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Analytica Chimica Acta xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

journal homepage: www.elsevier.com/locate/aca

Simultaneous determination of uric acid, xanthine, hypoxanthine and

caffeine in human blood serum and urine samples using
electrochemically reduced graphene oxide modified electrode
M. Amal Raj, S. Abraham John ∗
Centre for Nanoscience and Nanotechnology, Department of Chemistry, Gandhigram Rural Institute, Gandhigram 624 302, Dindigul, Tamilnadu, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Glassy carbon electrode was modi-

fied with graphene by self assembly
method.
 Modified electrode was character-
ized by Raman spectroscopy.
 Graphene modified electrode sep-
arated the voltammetric signals of
four purine derivatives.
 Selective and simultaneous deter-
mination of four purine compounds
were achieved.
 Practical application was demon-
strated in human blood serum and
urine samples.

a r t i c l e i n f o a b s t r a c t

Article history: This paper describes the fabrication of graphene on glassy carbon electrode (GCE) by electrochemical
Received 23 November 2012 reduction of graphene oxide (GO) attached through 1,6-hexadiamine on GCE and the simultaneous deter-
Received in revised form 3 February 2013 mination of structurally similar four purine derivatives using the resultant electrochemically reduced GO
Accepted 8 February 2013
(ERGO) modified electrode. The electrocatalytic activity of ERGO was investigated toward the oxidation
Available online xxx
of four important purine derivatives, uric acid (UA), xanthine (XN), hypoxanthine (HXN) and caffeine
(CAF) at physiological pH. The modified electrode not only enhanced the oxidation currents of the four
Keywords:
purine derivatives but also shifted their oxidation potentials toward less positive potentials in contrast
Graphene oxide
Electrochemical reduction
to bare GCE. Further, it successfully separates the voltammetric signals of the four purine derivatives in
Raman spectra a mixture and hence used for the simultaneous determination of them. Selective determination of one
Purine derivatives purine derivative in the presence of low concentrations other three purine derivatives was also realized
Human blood plasma at the present modified electrode. Using differential pulse voltammetry, detection limits of 8.8 × 10−8 M,
1.1 × 10−7 M, 3.2 × 10−7 M and 4.3 × 10−7 M were obtained for UA, XN, HXN and CAF, respectively. The
practical application of the modified electrode was demonstrated by simultaneously determining the
concentrations of UA, XN, HXN and CAF in human blood plasma and urine samples.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Purine bases and their derivatives play an important role in the

functioning of living systems [1]. Purines are involved in many
metabolic processes as cofactors which play key roles in biologi-
∗ Corresponding author. Tel.: +91 451 245 2371; fax: +91 451 245 3031. cal fundamental processes [2]. Several pathological conditions alter
E-mail addresses: abrajohn@yahoo.co.in, abrajohn@gmail.com (S.A. John). the levels of purine derivatives in blood serum and urine which are

0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aca.2013.02.017

Please cite this article in press as: M.A. Raj, S.A. John, Simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine
in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode, Anal. Chim. Acta (2013),
http://dx.doi.org/10.1016/j.aca.2013.02.017
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Chart 1. Structures of purine derivatives.

