A New Quantification Method Using Electrochemical Mass Spectrometry

Article abstract of DOI:10.1007/s13361-018-2116-6

Mass spectrometry-based quantification method has advanced rapidly. In general, the methods for accurate quantification rely on the use of authentic target compounds or isotope-labeled compounds as standards, which might be not available or difficult to synthesize. To tackle this grand challenge, this paper presents a novel approach, based on electrochemistry (EC) combined with mass spectrometry (MS). In this approach, a target compound is allowed to undergo electrochemical oxidation and then subject to MS analysis. The oxidation current recorded from electrochemistry (EC) measurement provides information about the amount of the oxidized analyte, based on the Faraday’s Law. On the other hand, the oxidation reaction yield can be determined from the analyte MS signal changes upon electrolysis. Therefore, the total amount of analyte can be determined. In combination with liquid chromatography (LC), the method can be applicable to mixture analysis. The striking strength of such a method for quantitation is that neither standard compound nor calibration curve is required. Various analyte molecules such as dopamine, norepinephrine, and rutin as well as peptide glutathione in low quantity were successfully quantified using our method with the quantification error ranging from ? 2.6 to +?4.6%. Analyte in a complicated matrix (e.g., uric acid in urine) was also accurately measured. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13361-018-2116-6

B American Society for Mass Spectrometry, 2019
J. Am. Soc. Mass Spectrom. (2019)
DOI: 10.1007/s13361-018-2116-6
RESEARCH ARTICLE
A New Quantification Method Using Electrochemical Mass
Spectrometry
Chang Xu,1 Qiuling Zheng,1 Pengyi Zhao,1,2 Joseph Paterson,1 Hao Chen1,2
1Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH
45701, USA
2Department of Chemistry & Environmental Science, New Jersey Institute of Technology, Newark, NJ 07102, USA
Abstract. Mass spectrometry-based quantifica-
tion method has advanced rapidly. In general,
the methods for accurate quantification rely on
the use of authentic target compounds or
isotope-labeled compounds as standards, which
might be not available or difficult to synthesize. To
tackle this grand challenge, this paper presents a
novel approach, based on electrochemistry (EC)
combined with mass spectrometry (MS). In this
approach, a target compound is allowed to un-
dergo electrochemical oxidation and then subject to MS analysis. The oxidation current recorded from electro-
chemistry (EC) measurement provides information about the amount of the oxidized analyte, based on the
Faraday’s Law. On the other hand, the oxidation reaction yield can be determined from the analyte MS signal
changes upon electrolysis. Therefore, the total amount of analyte can be determined. In combination with liquid
chromatography (LC), the method can be applicable to mixture analysis. The striking strength of such a method
for quantitation is that neither standard compound nor calibration curve is required. Various analyte molecules
such as dopamine, norepinephrine, and rutin as well as peptide glutathione in low quantity were successfully
quantified using our method with the quantification error ranging from − 2.6 to + 4.6%. Analyte in a complicated
matrix (e.g., uric acid in urine) was also accurately measured.
Keywords: Quantification, Mass spectrometry, Electrochemistry, Chromatography
Received: 20 October 2018/Revised: 19 November 2018/Accepted: 25 November 2018
calibration or isotope-labeled compound as a reference [9,
10]. To date, a variety of elegant quantitative MS methods
Introduction
ass spectrometry (MS) has become a widely used tech-
nique for characterization and identification of both
have been developed with great successes, such as isotope-
coded affinity tags (ICAT) [11–17], stable isotope labeling
with amino acids in cell culture (SILAC) [15, 18–21], isobaric
tags for relative and absolute quantification (iTRAQ) [22–25],
metal element chelated tags (MECT) [26], and isotope-coded
protein labeling (ICPL) [27], etc. However, standard or
isotope-labeled compounds that are needed for quantification
might not be available and sometimes their syntheses are costly
and time-consuming [28–31].
