Study of electrochemical reactions using nanospray desorption electrospray ionization mass spectrometry

Article abstract of DOI:10.1021/ac300916k

The combination of electrochemistry (EC) and mass spectrometry (MS) is a powerful analytical tool for studying mechanisms of redox reactions, identification of products and intermediates, and online derivatization/ recognition of analytes. This work reports a new coupling interface for EC/MS by employing nanospray desorption electrospray ionization, a recently developed ambient ionization method. We demonstrate online coupling of nanospray desorption electrospray ionization MS with a traditional electrochemical flow cell, in which the electrolyzed solution emanating from the cell is ionized by nanospray desorption electrospray ionization for MS analysis. Furthermore, we show first coupling of nanospray desorption electrospray ionization MS with an interdigitated array (IDA) electrode enabling chemical analysis of electrolyzed samples directly from electrode surfaces. Because of its inherent sensitivity, nanospray desorption electrospray ionization enables chemical analysis of small volumes and concentrations of sample solution. Specifically, good-quality signal of dopamine and its oxidized form, dopamine o-quinone, was obtained using 10 μL of 1 μM solution of dopamine on the IDA. Oxidation of dopamine, reduction of benzodiazepines, and electrochemical derivatization of thiol groups were used to demonstrate the performance of the technique. Our results show the potential of nanospray desorption electrospray ionization as a novel interface for electrochemical mass spectrometry research.

Full text of DOI:10.1021/ac300916k

Article
pubs.acs.org/ac
Study of Electrochemical Reactions Using Nanospray Desorption
Electrospray Ionization Mass Spectrometry
Pengyuan Liu,† Ingela T. Lanekoff,‡ Julia Laskin,*,‡ Howard D. Dewald,† and Hao Chen*,†
†Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Clippinger Laboratories, Ohio
University, Athens, Ohio 45701, United States
‡Chemical and Materials Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999 K8-88, Richland, Washington
99352, United States
S
* Supporting Information
ABSTRACT: The combination of electrochemistry (EC) and mass
spectrometry (MS) is a powerful analytical tool for studying mechanisms
of redox reactions, identification of products and intermediates, and online
derivatization/recognition of analytes. This work reports a new coupling
interface for EC/MS by employing nanospray desorption electrospray
ionization, a recently developed ambient ionization method. We demonstrate
online coupling of nanospray desorption electrospray ionization MS with a
traditional electrochemical flow cell, in which the electrolyzed solution
emanating from the cell is ionized by nanospray desorption electrospray
ionization for MS analysis. Furthermore, we show first coupling of nanospray
desorption electrospray ionization MS with an interdigitated array (IDA)
electrode enabling chemical analysis of electrolyzed samples directly from
electrode surfaces. Because of its inherent sensitivity, nanospray desorption
electrospray ionization enables chemical analysis of small volumes and concentrations of sample solution. Specifically, good-
quality signal of dopamine and its oxidized form, dopamine o-quinone, was obtained using 10 μL of 1 μM solution of dopamine
on the IDA. Oxidation of dopamine, reduction of benzodiazepines, and electrochemical derivatization of thiol groups were used
to demonstrate the performance of the technique. Our results show the potential of nanospray desorption electrospray ionization
as a novel interface for electrochemical mass spectrometry research.
oth electrochemistry (EC) and mass spectrometry (MS)
play an important role in analytical chemistry. The
In this study we demonstrate coupling of EC with MS using
nanospray desorption electrospray ionization.32−36 Nanospray
desorption electrospray ionization is a new ambient surface
ionization technique that enables sensitive analysis of both
solid32−34 and liquid samples35 on surfaces. The nanospray
desorption electrospray ionization source comprises two
capillaries, a primary capillary that supplies solvent to a surface,
to dissolve or extract analyte, and a second nanospray capillary
that removes the resulting analyte solution and generates
charged droplets at the MS inlet by nanoelectrospray.
B
combination of these two techniques (EC/MS) enables
identification and structural characterization of products and
intermediates of electrochemical reactions.1−4 Furthermore, EC
is often used for online analyte pretreatment or derivatization
for improved detection5,6 and MS/MS characterization.7 EC/
MS has been also used for mechanistic studies of redox
reactions of interest to biology,8−12 environmental science,13
and drug discovery.4,14
EC has been interfaced with MS using a variety of ionization
techniques including electron ionization (EI),15 thermospray
(TS),16 fast atom bombardment (FAB),17 and electrospray
ionization (ESI).18−22 In particular, EC/ESI-MS has found
extensive applications because ESI is a soft ionization method
and can be used to detect both labile compounds and large
biomolecules. The most recent coupling method is EC/liquid
sample desorption electrospray ionization (DESI)-
MS,11,12,23−26 which was developed by us in 2009. In DESI-
MS,27−30 an electrosonic spray ionization (ESSI) probe31 is
used to desorb and ionize molecules in the electrolyzed sample
solution. EC/DESI-MS coupling24 has the advantage of simple
instrumentation, fast signal response, and flexibility in the
selection of solvent systems for electrolysis.
