nitrosylated
peptide ion at m/z 619.8 displays a large relative intensity
change (decreased by 61%; using non-nitrosylated peptide
ion [LIFAGK + H]+ m/z 648.4 as the reference), whereas
other nitroso-free peptide ions have little relative intensity
change from –2% to +9% (Table 1S, Supporting Information)
probably because of the MS response fluctuation. Meanwhile,
the reduced peptide at m/z 605.3 with a mass shift of 29 Da
from the precursor ion was observed as a result of NO loss and
H addition from the nitrosylated insulin B (13-23).
Carbohydrate Oxidation
Electrochemical oxidation of carbohydrates such as glucose
has been of great interest over the years and shows potential
applications in areas including blood sugar sensor and fuel cell
research [53], but it has not been examined using online MS
techniques [54–57]. Previous electrochemical studies show
Figure 2d shows the acquired CID MS/MS spectrum of the
doubly charged peptide ion [M + 2H]2+ at m/z 619.8, which
again shows the facile NO loss to produce sulfur radical ion at
m/z 604.8. MS3 of this sulfur radical cation (Figure 2e) shows

1680
M. Lu et al.: Electrochemical Mass Spectrometry
.
-NO
604.8
100
50
619.8
(a)
(d)
EC reduction
EALYLVCGERG
EALYLVCNOGERG
619.8
700
619.8
604.8
[M+2H]2+
619.8
0
(b) Cell off
412.8
500
900
1100
.
100
50
-HS
100
b10+H2O2+
(e)
y
E A L Y L V C G E R G
b
50
y9
y6
b6 y7
700
b4
0
0
500
(c) -2V on
600
605.3
700
[M-NO+3H]2+
500
900
(f) 605.3
1100
605.3
100
y5
100
2+
b10
y6
b6-H2O
y7
y
E A L Y L V C G E R G
b
2+
50
0
y10
y4
412.8
50
0
619.8
b6
y8
b5
b7
b4
b9
y2 b3
y3
y9
b8
500
600
700
300
500
700
900
1100
m/z
Figure 2. (a) Scheme showing peptide before and after EC reduction; DESI-MS spectra acquired when a solution of 10 μM
nitrosylated insulin B (13-23) in water containing 0.5% HCOOH flowed through the electrochemical cell (b) with no potential applied
and (c) with applied potential of –2 V. CID MS/MS spectra of (d) doubly charged nitrosylated insulin B (13-23) [M + 2H]2+ (m/z 619.8)
before reduction, (e) the peptide radical ion at m/z 604.8, and (f) doubly charged reduction product at m/z 605.3
that glucose is poorly oxidized on gold surface in acidic solu-
tion because of the low concentration of OH adsorbed on a gold
electrode at low pH [54], but a gold electrode is attractive for
glucose oxidation in neutral and alkaline solutions as it shows
more negative oxidative potential for glucose and higher oxida-
tion current compared with other metal electrodes [57]. In this
study, the direct electrochemical oxidation of glucose in alkaline
solution with a gold electrode was performed and monitored
using online DESI-MS. Figure 3a shows the DESI-MS spectrum
acquired when a solution of 500 μM glucose in water containing
2 mM NaOH (pH 11) flowed through the thin-layer electrochem-
ical cell with no potential being applied. Without the cell ener-
gized, only the deprotonated glucose at m/z 179 was detected.
When an oxidation potential of 1.5 V was applied to the working
electrode of the cell, the deprotonated oxidation product, gluconic
acid, was observed at m/z 195. The CID of this oxidation product
ion was compared with the authentic gluconic acid sample, and
the result is shown in Figure 3c and d. An identical fragmentation
pattern showing consecutive losses of H2O and HCHO was
observed. This result confirms the structure of the
electrogenerated glucose oxidation product as gluconic acid.
Acidic and neutral pH solvents were also tested, and the results
are shown in Figure 3e–h. In stark contrast with alkaline pH, no
oxidation product was detected at either acidic or neutral pH,
which confirms that the lower pH does not favor glucose electro-
oxidation.