To tackle this challenge, we present a novel approach
using MS in combination with electrochemistry (EC) [32–
35] to solve this problem, with the goal of achieving accu-
rate chemical quantification without using any standards or
isotope-labeled compounds. Online coupling of EC with MS
is versatile and has been shown to have many applications
M
small and large molecules [1–5], due to its capability for
determining molecular weight (MW) and molecular structure
based on tandem mass analysis. Although MS is powerful in
qualitative analysis, accurate quantification by MS has chal-
lenges due to the fact that the MS signal fluctuates and the
signal intensity in a MS spectrum does not always correlate
well with the amount of analyte [6–8]. Therefore, accurate MS
quantification relies on using standard compounds for
Electronic supplementary material The online version of this article (https://
doi.org/10.1007/s13361-018-2116-6) contains supplementary material, which
is available to authorized users.
Correspondence to: Hao Chen; e-mail: hao.chen.2@njit.edu

C. Xu et al.: Quantification Using a Combined EC and MS
in drug metabolism study, protein structural analysis and
electrochemical reaction mechanism elucidation [36–45].
However, quantification based on electrochemical mass
spectrometry has not been reported so far, which is the
focus of this study. In our study, a target compound, if it
is electroactive, is first introduced to an electrochemical cell
for electrochemical redox conversion (oxidation or reduc-
tion) followed by MS detection. Electrochemical redox re-
action results in an electric current response. The integration
of the current peak over time provides information about the
amount of the oxidized/reduced analyte, based on the Fara-
day’s Law. According to the Faraday’s law, the charge Q,
the total electric charge responsible for oxidizing/reducing
analyte substance in coulombs in the redox reaction, is
directly proportional to quantity of the oxidized/reduced
substance: Q=nzF, where n is the moles of analyte, Z is
the number of electrons transferred per molecule for the
redox reaction, and F is the Faraday constant (9.65 × 104
C/mol). Q can be directly measured from the integration of
Faradaic current over time. The moles of the analyte that is
oxidized or reduced can be calculated as n=Q/zF.
On the other hand, the electroactive species shows reduced
intensities in the acquired MS spectra upon oxidation/reduc-
tion, and the relative MS intensity change upon redox reaction,
Δi, can reflect the redox conversion yield. In this study, Δi can
be measured in two ways. From the acquired MS spectra before
and after electrolysis, Δi can be the relative change of the target
analyte peak intensity (relative to a reference peak) upon elec-
trolysis, as illustrated in Scheme 1a. Alternatively, Δi can be the
relative change of the target analyte peak area in the extracted
ion chromatogram (EIC) upon electrolysis, as illustrated in
Scheme 1b. Indeed, in a separate experiment, we confirmed
that the EIC peak area is proportional to the sample concentra-
tion (see discussion in the Supporting Information Figure S1).
Thus, the amount of analyte converted, in combination with the
conversion yield, can be used to calculate the total amount of
analyte. In other words,
Experimental Section
Chemicals
Dopamine (DA) hydrochloride, (−)-norepinephrine (NE), rutin
(RN) hydrate (≥ 94% purity), and uric acid (UA) were purchased
from Sigma-Aldrich (St. Louis, MO). Glutathione (GSH) (the
reduced form) was purchased from TCI America (Portland,
OR). Formic acid and acetonitrile were obtained from Fisher
Chemical (Fair Lawn, NJ), and deionized water used for sample
preparation was obtained using a Nanopure Diamond Barnstead
purification system (Barnstead International, Dubuque, IA).
Apparatus
A Waters ultra-performance liquid chromatography (UPLC,
Milford, MA, USA) system equipped with a C18 column was
coupled with an electrochemical thin-layer flow cell (Scheme
2). The thin-layer cell used was an Antec ReactorCell™ (Antec
BV, Neitherland) equipped with a Magic Diamond (boron
doped diamond) disc electrode (i.d., 8 mm) as the working
electrode (WE) used for oxidation process. A HyREF™ elec-
trode was used as the reference electrode (RF) and carbon-
loaded PTFE was used as a counter electrode (CE). A positive
potential (ranging from + 1.2 to + 1.3 V) was applied to the WE
electrode for oxidation of the LC-separated target compounds.