Nanospray desorption electrospray ionization MS has been
used for the analysis of organic and biological molecules
deposited onto solid supports. Examples of nanospray
desorption electrospray ionization MS applications include
analysis of ambient organic aerosol samples,33,36 imaging of
biological tissues,34 and analysis of liquid petroleum samples.35
It has been demonstrated that nanospray desorption electro-
spray ionization is a soft ionization technique that can be used
to study labile molecules. Similar to DESI, chemically labile
compounds are preserved during nanospray desorption electro-
Received: April 6, 2012
Accepted: June 8, 2012
Published: June 8, 2012
© 2012 American Chemical Society
5737
dx.doi.org/10.1021/ac300916k | Anal. Chem. 2012, 84, 5737−5743

Analytical Chemistry
Article
Figure 1. Online coupling of an electrochemical flow cell with nanospray desorption electrospray ionization MS. (a) Scheme showing the
configuration of the EC/nanospray desorption electrospray ionization MS with an electrochemical flow cell. Nanospray desorption electrospray
ionization MS spectra acquired when the dopamine solution flowed through the thin-layer electrochemical cell with an applied potential of (b) 0.0
and (c) 1.5 V. (d) EIC of m/z 152 acquired when the dopamine solution flowed through the thin-layer electrochemical cell.
spray ionization analysis,33,37 which is important for detection
of labile intermediates in EC reactions. In addition, its high
sensitivity32 and the ability to analyze small volumes (<1 μL) of
liquid samples without any sample pretreatment makes it
suitable for direct analysis of electrolyzed solutions on electrode
surfaces. The ability to perform such measurements is
important for characterization of products and intermediates
formed in electrochemical reactions.
Two different types of electrochemical cells were used in this
study, an electrochemical flow cell and an interdigitated array
(IDA)38−41 electrode. Electrochemical flow cells are widely
used in EC/ESI-MS and EC/DESI-MS coupling in which
sample solutions electrolyzed in the flow cell are analyzed using
MS.9,21 EC measurements using IDA electrodes rely on a
steady-state current generated between two closely spaced
electrodes printed on an insulating surface. To the best of our
knowledge, coupling of IDA electrodes with MS is demon-
strated for the first time in this study. IDA electrodes are
characterized by rapid response time and require only a small
volume of the analyte sample solution. By coupling IDA
electrodes with nanospray desorption electrospray ionization
MS, MS signal can be obtained directly from a small droplet of
sample solution (e.g., 10 μL) on the electrode, as demonstrated
in this study.
electrode is 2 mm ×10 μm (length × width), and the interval
between two adjacent electrodes is 5 μm (Figure 2a and Figure
S1a in the Supporting Information). The whole IDA electrode
was mounted in a USB-like connecting cable provided by the
manufacturer (ALS, Tokyo, Japan). A potentiostat (CV-27
model, BAS, West Lafayette, IN) was used to apply potentials
to the electrochemical flow cell and the IDA electrode.
Samples were analyzed using either a Finnigan LTQ/
Orbitrap mass spectrometer (Thermo Electron, Bremen,
Germany) or a Bruker HCTultra ion trap mass spectrometer
(Bruker Daltonics, Bremen, Germany) both equipped with a
custom-designed nanospray desorption electrospray ionization
source.32 The electrochemical flow cell or IDA electrode was
mounted onto a nanospray desorption electrospray ionization
custom-built sample holder. Fused-silica capillary (50 μm i.d.,
183 μm o.d., Polymicro Technologies, L.L.C., Phoenix) was
used to make both the primary and the nanospray capillaries.
The capillaries were independently positioned using two XYZ
stages. The solvent injected through the primary capillary for
extraction was a mixture of MeOH/H2O/HOAc (50:50:1 by
volume). The typical flow rate of this solvent was 2 μL/min.
The solvent flow rate was maintained by carefully positioning
one end of the primary and the nanospray capillaries relative to
each other forming a solvent bridge (Figures 1a and 2a). The
other end of the nanospray capillary was pointed to the heated
capillary of the mass spectrometer. A high voltage of 2−3 kV
was applied to the primary capillary, when using the LTQ/
Orbitrap, or to the MS inlet, when using the ion trap, for
nanospray ionization.