Two other disaccharides, maltose and cellobiose, in alkaline
solution were also examined using our online EC/DESI-MS
method, and the results are shown in Figure 4S (Supporting
Information). As shown in Figure 4S-a, without the cell turned
on, only the deprotonated maltose [maltose-H]– at m/z 341 was
observed. With 1.5 V being applied to the cell, the
deprotonated maltobionic acid (m/z 357), the major oxidation
product of maltose, was detected successfully. CID of m/z 357
is shown in Figure 4S-c, in which the losses of C5H12O4,
C5H12O6, C6H12O6, water, and further losses of HCHO and
CO were observed. Another disaccharide cellobiose was also
tested and gave results similar to those observed with maltose
(Figure 4S-d, S-e, and S-f, Supporting Information).
Note that these experiments represent some of the very few
reported examples of using online MS techniques to investigate
electrochemical reactions in alkaline pH conditions [58–60]
(one reason for this is that alkaline pH does not favor the
formation of positive ions by MS). In the case of using DESI-
MS, the electrolysis pH does not affect the ionization as the
DESI spray pH could be adjusted depending on the ionization
mode in need [38].
Carbamazepine Oxidation
Carbamazepine electro-oxidation in water was also examined
using EC/DESI-MS in our study. Carbamazepine is a tricyclic

M. Lu et al.: Electrochemical Mass Spectrometry
1681
100
50
100
50
179.1
[Glucose-H]-
215.0
(a) 1 mM NaOH,
Cell off
(e) 2 mM NaCl with 0.5%
HCOOH, Cell off
[Glucose-H]-
[Glucose+Cl]-
179.1
0
0
180
[Gluconic acid-H]-
190
160
170
160
180
220
240
200
195.0
100
(f) 2 mM NaCl with 0.5%
(b) 1 mM NaOH,
Cell on
100
215.0
HCOOH, Cell on
[Glucose+Cl]-
OH OH
O
HO
O
50
179.1
[Glucose-H]-
179.1
OH OH
50
0
0
180
190
160
170
160
180
220
240
200
195.0
O
(c) m/z 195 Standard
100
100
215.0
OH OH
O
HOCH2COO-
75.1
(g) 2 mM NaCl, Cell off
[Glucose+Cl]-
HO
129.1
OH OH
-H2O
177.1
-HCHO
50
0
50
0
[Glucose-H]-
179.1
99.1
-2H2O
159.1
-HCHO
80
100
120
140
180
160
220
160
180
240
200
m/z 195 EC product
(d)
75.1
100
100
215.0
(h) 2 mM NaCl, Cell on
[Glucose+Cl]-
129.1
195.0
[Glucose-H]-
179.1
50
0
50
99.1
100
159.1 177.1
180
0
80
120
140
220
160
160
180
240
200
m/z
Figure 3. DESI-MS spectra acquired when a solution of 500 μM glucose in water containing 1 mM NaOH flowed through the thin-
layer electrochemical cell at a rate of 5 μL/min with an applied potential of (a) 0.0 V, and (b) 1.5 V. CID MS/MS spectra of m/z 195
generated (c) by ESI of authentic gluconic acid sample, and (d) by electro-oxidation of glucose. DESI-MS spectra acquired when a
solution of 500 μM glucose in water containing 2 mM NaCl in 0.5% HCOOH with applied potential of (e) 0.0 V, and (f) 1.5 V; DESI-MS
spectra acquired when a solution of 500 μM glucose in water containing 2 mM NaCl with applied potential of (g) 0.0 V, and (h) 1.5 V
compound used as an antiepileptic drug in the treatment of
epilepsy and bipolar disorders [61]. More than 30 carbamaze-
pine metabolites have been detected in the human body, some of
which are pharmacologically active or genotoxic [62]. As shown
in Scheme 2S-a (Supporting Information) [63], carbamazepine is
mainly metabolized via three pathways in the liver. The primary
route is the production of the carbamazepine-10,11-epoxide; a