Mass spectrum data was collected using a high-resolution Q-
Executive Plus hybrid quadrupole-Orbitrap mass spectrometer
(Thermo Scientific, San Jose, CA). The oxidation current re-
sponse was monitored and recorded by using a ROXY™
potentiostat (Antec BV, Neitherland). The electric current peak
area was integrated by importing the current data point to
software OriginPro 8.0, for calculation of the total electric
charge Q involved in oxidation reaction. The eluate flowing
out of the cell was either collected and subsequently analyzed
using nano-electrospray ionization mass spectrometry (nano-
ESI-MS, Scheme 2a) or directly monitored online using liquid
sample desorption spray ionization mass spectrometry [50]
(LS-DESI-MS, Scheme 2b).
Total amount of analyte
=(amount of the oxidized/reduced analyte)/(the oxidation/
reduction yield)
For nano-ESI-MS analysis, the emitter tip was pulled using
a laser puller (Model P-2000, Sutter Interment, Novato, CA).
The sample injection flow rate for nano-ESI ionization was
2 μL/min with + 3.5 kV potential applied to the nano-ESI
emitter. Nano-ESI-MS spectra were obtained based on averag-
ing of 50 scans. For online MS analysis, a modified version of
liquid sample DESI configuration was adopted (Scheme 2b)
and the DESI probe consisted of a piece of inner fused silica
capillary (2.6 cm) and a piece of a concentric outer PEEK
capillary (2.9 cm). Another piece of fused silica capillary was
connected with the electrochemical flow cell in one end and
inserted into the PEEK capillary in the other end via a small
hole drilled near the outlet of the PEEK capillary (Scheme 2b).
This way the sample flowing out of the cell can be introduced
into the PEEK capillary in which the sample can be ionized via
mixing with the solvent spray coming out of the inner fused
silica capillary. Unlike our previously reported LS-DESI con-
figuration [50, 51], the sample capillary outlet and the DESI
=(Q/zF)/Δi
=Q/(zFΔi)
Unlike traditional coulometric approach, analyte does not
need to undergo complete oxidation or reduction conversion in
our approach, which can be advantageous as 100% conversion
is often practically difficult to achieve. No limitation to a full
redox conversion also allows fast electrolysis, as demonstrated
in this study that electrolysis can be performed during the time
of sample elution from liquid chromatography (LC). The ca-
pability of coupling LC with EC/MS [46] for quantification
further makes this method applicable for analysis of both pure
compounds and mixtures. In literature, electrochemical detec-
tion coupled with LC is a known technique for mixture analy-
sis, where electrochemical oxidation or reduction of LC-
separated compounds generates electric current for detection
or quantification purpose [47–49].

C. Xu et al.: Quantification Using a Combined EC and MS
Scheme 1. Two ways to measure the conversion yield of the target molecule during electrolysis based on MS signal changes: (a)
based on the relative ion signal change (relative to a reference peak); (b) based on the EIC peak area changes
probe in this new DESI set-up do not need to be aligned so that
the operation of DESI source becomes easy. N2 was used as the
nebulization gas (160 psi) and the spray solvent for DESI was
CH3OH/H2O/HOAc (50:50:1 by volume) and infused at the
flow rate of 10 μL/min. To measure the relative area change for
target EIC peak before and after electrolysis, the integration
time window (t, Scheme 1b) was kept the same.
After sample was injected into the LC/EC instrument, the
electrochemical cell was either turned off or on. As the sample
flowed out of the electrochemical cell, the eluate was collected.
The collected un-oxidized and oxidized sample solutions were
adjusted to the same volume (e.g., 100 μL) and then spiked with
1 μL of arginine (spiked as a reference compound, the final
arginine concentration was 10 μM), for subsequent nano-ESI-
MS analysis. The comparison in the recorded MS sample signals
(relative to the spiked arginine) for un-oxidized and oxidized
samples provided information about the oxidation reaction yield.