EXPERIMENTAL SECTION
■
Apparatus. Both a commercial thin-layer electrochemical
flow cell (BAS, West Lafayette, IN) and an IDA electrode
(ALS, Tokyo, Japan) were used in EC/nanospray desorption
electrospray ionization MS experiments. The thin-layer electro-
chemical flow cell is composed of a glassy carbon working
electrode (WE, 6 mm diameter, BAS, West Lafayette, IN)
embedded in PEEK, a stainless steel auxiliary electrode (AE)
which is separated from the WE by a Teflon gasket (0.01 in.
thick), and a Ag/AgCl (3 M NaCl) reference electrode (RE)
contacting the sample solution through a small hole in the AE
block. The flow rate of sample solutions flowing through the
electrochemical cell was 5 μL/min. The carbon IDA electrode
comprises 65 pairs of interdigitated working electrodes. Each
RESULTS AND DISCUSSION
■
Online Coupling of an Electrochemical Flow Cell with
MS by Nanospray Desorption Electrospray Ionization.
Figure 1a shows a schematic drawing of the online coupling of a
thin-layer electrochemical flow cell with nanospray desorption
electrospray ionization MS. In this coupling, a piece of PEEK
capillary tube was mounted on a plastic plate as a conduit to
generate a flow pathway for the solution emanating from the
electrochemical flow cell. Ionization of the solution was
5738
dx.doi.org/10.1021/ac300916k | Anal. Chem. 2012, 84, 5737−5743

Analytical Chemistry
Article
cable, was mounted on the custom-built nano-DESI sample
holder. An amount of 10 μL of the analyte solution was
sufficient to cover all four IDA electrodes (including two WEs,
one CE, and one RE). The IDA electrode was connected to a
potentiostat which was employed to apply potentials to the
electrodes to trigger oxidation or reduction.
Analysis was conducted using a Bruker Ion Trap MS
equipped with a custom-designed nanospray desorption
electrospray ionization source. In this experiment, the electrical
field gradient was created by applying a negative high voltage of
3 kV to the heated glass capillary while the primary capillary
was grounded. A 10 μL droplet containing 20 μM dopamine in
MeOH/H2O/HOAc (90:10:1, by volume) was placed on the
IDA electrode. A mixture of 90:10:1 (v/v/v) MeOH/H2O/
HOAc was injected through primary capillary to pick up the
electrolyzed dopamine solution from the IDA surface. The
acquired nanospray desorption electrospray ionization mass
spectra are shown in Figure S1 (Supporting Information).
Initially, the protonated dopamine peak was observed in the
spectrum. After applying a 1.5 V potential, the oxidation
product ion of m/z 152 was observed. The EIC of m/z 152
shown in Figure 2b shows an increase in the abundance of the
oxidation product ion as a result of turning on the IDA
electrode. This presents a first demonstration of the successful
online coupling of EC/nanospray desorption electrospray
ionization MS using an IDA electrode.
Off-Line Coupling of an IDA Electrode with MS by
Nanospray Desorption Electrospray Ionization. It should
be noted that the online coupling of MS with an IDA electrode
described in the previous section was performed by applying a
high voltage to the mass spectrometer inlet. However, most MS
instruments are configured such that the inlet is held at a low
potential while the high voltage is applied to the electrospray
needle. In this study, such source configuration was examined
using an LTQ/Orbitrap instrument. In this system, the
electrical field gradient for nanospray desorption electrospray
ionization experiments was generated by applying a high
voltage of 3 kV to the primary capillary while keeping the
heated inlet at a low potential (<100 V). However, no MS
signal from an IDA electrode was obtained in this
configuration. In fact, MS signal disappeared immediately
when the IDA electrode and nanospray desorption electrospray
ionization were turned on simultaneously indicating that the
electrical circuit generated by addition of the potentiostat
interferes with the field gradient necessary for the ionization of
the analyte. An alternative configuration, which allows off-line
coupling, was tested by turning on the IDA electrode for a
certain period of time and then disconnecting it from the
potentiostat prior to nanospray desorption electrospray
ionization MS analysis of the electrolyzed sample solution.
Off-line EC/nanospray desorption electrospray ionization
coupling with an IDA electrode was first demonstrated using
oxidation of dopamine (Scheme 1). A 10 μL droplet of 1 μM
dopamine solution, in which only 10 pmol of dopamine was
present, was placed on the IDA electrode surface and analyzed
with nanospray desorption electrospray ionization MS. Before
applying the voltage to the electrode, the protonated dopamine
(m/z 154) and its main fragment ion (m/z 137) were observed
in the spectrum, whereas the oxidation product ion was absent
(Figure S2a, Supporting Information). The oxidation product
at m/z 152 was observed in the spectrum (Figure S2b,
Supporting Information) after 1.5 V was applied to the IDA
electrode for 1 min. The signal response was measured for
Figure 2. Online coupling of the IDA electrode with nanospray
desorption electrospray ionization MS. (a) Scheme showing the
configuration of the EC/nanospray desorption electrospray ionization
MS (Bruker Ion Trap). (b) EIC of m/z 152 in nanospray desorption
electrospray ionization MS analysis of 10 μL of 20 μM dopamine
droplet on the IDA electrode.
obtained by bringing the nanospray desorption electrospray
ionization probe (i.e., the primary and nanospray capillaries) in
contact with the solution. The elemental compositions of the
observed ions were confirmed using accurate mass measure-
ments performed using the LTQ/Orbitrap instrument.