second route is the formation of hydroxylated compounds. The
third route is the formation of iminostilbene. While in the
leucocytes with strong oxidizing properties, carbamazepine
237.1
100
(a) Cell off
N
NH3
O
+
50
0
CHO
208.1
237.1
N
H+
(b) Cell on
100
180.1
CHO
N
COOH
H+
[208+H2O]
226.1
O
N
N+
50
0
H 224.1
N
H2
O
NH3
+
253.1
196.1
200
220
180
240
260
280
m/z
Figure 4. DESI-MS spectra acquired when a solution of 200 μM carbamazepine in 20 mM HCOONH4 in H2O flowed through the
thin-layer electrochemical cell at a rate of 40 μL/min with an applied potential of (a) 0.0 V, and (b) 1.3 V

1682
M. Lu et al.: Electrochemical Mass Spectrometry
O
CHO
N
N
N
NH2
NH2
O
O
O
NH2
MW 236
MW 252
1
MW 252
2
CHO
N
O
N
H
N
MW 207
3
MW 179
MW 195
4
5
Scheme 2. Proposed electrochemical oxidation pathway for carbamazepine
could be converted to various metabolites including an interme-
diate aldehyde, 9-acridine carboxaldehyde, acridine, and
acridone (Scheme 2S-b, Supporting Information) [40, 63]. The
determination of carbamazepine and its metabolites in biological
-C2H4
m/z 180 standard
152.1
N
100 (d)
H+
(a)
180.1
m/z 253 EC product
100
50
210.1
CHO
N
-NH=C=O
128.1
77.1 101.1
100
0
-NH3
236.1
50
150
200
O
NH3
152.1
100
253.1
m/z 180 EC product
(e)
180.1
180.1
-HCHO
128.1
0
77.1
101.1
100
240
160
180
220
200
0
50
150
200
-CO
180.1
-CH2=NH
167.1
m/z 208 standard
(b)
CHO
m/z 196 standard
(f)
100
100
O
N
H+
N
H2
+
208.1
196.1
200
0
50
0
100
200
150
100
150
50
180.1
167.1
100
100
(g) m/z 196 EC product
m/z 208 EC product
(c)
208.1
196.1
200
0
0
50
100
150
50
100
200
150
m/z
Figure 5. CID MS/MS spectra of the protonated electrochemical oxidation products of (a) m/z 253, (c) m/z 208, (e) m/z 180, (g) m/z 196,
and the protonated standard compounds of (b) 9-acridine carboxaldehyde (m/z 208), (d) acridine (m/z 180), and (f) acridone (m/z 196)

M. Lu et al.: Electrochemical Mass Spectrometry
1683
fluids using chromatography [64, 65] and electrochemical de-
tection [61, 66, 67] was reported. However, these methods lack
chemical specificity. EC/MS is a valuable instrumental method
to mimic metabolic pathways of drug compounds since most
metabolism pathways involve redox reactions. Electrochemical
simulation is a fast method and can potentially provide metabolic
information comparable to an in vitro study [68–71]. Although
most EC/MS work use organic solvent to facilitate MS detection,
it would make more sense to use water as the electrolysis solvent
for mimicking drug metabolism in aqueous media. In a previ-
ously reported study of the electrochemical behavior of carba-
mazepine, initially a one-electron oxidation of carbamazepine
was observed to form a radical that dimerizes rapidly; the dimer
itself can then undergo further oxidation [72].
Figure 4 shows the experimental results for online EC/
DESI-MS monitoring of carbamazepine electro-oxidation.
First, EC/DESI-MS was tested for a 200 μM carbamazepine
in 20 mM HCOONH4 in H2O (pH 6.8) using a flow rate of
40 μL/min. With the electrochemical cell not energized, only
the protonated carbamazepine (m/z 237) was detected with high
intensity (Figure 4a). When a positive potential of 1.3 V was
applied to the working electrode of the cell, the intensity of m/z
237 ion decreased and other ions of m/z 180, 196, 208, 224,
226, and 253 were detected (Figure 4b).