LC/EC with Offline MS Analysis For DA and NE analysis, an
isocratic elution program was employed using 95% A for 10 min
(mobile phase A: water containing 0.1% formic acid and mobile
phase B: ACN containing 0.1% formic acid). The mobile phase
flow rate was 100 μL/min, and the sample injection volume was
6 μL for DA and 3 μL for NE, respectively. Both DA and NE
samples were prepared in ACN/H2O (5:95 by volume) containing
0.1% formic acid. Control data were also collected using ACN/
H2O (5:95 by volume) containing 0.1% formic acid without DA
or NE as a blank sample. + 1.3 V was applied for DA oxidation,
and + 1.2 V was applied for NE oxidation.
LC/EC with Online MS Analysis Rutin and GSH were ana-
lyzed using online LC/EC/DESI-MS apparatus, and the mobile
phase flow rate was set as 300 μL/min. An isocratic elution
program using 70% A for 10 min was used for rutin, and 80%
A isocratic elution program for 10 min was used for GSH.
Rutin was dissolved in DMSO first and then diluted with a
solvent containing 30% ACN and 0.1% formic acid. GSH
solution was directly prepared using a solvent containing
Apparatus
Scheme 2. Schematic drawing of our chemical quantification apparatus using LC and EC with (a) offline MS detection and (b)
online MS detection

C. Xu et al.: Quantification Using a Combined EC and MS
20% ACN and 0.1% formic acid. The injected concentrations
of rutin and GSH samples were 50 μM and 200 μM, respec-
tively; the injection volume for both rutin and GSH was 6 μL.
Quantitation of urine acid in urine sample was also
attempted using our method. A urine sample was first filtrated
using 0.2 μm WWPTFE membrane, and then diluted 8 times
using water containing 0.1% formic acid. In the LC/EC/DESI-
MS analysis of urine sample, + 1.3 V was applied to the cell for
uric acid oxidation, and the mobile phase flow rate for the
UPLC separation was 300 μL/min. The injection urine sample
volume was 6 μL. The elution program was 100 to 95% A in
the first 4 min, and 95 to 70% A in the following 2 min.
The measurement result for uric acid using LC/EC/DESI-
MS was compared with traditional LC/UV-Vis method. In the
LC/UV-Vis experiment, a Surveyor HPLC System equipped
with Surveyor PDA detector (Thermo Finnigan, San Diego,
CA) was used. The detection wavelength used was 280 nm.
The column used was a reversed phase C18 column (4.6 mm ×
150 mm, Waters, Milford, MA). Standard uric acid solutions
(pH was adjusted to 7 using ammonia) were prepared in order
to generate the calibration curve. The elution program was
100% A for 3 min, then 100% A to 40% A for 7 min, 40% A
to 100% A for 5 min, and finally 100% A for 10 min.
compound, injected for nano-ESI-MS analysis. The ion signal
of DA (relative to arginine) with electrolysis, in comparison to
that without electrolysis, provides the oxidation yield. In the
collected nano-ESI-MS spectrum of DA sample without elec-
trolysis (Figure 1e), the protonated arginine and protonated DA
were detected at m/z 175 and 154, respectively. In contrast,
dopamine quinone (DQ), an oxidized DA product, was seen at
m/z 152 in the nano-ESI-MS spectrum of DA sample with
electrolysis (Figure 1f). The ratio of [m/z 154]/[m/z 175] in
Figure 1f dropped by 18.6% (based on triplicate measurement,
see data in Table S2, Supporting Information), in comparison
to the ratio of [m/z 154]/[m/z 175] in Figure 1e, indicating that
the oxidation yield was 18.6%. Based on the oxidation yield
(18.6%) and the amount of oxidized DA (56.1 pmol), the
amount of the injected DA measured by our method was
301.5 pmol. In comparison to the actual injection amount of
DA (6 μL × 50 μM = 300 pmol), our quantification measure-
ment error was only 0.5%.