Dopamine, a compound traditionally used for the character-
ization of EC, EC/MS, or liquid chromatography (LC)/EC
systems,42,43 was chosen for testing the online coupling of the
flow cell with nanospray desorption electrospray ionization MS.
Dopamine can be electrochemically oxidized to dopamine o-
quinone (DQ) via losses of two electrons and two protons. The
oxidation mechanism is shown in Scheme 1.
Scheme 1. Oxidation Mechanism of Dopamine
In the experiment, when 0.1 mM dopamine solution flowed
through the electrochemical flow cell without any potential
applied, a protonated peak of dopamine was observed in the
spectrum at m/z 154 (Figure 1b). Another dominant peak at
m/z 137 was produced, as a result of the loss of NH3 from the
protonated dopamine. When the flow cell was turned on with a
1.5 V potential applied to the WE, a peak at m/z 152
corresponding to the protonated DQ, the oxidation product of
dopamine, appeared in the spectrum (Figure 1c). The fragment
ion at m/z 123 produced by the loss of CH2NH from m/z
152 was also detected. The errors between the observed m/z
values and the calculated values are within 6 ppm (Table S1,
Supporting Information), indicating good mass accuracy and
confirming the identity of these assigned ions. Extracted ion
chromatogram (EIC) of m/z 152 (Figure 1d) shows a good
MS response to the potential applied, confirming that the
oxidation product of dopamine generated via electrochemical
oxidation can be directly ionized and transferred from solution
into the gas phase for MS detection by nanospray desorption
electrospray ionization. These results clearly show a successful
online coupling of a thin-layer electrochemical flow cell with
nanospray desorption electrospray ionization MS.
Online Coupling of an IDA Electrode with MS by
Nanospray Desorption Electrospray Ionization. Figure 2a
shows a schematic drawing of the nanospray desorption
electrospray ionization coupling with an IDA electrode. The
IDA electrode, which was placed in a USB-like connecting
5739
dx.doi.org/10.1021/ac300916k | Anal. Chem. 2012, 84, 5737−5743

Analytical Chemistry
Article
Figure 3. Off-line nanospray desorption electrospray ionization MS analysis of a 10 μL droplet containing 20 μM CPZ on the IDA electrode (a)
before and (b) after applying 1.5 V potential using the LTQ/Orbitrap. The proposed product ions are labeled in panel b.
different concentrations of dopamine in the droplet (10, 100,
and 1000 μM). A background peak at m/z 159 was used as an
internal standard for comparing the relative abundance of the
oxidation product ion peak at m/z 152 which shows a very
good linear correlation with the dopamine concentration (R2 =
0.9905, Figure S3, Supporting Information). Because the
concentration of DQ is not known, we determined the limit
of detection (LOD) of DQ based on the initial dopamine
concentration. Using this approach, the LOD of 6.8 pmol
(initial dopamine concentration) was estimated by extrapolat-
ing the linear plot shown in Figure S3 (Supporting
Information) to the point where the intensity of the peak at
m/z 152 is 3 times higher than the background. The linearity of
the observed signal with concentration indicates that EC/
nanospray desorption electrospray ionization MS could be used
for quantitative analysis. Further, by comparing to previous
EC/ESI-MS44,45 and EC/DESI-MS24 studies, which typically
use concentrations of dopamine in the millimolar or
submillimolar range, our data clearly show the advantage of
the EC/nanospray desorption electrospray ionization MS
experiments that enable sensitive characterization of small
amounts of material directly from the IDA electrode.
Both online and off-line nanospray desorption electrospray
ionization MS analysis of solutions on IDA electrodes described
in this study enable in situ analysis of the electrolyzed solutions
on electrode surfaces. In situ analysis in electrochemistry is
appealing because it provides insights on the mechanisms of
electrochemical reactions in complex electrode processes.
Furthermore, some short-life intermediates or early transition
states could be discovered by in situ analyses. To date, several
EC/MS methods, including both EC/ESI-MS8,46 and EC/
DESI-MS,23,47 have been used to directly analyze electrode
surfaces. In the first method of EC/ESI-MS,46 a coaxial capillary
set is employed to introduce the sample solution to specific
position on the working electrode for electrolysis and then to
transfer the electrolyzed solution through the inner capillary to
ESI-MS. Imaging of the electrode surface was achieved by
employing this method. Similar EC/ESI-MS-based design
involving a liquid microjunction surface sampling probe
(LMJ-SSP) was reported.8 A second direct analysis method,
an EC/DESI-MS method,23 was developed using a tubular
electrode flow-through cell, in which the electrolyzed solution
emanating from the cell was directly ionized on the surface of
the anode for MS analysis. A third EC/DESI-MS method47 was
developed for the analysis of a small volume of an analyte
solution. In this technique, EC/MS analysis is performed from
a small droplet of sample solution held by a pair of electrodes.