The electro-oxidation mechanism of carbamazepine is pro-
posed in Scheme 2. First, carbamazepine undergoes epoxida-
tion to generate carbamazepine-10,11-epoxide 1, which isom-
erizes to an intermediate aldehyde 2 via ring contraction. The
detected ion of m/z 253 appears to be unstable as it could not be
detected with lower sample injection flow rate through electro-
chemical cell during the experiment. This excludes the possi-
bility that the observed m/z 253 is from the stable species
carbamazepine-10,11-epoxide, which is commercially avail-
able. Instead, we propose that it is the protonated intermediate
aldehyde 2. CID of m/z 253 yields the fragment ions of m/z
236, 210, and 180 by consecutive losses of NH3, NHCO, and
HCHO, consistent with the assigned structure (Figure 5a). As
proposed in Scheme 2, loss of the CONH2 substituent from 2
affords 9-acridine carboxaldehyde 3 (detected at m/z 208).
Subsequent loss of CO gives rise to acridine 4 (detected at
m/z 180). Acridine can then be further oxidized to form the
corresponding acridone 5 (detected at m/z 196). The compari-
son of MS/MS spectra between authentic compounds and the
assigned products, 9-acridine carboxaldehyde, acridine and
acridone, is also shown in Figure 5. The protonated 9-acridine
carboxaldehyde (m/z 208) dissociates into m/z 180 by loss of CO
(Figure 5b and c). The protonated acridine (m/z 180) dissociates
into m/z 152 by loss of C2H4. CID of the acridone ion (m/z 196)
yields fragment ion of m/z 167 by loss of CH2=NH (Figure 5f
and g). The fragmentation pathways observed for the
electrogenerated products are the same as those of authentic
compounds, which supports the proposed structures and thereby
the proposed oxidative reaction mechanism shown in Scheme 2.
As shown in Figure 4b, there are another two new ions observed
with m/z 224 and 226. It is likely that m/z 224 results from
oxidation of 9-acridine carboxaldehyde 3 to carboxylic acid.
The m/z 226 might be a water adduct of m/z 208. The
observed electrochemical oxidation of carbamazepine in
this study appears to be similar to the reported oxidative
metabolic pathway for carbamazepine in leucocytes
(Scheme 2S-b, Supporting Information), providing an
additional example showing that electrochemical mass
spectrometry could be used for in vivo drug metabolism
simulation.
Conclusions
Electrochemical reactions of several organic compounds with
biological relevance in aqueous solution, including the reduc-
tion of nitrosothiols and the oxidation of carbohydrates and
carbamazepine, were examined using EC/DESI-MS. Several
interesting findings were uncovered in this study. First, it was
found that electroreduction of nitrosothiols leads to free thiols
involving characteristic mass decrease of 29 Da and a large
intensity drop, providing a quick way to identify nitrosylated
species. Also, the reduced nitrosylated peptide gives more
sequence information upon CID. For oxidation of glucose,
maltose, and cellobiose in alkaline solution, the corresponding
carboxylic acid products were detected successfully. In con-
trast, no oxidation products were observed at neutral or acidic
pH. For carbamazepine electrochemical oxidation, a similar
oxidative metabolism pathway as in leucocytes was revealed
using EC/DESI-MS. The mechanistic information of these
redox reactions revealed by EC/DESI-MS would be of value
in the nitroso-proteome research and carbohydrate/drug meta-
bolic studies. In addition, the study also suggests that EC/
DESI-MS is suitable for investigating different electrochemical
reactions in aqueous solution, at both acidic and alkaline pHs.
Acknowledgments
The authors acknowledge support for this work by NSF Career
Award (CHE-1149367), NSF (CHE-1455554), NNSFC
(21328502), and the Merck New Technology Research
Licensing Committee (NTRLC).
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