(-)-Norepinephrine (NE), another neurotransmitter com-
pound, was tested, too. The NE is known to undergo electro-
chemical oxidation via a two-electron transfer reaction to form
norepinephrine o-quinone (NQ, Scheme 3b). Figure S2b-d
(Supporting Information) displays the electric current diagram
in which it showed a sharp peak at 1.6 min resulting from the
NE oxidation. In contrast, this peak was absent in the control
experiment of using blank sample (Figure S2a, Supporting
Information). Integration of the oxidation peak showed the
amount of oxidized NE to be 22.3 pmol (Table S3,
Supporting Information). On the other hand, the oxidation
yield was measured to be 7.37% by MS analysis of the NE
compound before and after electrolysis (Table S3). Therefore,
the measured amount of NE was 302.7 pmol. In comparison to
the injection amount of 300 pmol (3 μL of 100 μM of NE was
injected for analysis), our measurement error was only 0.9%.
The results for the DA and NE measurements show that our
electrochemical mass spectrometry is feasible for chemical
quantification, without using standard analyte. Encouraged by
this result, we further proceeded to test more organic com-
pounds such as rutin, as discussed below. However, we rea-
soned that online MS analysis would expedite the analysis
process and thereby took online MS analysis protocol.
Results and Discussion
LC/EC with Offline MS Analysis
To demonstrate the viability of our method, several organic
molecules were chosen as test samples. Dopamine (DA), a
known neurotransmitter, was tested first. The DA and other
compounds tested in this study are representative electroactive
species and they were often used to evaluate new electrochem-
ical analysis methods. DA is known to undergo electrochemi-
cal oxidation via a two-electron transfer reaction to produce
dopamine o-quinone (DQ, Scheme 3a). In our experiment,
50 μM of DA in ACN/H2O (5:95 by volume) containing
0.1% formic acid was injected into LC and then flowed through
the electrochemical cell.
Figure 1a displays the current response for a blank sample
(solvent only). A tiny peak was noted at 3.0 min, probably due
to oxidation of impurities in the blank sample (Figure 1a).
When DA sample was injected for oxidation (oxidation poten-
tial + 1.3 V), a sharp electric current peak at 3.2 min was seen
(Figure 1b–d). In the triplicate measurements, the DA peak
retention times and integrated areas were consistent (the total
electric charge of Q involved in oxidation was measured to be
1.08~1.09 × 10−5 C, Figure 1b–d and Table S2, Supporting
Information). By integrating the measured current areas, the
calculated amount of the oxidized dopamine on average was
56.1 pmol.
LC/EC with Online MS Analysis
Rutin, a glycoside of the flavonoid quercetin containing two
glucose units (structure shown in Scheme 3c), was examined.
Rutin is known to undergo a two-electron electrochemical
oxidation at 3′ and 4′ phenol groups to form rutin quinone
(RQ) by losing two H atoms. Indeed, before electrolysis (Fig-
ure 2a), the protonated rutin was seen at m/z 611. After elec-
trolysis (Figure 2b), a peak at m/z 609 was observed, corre-
sponding to the oxidized rutin product RQ. Figure 2c, d shows
the EIC (m/z 611, the protonated rutin) of 50 μM rutin with an
injection volume of 6 μL (injected amount: 300 pmol) at the
mobile flow rate of 300 μL/min with the applied potential of
0 V and + 1.3 V, respectively, in which the rutin peak was
On the other hand, the oxidation yield was measured by MS
analysis of samples before and after electrolysis. In this exper-
iment, DA was collected after it flowed through LC and the
electrochemical cell, spiked with arginine as a reference

C. Xu et al.: Quantification Using a Combined EC and MS
LC/EC with offline MS analysis
(a)
(b)
(c)
(d)
(e)
Scheme 3. Equations showing the electrochemical oxidation of a) DA, b) NE, c) RN, d) GSH, and e) UA
observed at 1.4 min. The integrated area for the peak shown in
Figure 2d was smaller by 2.5%, in comparison with that of the
peak shown in Figure 2c, indicating that the oxidation yield for
rutin was 2.5% (see data in Table S4, Supporting Information).
On the other hand, the rutin oxidation current peak was detect-
ed, as shown in Figure 2f–h (Figure 2e shows the background
current diagram for blank solvent sample as a contrast). Based
on the integration of the current peak area (Figure 2f–h), the
amount of the oxidized rutin on average was calculated to be
7.3 pmol. Therefore, our measured amount of rutin was
7.3 pmol/2.5% = 292 pmol, which was close to the injection
amount of 300 pmol with the measurement error to be − 2.6%.