Following the electrolysis, the droplet is blown into the MS by
a DESI spray beam for subsequent analysis. However, this
method is limited by the lack of continuous ion signal due to
the usage of nebulizing gas. In contrast, nanospray desorption
electrospray ionization analysis of one droplet of solution
generates continuous MS signal that lasts for minutes. In the
next sections we describe the results of several proof-of-
principle experiments that demonstrate the utility of nanospray
desorption electrospray ionization MS for in situ character-
ization of the products of electrochemical reactions.
Applications of Coupling of an IDA Electrode with MS
by Nanospray Desorption Electrospray Ionization.
Following the successful demonstration of coupling of
nanospray desorption electrospray ionization with the IDA
electrode using oxidation of dopamine, other types of oxidation
and reduction reactions were examined using this approach.
Both online and off-line methods were used in these
experiments. Because analysis using LTQ/Orbitrap enables
unambiguous identification of products based on the accurate
mass measurement, in the following section we report the
results obtained using off-line analysis (analogous results could
be obtained by online characterization using the Bruker Ion
Trap MS instrument).
Oxidation of Chlorpromazine. Chlorpromazine (CPZ), one
of the most important compounds in the phenothiazine class of
drugs that is widely used in the treatment of schizophrenia,
readily undergoes oxidation in an acidic medium. The oxidation
mechanism of CPZ is well-studied,12,48,49 which involves a one-
electron oxidation process resulting in formation of a radical
cation intermediate followed by subsequent chemical reactions
with solvents. In this study, 10 μL of 20 μM CPZ solution was
placed on the IDA electrode surface and analyzed by nanospray
desorption electrospray ionization MS. The protonated peak of
CPZ was observed at m/z 319, and no oxidation product ions
were observed prior to applying voltage to the electrode
(Figure 3a). After applying 1.5 V to the IDA electrode for 1
min, ions corresponding to both water (m/z 335) and
methanol (m/z 349) derivatives of CPZ were observed in the
spectrum (Figure 3b). The elemental compositions of these
compounds were confirmed using accurate mass measurement
in an LTQ/Orbitrap (Table S3, Supporting Information).
5740
dx.doi.org/10.1021/ac300916k | Anal. Chem. 2012, 84, 5737−5743

Analytical Chemistry
Article
Scheme 2. Reduction Mechanism of Flunitrazepam
Figure 4. Off-line nanospray desorption electrospray ionization MS analysis of 10 μL of 20 μM flunitrazepam droplet on the IDA electrode (a)
before and (b) after applying −1.5 V potential using the LTQ/Orbitrap.
Reduction of Flunitrazepam and Clonazepam. Electro-
chemical reduction was performed using flunitrazepam as a
model system. Flunitrazepam is a potent sedative and powerful
hypnotic used in the treatment of insomnia around the world.
The reduction mechanism of flunitrazepam summarized in
Scheme 2 has been extensively studied.50,51 Cyclic voltammetry
studies indicated that the first step in the reduction reaction
(reaction 2 in Scheme 2) results in conversion of the nitro
group into a nitroso group.51 This reaction was observed at a
relatively low potential of −0.16 V when using a dropping
mercury electrode (DME) at pH of 4.0. A simultaneous
reduction of the nitroso group into an N-hydroxyl group
(reaction 3 in Scheme 2) was also reported in that study. The
second step in the reduction process corresponding to the
reduction of the azomethine group (reaction 4 in Scheme 2)
was observed at a more negative potential. Specifically, it was
reported51 that the azomethine group was reduced at a
potential of −0.73 V when using a DME at pH of 4.0. We
have recently examined the reduction of flunitrazepam using
EC/DESI-MS.12 In that study, we observed that the nitro group
undergoes the reduction via nitroso, N-hydroxyl intermediates
to the final product amine. Similarly, in this study, all reduction
products of reactions 2−5 (Scheme 2) were observed. In
details, a 10 μL flunitrazepam solution (20 μM) was placed on
the IDA electrode surface with no potential applied to the IDA
electrode and analyzed by nanospray desorption electrospray
ionization MS. The dominant peak corresponding to the
protonated flunitrazepam at m/z 314 was observed in the
nanospray desorption electrospray ionization spectrum of the
analyte solution (Figure 4a). After a negative potential of −1.5
V was applied to the IDA electrode for 1 min, the intermediates
and products of the reduction reaction were observed as
protonated species at m/z 298, 300, 302, and 286 (Figure 4b),