biochemical indicators for several diseases [2]. The levels of uric
acid (UA), oxypurines such as xanthine (XN) and hypoxanthine
(HXN) and trimethylxanthine derivative, caffeine (CAF) (Chart 1)
may provide sensitive indicators for physiological diseases [3,4].
UA, XN and HXN are present in body fluids by the metabolism
of purine derivatives whereas CAF is introduced into the body
fluids by consuming tea, coffee and coca cola [5]. UA is a prod-
uct of the metabolic breakdown of purine nucleotides and the
abnormal level of UA results in several diseases like gout, hyper-
uricaemia and pneumonia [6]. In normal serum, UA levels are less
than 420 ␮mol L−1 in men and 330–360 ␮M L−1 for women [7]. XN
is an important intermediate of the purine metabolism and the
abnormality of XN leads to xanthinuria and its therapeutic level
is 10–20 mg mL−1 in blood [8]. HXN is found as a minor purine base
in tRNA and also a product in the metabolism of purine nucleotides
[9]. It is an important factor to know the fish freshness [10]. CAF, a
methyl derivative of xanthine, is a drug which has many physiolog-
ical effects such as stimulant to central nervous system and cardiac
muscles, dieresis and positive effect on cardiovascular system [11].
Several epidemiological studies show that CAF consumption and
plasma UA level were related to the incidence of some neurode-
generative diseases [12,13]. Determination of XN and UA levels
is important for diagnosing gout and hyperuricaemia [14]. Since
several physiological properties are related to the concentrations
of these four structurally similar purine derivatives, simultaneous
determination of them is very important in the clinical point of
view. Several reports were available in the literature for the simul-
taneous determination of UA, XN and HXN [15–23] and individual
determination of CAF [24–29]. However, to the best of our knowl-
edge, no report is available in the literature for the simultaneous
determination of UA, XN, HXN and CAF. Hence, the objective of
the present study is to simultaneously determine the four purine
derivatives using the graphene modified glassy carbon electrode
(GCE).
Among the different allotropes of carbon, graphene receives
huge attention due to its unique thermal, electrical and mechan-
ical properties which originate from the two dimensional, single
atom thick structure of graphene [30,31]. The unique proper-
ties of graphene make them to be used in supercapacitors [32],
fuel and solar cells [33] and biosensors [34,35]. The high spe-
cific surface area, besides high conducting nature of graphene
layers, makes them to be used as a novel material for the fab-
rication of electrodes [36]. Recently, researchers use graphene
to fabricate electrodes for the electrochemical sensing of metal
ions [37], biomolecules [38] and toxic chemicals [39]. Most of the
researchers use drop casting method for the fabrication of graphene
on different electrode substrates [40–43]. Self-assembly is the best
method to fabricate graphene films with desirable thickness on
electrode substrates [44]. In the present study, the precursor of
graphene, graphene oxide (GO), was electrostatically assembled
on GCE through diamine linker. The electrostatically assembled
GO was electrochemically reduced at more negative potential to
retain the aromatic backbone of graphene and used for the elec-
trochemical sensing of structurally similar purine derivatives. Due
to facile electron transfer reaction at ERGO films, the modified
electrode separates the voltammetric signals of the four analytes
with enhanced peak currents compared to bare GCE and also pre-
vents the surface fouling effect caused by the oxidation products
of purine derivatives. The practical application of the present mod-
ified electrode was demonstrated by simultaneously determining
the concentrations of UA, XN, HXN and CAF in human blood serum
and urine samples.