We also tested peptide quantification using our approach,
based on the electrochemical oxidation conversion of thiols
into disulfides. Glutathione (GSH) was chosen as the test
example. GSH is a thiol-containing peptide and it can be
oxidized into glutathione disulfide (GSSG, Scheme 3d). In-
deed, before electrolysis (Figure S3c, Supporting Information),
the protonated GSH was detected at m/z 308. After electrolysis
(Figure S3d), a peak at m/z 307 was observed, corresponding to
+ 2 ion of GSSG product. Figure S3a and S3b show the EIC
(m/z 308, the protonated GSH) of 200 μM GSH with an
injection volume of 6 μL (injected amount: 1200 pmol) with
the applied potential of 0 V and + 1.3 V, respectively. The
integrated area for the peak shown in Figure S3b was smaller
by 16.0%, in comparison with that of the peak shown in
Figure S3a, indicating that the oxidation yield for GSH was
16.0% (see data in Table S5, Supporting Information). On the
other hand, the GSH oxidation current peak was detected, as
shown in Figure S3f, S3 g and S3 h (Figure S3e shows the
background current diagram for blank solvent sample as a
contrast). Based on the integration of the current peak area,

C. Xu et al.: Quantification Using a Combined EC and MS
DA
DA
DA
DA, 1st injection
DA, 2nd injection
DA, 3rd injection
a) Blank
(a)
(b)
(c)
(d)
11
1
0
1
0
1
0
Area 1.08x10-5
C
Area 1.08x10-5
C
Area 1.09x10-5
C
00
-1
-1
-1
-1
-1
0
3
6
0
0
3
6
4
0
3
6
0
0
3
6
4
0
2
4
0
2
4
2
2
Time (min)
Time (m in)
Time (m in)
Time (min)
Time (min)
TTimimee(m(mini)n)
Time (min)
175.11868
Ref peak
[Arg+H]+
100
DA, Cell off
(e)
50
[DA+H]+
154.08598
0
150
154
158
158
162
166
170
170
174
175.11866
100
DA, Cell on
(f)
x5
50
0
[DQ+H]+
152.07036
154.08596
166
150
154
174
162
m/z
Figure 1. (a) Electric current response due to the blank sample. (b–d) Electrochemical current responses to the oxidation of DA
sample in triplicate measurements. Nano-ESI-MS spectra of dopamine with applied potential of (e) 0 V; and (f) + 1.3 V, the resulting
DQ product peak is seen at m/z 152 in f
the amount of the oxidized GSH on average was calculated to
be 200.9 pmol. Therefore, our measured amount of GSH was
1256 pmol, which was close to the injection amount of
1200 pmol with the measurement error to be 4.6%.
The results above show that it is feasible to use our method
for chemical quantification using online LC/EC/MS technique.
In particular, the success for GSH quantification suggests the
possibility of using our approach for proteins carrying thiols or
[RN+H]+
[RN+H]+
611.15968
611.15976
100
100
b) Cell on
a) Cell off
×10
50
50
[RQ+H+]
609.16498
0
0
608.5
609.5
611.5
612.5
6m10/.5z
608.5
609.5
610.5
611.5
612.5
m/z
[RN+H]+
[RN+H]+
100
50
0
100
50
0
d) Ruꢀn, Cell on
c) Ruꢀn, Cell off
0.2
0.6
1.0
1.4
1.8
0.2
0.6
1.0
Time (min)
1.4
1.8
Time (min)
h) Ruꢀn, 3rd injecꢀon
g) Ruꢀn, 2nd injecꢀon.