corresponding to the products in the stepwise reduction
reaction 2−5 in Scheme 2, respectively. The observed product
distribution is slightly different from the results obtained using
EC/DESI-MS12 by us. Specifically, in our recent study we
found the nitro group was reduced to nitroso group, giving the
product at m/z 298 and the nitroso group was further reduced
to N-hydroxyl group, resulting in the formation of a product at
m/z 300. However, the reduction of the N-hydroxyl group
occurred directly without the reduction of the azomethine
group, resulting in formation of a product at m/z 284. In
contrast, in this study we observe a stepwise reduction of
azomethine group and N-hydroxyl group resulting in the
formation of the products at m/z 302 and 286 that were not
observed using DESI-MS.12 Although both experiments used
the same glassy carbon WE and the same potential was applied
to the electrode, different REs were used in these experiments,
which could contribute to the observed difference in the
reduction mechanism. Specifically, in this study we used a
glassy carbon RE, whereas the EC/DESI-MS study used a
HyREF RE. Alternatively, differences in the pH of the
electrolyzed solutions could be responsible for the differences
in the reduction mechanism. The pHs of the starting solutions
were slightly different. Specifically, in this study we used
MeOH/H2O/HOAc (50:50:1, v/v), whereas the EC/DESI-MS
study used MeOH/H2O (1:1, v/v) containing 0.5% formic acid
and 20 mM NH4OAc. Furthermore, it is likely that the pH
change for the reduction of a stationary phase droplet in this
study is larger than that for a flowing solution in the previous
EC/DESI-MS study as verified in a separate experiment (see
the detailed discussion in the Supporting Information). Indeed,
the formation gas bubble was observed in this study, probably
due to the conversion of proton to hydrogen gas. Similar results
were obtained for another benzodiazepine compound,
clonazepam (Figure S4, Supporting Information), in which
product ions from stepwise reduction of nitro, nitroso,
azomethine, and N-hydroxyl groups were observed in the
spectrum. These observations are in a good agreement with
5741
dx.doi.org/10.1021/ac300916k | Anal. Chem. 2012, 84, 5737−5743

Analytical Chemistry
Article
previous EC studies.50,51 In an earlier EC/ESI-MS study,52 N-
hydroxyl and amine intermediates were observed in the
reduction of nitrobenzene but the nitroso intermediate was
not detected.
Electrochemical Derivatization of Thiols. Online derivatiza-
tion is an important application of selective chemical analysis by
EC/MS. In this study, online derivatization was demonstrated
using dopamine and cysteine. DQ can serve as a derivatizing
reagent24,53,54 for cysteine, peptides, or proteins which contain
free thiol groups (Scheme 3). First, cysteine tagging by DQ was
152), and the derivatized cysteine (m/z 273) were present in
the spectrum. The EIC (Figure 5c) displays the process of
derivatization: the intensity of m/z 152 decreases with time
while the abundance of m/z 273 increases. Similar results were
obtained when cysteine was replaced by a tripeptide,
glutathione (GSH, data not shown). Reaction of GSH with
DQ formed on the IDA electrode resulted in the formation of
DQ-tagged GSH, confirming the successful derivatization.
CONCLUSIONS
■
In summary, we have demonstrated the first online and off-line
coupling of EC with MS using the new interface, nanospray
desorption electrospray ionization. Various electrochemical
reactions including dopamine oxidation, CPZ oxidation,
flunitrazepam and clonazepam reductions, as well as EC-
triggered derivatizations were successfully examined using the
EC/nanospray desorption electrospray ionization MS method.
On the basis of this study, several interesting characteristics of
this new coupling method have been revealed. First, in
comparison with previously reported EC/MS techniques,
EC/nanospray desorption electrospray ionization MS em-
ployed in this study has the advantage of high sensitivity and
the capability of analyzing small volumes of electrolyzed
solution. This capability is important in analyzing trace
amounts of biological samples. Second, we demonstrated for
the first time the utility of EC/nanospray desorption electro-
spray ionization MS for direct analysis of electrolyzed solutions
on IDA electrodes. Similar to the reported EC/MS interface
using liquid sample desorption electrospray ionization,24 this
new interface of nanospray desorption electrospray ionization
allows in situ analysis of the electrolyzed solutions and could
have flexibility in the selection of solvents for electrolysis.