f) Ruꢀn, 1st injecꢀon
e) Blank
RN
RN
RN
0.2
Area
Area
Area
1.41×10-6
C
1.40×10-6
C
1.43×10-6
C
0
-0.1
0
1
2
0
2
1
2
0
1
2
0
1
Time (min)
Time (min)
Time (min)
Time (min)
Figure 2. Online DESI-MS spectra of rutin when the applied potential was (a) 0 V and (b) +1 .3 V. the peak of the oxidation product of
rutin, RQ, was seen at m/z 609 in (b). EIC of rutin recorded when the applied potential was (c) 0 V and (d) + 1.3 V. electric current
responses (e) due to the blank solvent and (f–h) the oxidation of rutin sample in triplicate measurements

C. Xu et al.: Quantification Using a Combined EC and MS
disulfide bonds. For example, a cysteine-containing protein
could be digested enzymatically into peptides carrying thiol
groups, which can be analyzed using our method. Such an
investigation is underway and will be reported on due course.
Since our method involves the use of LC, we reasoned that it
could be used for direct quantitation of chemical existing in a
mixture. We therefore chose the uric acid (UA) analysis as an
example to demonstrate such strength of our method.
was 2399 pmol. In consideration of the fact that the injection
volume of urine was 6 μL and the dilution factor was 8, the
concentration of uric acid in the raw urine was determined to be
3.2 mM. This measured result is in line with the report that the
normal level of uric acid is 1.5–4.4 mM in urine [54].
To further check our measurement accuracy, the sample was
also measured using LC/UV-Vis method, a traditional “gold”
standard for quantification. In a separate experiment, 6 μL of the
diluted urine sample was injected into LC/UV-Vis instrument and
the elution of uric acid was monitored by UV-Vis detector. The
concentration of uric acid in the diluted urine sample was quan-
tified to be 385.6 μM, based on a calibration cure acquired by
injecting standard uric acid sample solutions for the LC/UV
analysis (Figure S4, Supporting Information). That means that
the injected urine sample contains 385.6 μM × 6 μL = 2314 pmol,
which only differed from the value of 2399 pmol as measured by
our LC/EC/MS method by 3.7%, emphasizing the accuracy of our
proposed approach.
Furthermore, we spiked the urine sample with known amount
of uric acid and then re-analyze the sample to test the accuracy of
our measurement. Based on the measured mentioned above, 6 μL
of the diluted urine sample contained 2399 pmol of uric acid. We
further spiked the same urine sample with 600 pmol of standard
uric acid and measured the spiked sample with our LC/EC/DESI-
MS method. The uric acid in the spiked sample was measured to
be 3038 pmol, which is fairly close to the sum of original uric acid
in urine and the added standard (2999 pmol), with the deviation of
1.3%, validating the uric acid quantification in urine by our
method. Table S7 (Supporting Information) summarizes the
Urine Analysis
Elevated uric acid level in blood can lead to its aggregation in
joints and trigger gout. Uric acid is also related to kidney
disease [52]. Therefore, it is crucial to determine the uric acid
level in urine. Urine acid is known to undergo a two-electron
electrochemical oxidation to generate a diimine (Scheme 3e)
[53]. In our experiment, raw urine sample was diluted by
eightfold using solvent and then injected into LC/EC/MS for
analysis. Uric acid was eluted out at 2 min with a gradient
elution program (Figure 3a, b) and was detected as an ion of
m/z 169 via protonation. The electric current response due to
the uric acid oxidation is shown in Figure 3d–f, in which a
sharp peak at 2 min was observed. In contrast, this oxidation
current peak was absent in the blank solvent sample
(Figure 3c). Integration of the electric current peak tells that
the oxidized amount of uric acid on average was 109.7 pmol
(Table S6, Supporting Information). Meanwhile, the compari-
son of the EIC peaks shown in Figure 3a, b shows the oxidation
yield of 4.6% (Table S6, Supporting Information). This sug-
gests that the amount of uric acid in the diluted urine sample
Figure 3. EIC spectra of the urine sample (m/z 169, the protonated uric acid) with the applied potential of (a) + 1.3 V and (b) 0 V.
electric current response due to (c) the blank sample and (d–f) the oxidation of UA in the urine sample in triplicate measurements

C. Xu et al.: Quantification Using a Combined EC and MS
experimental result of this spiking experiment. This result con-
firms that our method is applicable to mixture analysis.
References
The amount of sample used in the experiments above varied
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