Despite the possible voltage conflict involved in coupling MS
with EC cells, the use of this new interface will extend the
applications of electrochemical mass spectrometry, especially in
Scheme 3. Derivatization Mechanism of Cysteine with DQ
tested. The online derivatization experiment was performed by
infusing cysteine solution (10 μM) through the primary
capillary of the nanospray desorption electrospray ionization
probe. An amount of 10 μL of dopamine (10 μM) was placed
on the IDA electrode. Before a voltage was applied to the IDA
electrode, both protonated cysteine and protonated dopamine
were observed in the spectrum (Figure 5a). Oxidation of
dopamine was performed by applying a 1.5 V potential to the
dopamine droplet placed on an IDA electrode for 1 min. As
discussed earlier, oxidation of dopamine produces DQ that may
react with cysteine. Cysteine solution was infused through the
primary capillary after the IDA electrode was disconnected
from the potentiostat. Figure 5b shows that after oxidation,
both ions of the oxidation product of dopamine, DQ (m/z
Figure 5. Nanospray desorption electrospray ionization MS derivatization of 10 μM cysteine with 10 μL of 10 μM dopamine droplet on the IDA
electrode (a) before and (b) after applying 1.5 V potential using the LTQ/Orbitrap. (c) EIC of m/z of 152 and 273 in panel b.
5742
dx.doi.org/10.1021/ac300916k | Anal. Chem. 2012, 84, 5737−5743

Analytical Chemistry
Article
(23) Miao, Z.; Chen, H. J. Am. Soc. Mass Spectrom. 2009, 20, 10−19.
(24) Li, J.; Dewald, H. D.; Chen, H. Anal. Chem. 2009, 81, 9716−
9722.
directly studying the redox reaction mechanisms on electrode
surfaces.
(25) Zhang, Y.; Yuan, Z.; Dewald, H. D.; Chen, H. Chem. Commun.
2011, 47, 4171−4173.
ASSOCIATED CONTENT
* Supporting Information
Experimental details and other supporting spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
(26) Zhang, Y.; Cui, W.; Zhang, H.; Dewald, H. D.; Chen, H. Anal.
Chem. 2012, 84, 3838−3842.
́
(27) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science
2004, 306, 471−473.
(28) Huang, G.; Li, G.; Duncan, J.; Ouyang, Z.; Cooks, R. G. Angew.
Chem., Int. Ed. 2010, 50, 2503−2506.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Julia.Laskin@pnnl.gov (J.L.); chenh2@ohio.edu
(H.C.).
■
(29) Harris, G. A.; Galhena, A. S.; Fernan
2011, 83, 4508−4538.
́
dez, F. M. Anal. Chem.
(30) Wiseman, J. M.; Ifa, D. R.; Song, Q.; Cooks, R. G. Angew. Chem.,
Int. Ed. 2006, 45, 7188−7192.
́
(31) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Anal.
Notes
Chem. 2004, 76, 4050−4058.
The authors declare no competing financial interest.
(32) Roach, P. J.; Laskin, J.; Laskin, A. Analyst 2010, 135, 2233−
2236.
ACKNOWLEDGMENTS
■
(33) Roach, P. J.; Laskin, J.; Laskin, A. Anal. Chem. 2010, 82, 7979−
7986.
P.L. and H.C. acknowledge support from NSF (CHE-0911160)
and NSF Career (CHE-1149367) funds. J.L. and I.T.L.
acknowledge support from the Division of Chemical Sciences,
Geosciences & Biosciences, Office of Basic Energy Sciences of
the U.S. Department of Energy (US DOE). The research
presented here was performed at the W. R. Wiley Environ-
mental Molecular Sciences Laboratory (EMSL)a national
scientific user facility sponsored by the US DOE’s Office of
Biological and Environmental Research and located at the
Pacific Northwest National Laboratory (PNNL). PNNL is a
multiprogram national laboratory operated for DOE by
Battelle.
(34) Laskin, J; Heath, B. S.; Roach, P. J.; Cazares, L. H.; Semmes, O.
J. Anal. Chem. 2012, 84, 141−148.
(35) Eckert, P. A.; Roach, P. J.; Laskin, A.; Laskin, J. Anal. Chem.
2012, 84, 1517−1525.
(36) Nguyen, T. B.; Lee, P. B.; Updyke, K. M.; Bones, D. L.; Laskin,
J.; Laskin, A.; Nizkorodov, S. A. J. Geophys. Res. 2012, 117, D01207.
DOI: 10.1029/2011JD016944.
(37) Laskin, J.; Laskin, A.; Roach, P. J.; Slysz, G. W.; Anderson, G. A.;
Nizkorodov, S. A.; Bones, D. L.; Nguyen, L. Q. Anal. Chem. 2010, 82,
2048−2058.
(38) Niwa, O.; Xu, Y.; Halsall, H. B.; Heineman, W. R. Anal. Chem.
1993, 65, 1559−1563.
(39) Morita, M.; Niwa, O.; Horiuchi, T. Electrochim. Acta 1997, 42,
3177−3183.
REFERENCES
■
(40) Niwa, O.; Morita, M.; Tabei, H. Anal. Chem. 1990, 62, 447−
(1) Volk, K. J.; Yost, R. A.; Brajter-Toth, A. Anal. Chem. 1992, 64,
21A−33A.
452.
(41) Maness, K. M.; Terrill, R. H.; Meyer, T. J.; Murray, R. W.;
Wightman, R. M. J. Am. Chem. Soc. 1996, 118, 10609−10616.
(42) Luo, H.; Shi, Z.; Li, N.; Gu, Z.; Zhuang, Q. Anal. Chem. 2001,
73, 915−920.
(43) Aoki, A.; Matsue, T.; Uchida, I. Anal. Chem. 1990, 62, 2206−
2210.
(2) Karst, U. Angew. Chem., Int. Ed. 2004, 43, 2476−2478.
(3) Baltruschat, H. J. Am. Soc. Mass Spectrom. 2004, 15, 1693−1706.
(4) Permentier, H. P.; Bruins, A. P.; Bischoff, R. Mini-Rev. Med. Chem.
2008, 8, 46−56.
(5) Lunte, S. M. Trends Anal. Chem. 1991, 10, 97−102.
(6) Fosdick, S. E.; Crooks, R. M. J. Am. Chem. Soc. 2012, 134, 863−
866.
(44) Mautjana, N. A.; Looi, D. W.; Eyler, J. R.; Brajter-Toth, A.
Electroanalysis 2010, 22, 79−89.
(45) Kertesz, V.; Van Berkel, G. J. J. Mass Spectrom. 2001, 36, 204−
210.
(46) Modestov, A. D.; Srebnik, S.; Lev, O.; Gun, J. Anal. Chem. 2001,
73, 4229−4240.
(47) Chen, H.; Li, J.; Miao, Z.; Zhang, Y. U.S. Patent US 2010/
0258717 A1.
(48) Takamura, K.; Inoue, S.; Kusu, F.; Oyama, N. Bull. Chem. Soc.
Jpn. 1985, 58, 987−990.
(49) Hayen, H.; Karst, U. Anal. Chem. 2003, 75, 4833−4840.
(50) Richter, P.; Morales, A.; Lahsen, J. Analyst 1990, 115, 409−411.
(51) Smyth, W. F.; Smyth, M. R.; Groves, J. A.; Tan, S. B. Analyst
1978, 103, 497−508.
(7) Pretty, J. R.; Duckworth, D. C.; Van Berkel, G. J. Anal. Chem.
1998, 70, 1141−1148.
(8) Van Berkel, G. J.; Kertesz, V. Rapid Commun. Mass Spectrom.
2009, 23, 1380−1386.
(9) Deng, H.; Van Berkel, G. J. Electroanalysis 1999, 11, 857−865.
(10) Jurva, U.; Wikstrom, H. V.; Weidolf, L.; Bruins, A. P. Rapid
̈
Commun. Mass Spectrom. 2003, 17, 800−810.
(11) Zhang, Y.; Dewald, H. D.; Chen, H. J. Proteome Res. 2011, 10,
1293−1304.
(12) Lu, M.; Wolff, C.; Cui, W.; Chen, H. Anal. Bioanal. Chem. 2012,
403, 355−365.
(13) Hoffman, T.; Hofmann, D.; Klumpp, E.; Kuppers, S. Anal.
̈
Bioanal. Chem. 2011, 399, 1859−1868.
(52) Lu, W.; Xu, X.; Cole, R. B. Anal. Chem. 1997, 69, 2478−2484.
(53) Zhang, F.; Dryhurst, G. Bioorg. Chem. 1995, 23, 193−216.
(54) Nicolis, S.; Zucchelli, M.; Monzani, E.; Casella, L. Chem.Eur.
J. 2008, 14, 8661−8673.
(14) Lohmann, W.; Karst, U. Anal. Bioanal. Chem. 2008, 391, 79−96.
(15) Bruckenstein, S.; Gadde, R. R. J. Am. Chem. Soc. 1971, 93, 793−
794.
(16) Hambitzer, G.; Heitbaum, J. Anal. Chem. 1986, 58, 1067−1070.
(17) Bartmess, J. E.; Phillips, L. R. Anal. Chem. 1987, 59, 2012−2014.
(18) Zhou, F.; Van Berkel, G. J. Anal. Chem. 1995, 67, 3643−3649.
(19) Bond, A. M.; Colton, R.; D’Agostino, A.; Downard, A. J.;
Traeger, J. C. Anal. Chem. 1995, 67, 1691−1695.
(20) Xu, X.; Lu, W.; Cole, R. B. Anal. Chem. 1996, 68, 4244−4253.
(21) Deng, H.; Van Berkel, G. J. Anal. Chem. 1999, 71, 4284−4293.
(22) Regino, M. C. S.; Brajter-Toth, A. Electroanalysis 1999, 11, 374−
379.
5743
dx.doi.org/10.1021/ac300916k | Anal. Chem. 2012, 84, 5737−5743