Please cite this article in press as: M.A. Raj, S.A. John, Simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine
in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode, Anal. Chim. Acta (2013),
http://dx.doi.org/10.1016/j.aca.2013.02.017
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2. Experimental
2.1. Chemicals
Graphite powder and 1,6-hexadiamine were purchased from
Alfa Aesar. Ascorbic acid (AA), uric acid (UA), xanthine (XN),
hypoxanthine (HXN) and caffeine (CAF) were purchased from
Sigma–Aldrich and were used as received. 0.2 M phosphate buffer
(PB) solution was prepared using Na2 HPO4 and NaH2 PO4 . All other
chemicals were of analytical grade and were used as received.
Double distilled water was used for preparing all solutions. The
human blood serum and urine samples were collected from Maha-
lakshmi Clinical Laboratory, Chinnalapatti. The blood serum and
urine were diluted to 10 times and 25 times, respectively using PB
solution.
2.2. Instrumentation
The electrochemical measurements were carried out with CHI
electrochemical workstation (Model 643B, Austin, TX, USA). Elec-
trochemical measurements were performed in a conventional
two-compartment, three-electrode cell with GCE as a working elec-
trode, platinum wire as a counter electrode and NaCl saturated
Ag/AgCl as a reference electrode. For differential pulse voltam-
metry (DPV) measurements, amplitude of 0.05, pulse width of
0.06, sample width of 0.02 and pulse period of 0.2 were used. All
the electrochemical experiments were carried out under nitrogen
atmosphere at room temperature.
2.3. Fabrication of GO modified electrode
Initially, GO was synthesized using the Hummer’s method with
slight modification [45]. The well cleaned GCE was immersed into
1 mM solution of 1,6-hexadiamine (HDA) for 8 h. The HDA modified
electrode was rinsed with water and subsequently immersed into
exfoliated GO solution (1 mg mL−1 ) for 12 h. Then, the GO modi-
fied electrode was electrochemically reduced in PB solution (pH 7)
to retain the aromatic lattice of graphene. Scheme S1 illustrates
the schematic representation for the modification of GCE with
ERGO.
3. Results and discussion
3.1. Electrochemical reduction of self-assembled GO on GCE
For the fabrication of GO on GCE, 1,6-hexadiamine (HDA)
was first introduced on GCE by the self-assembly method. The
mechanism of amine attachment on GCE follows the Michael’s
nucleophilic addition of amine group on the olefinic bond present
in GCE [46]. The GO was assembled on HDA modified GCE by
the electrostatic interaction between the negatively charged GO
and the positively charged amine terminal of HDA. The surface
attached GO was electrochemically reduced to remove the oxy-
gen functional groups present on the surface of GO by cycling the
potential between 0 and −1.4 V for 15 cycles (Fig. S1). Prior to
the electrochemical reduction of GO, PB solution was completely
saturated with nitrogen gas to remove the dissolved oxygen. In
the first cycle, GO modified electrode shows a cathodic peak at
−1.15 V which might be due to the reduction of oxygen functional
groups. The reduction current decreases markedly in the second
cycle and on subsequent cycles, the cathodic wave was completely
disappeared indicating the complete reduction of GO. Due to the
removal of oxygen functional groups by electrochemical reduction,
aromatic backbone of graphene must be regenerated on the surface
of GCE.
Please cite this article in press as: M.A. Raj, S.A. John, Simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine
in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode, Anal. Chim. Acta (2013),
http://dx.doi.org/10.1016/j.aca.2013.02.017
3
Intensity (a.u)
b
a
1000 1500 2000
Wavenumber (cm –1)
Fig. 1. Raman spectra obtained for (a) ITO/HDA/GO and (b) ITO/HDA/ERGO plates.
3.2. Characterization of GO and ERGO electrodes by Raman
spectroscopy
Raman spectroscopy is a powerful technique to characterize car-
bon based materials [47]. The attachment of GO followed by its
electrochemical reduction was confirmed by Raman spectroscopy.
For Raman spectral measurements, GO and ERGO modified ITO
plates prepared by the same procedure were used. Fig. 1 shows the
Raman spectra of GO modified ITO plate before and after the elec-
trochemical reduction. GO modified electrode shows two peaks at
1344 cm−1 and 1588 cm−1 corresponding to D and G peaks of GO
(curve a). After the electrochemical reduction, the D and G peaks
were obtained at 1341 cm−1 and 1582 cm−1 , respectively (curve b).
A small shift in the G band after electrochemical reduction is due
to regeneration of aromatic lattice of graphene [48]. Moreover, the
intensity ratio of D and G band was increased from 1.01 to 1.39 after
the electrochemical reduction indicating a decrease in the average
size of sp2 domain [49]. This confirms the successful attachment
of GO on electrode surface by electrostatic assembly and its elec-
trochemical reduction removed the oxygen functional groups from
the surface of GO.
3.3. Electrocatalytic activity of ERGO modified electrode toward
purine derivatives
It is well known that graphene sheets show good electrical
conductivity [50]. Hence, an attempt is made to determine four
purine derivatives using the ERGO modified electrode. Fig. S2 shows
the CVs obtained for 0.5 mM UA at bare GC, GC/HDA/GO and
GC/HDA/ERGO electrodes in 0.2 M PB solution (pH 7.2). The oxi-
dation of UA was observed at 0.32 V (curve a: solid line). After 8
cycles, decrease in peak current and shifting of oxidation poten-
tial were observed at bare GCE (curve a: dotted line). On the other
hand, a slight enhancement of the oxidation current was observed
at GC/HDA/GO electrode (curve b: solid line). The increase in UA
oxidation current at GO modified electrode compared to bare GCE
might be due to the hydrogen bonding interaction between sec-
ondary amine groups of UA and the oxygen functional groups
present on the surface of GO. The oxidation peak was not stable
at GO modified electrode for further potential cycles (curve b: dot-
ted line). The insulating nature of GO at GCE electrode does not
prevent the surface fouling caused by the oxidation products of
UA. In the case of GC/HDA/ERGO electrode, the oxidation of UA was
observed at less positive potential (0.30 V) with enhanced oxida-
tion current when compared to bare and GO modified GCEs (curve
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Fig. 2. CVs obtained for the oxidation of 0.5 mM XN at (a) bare GC (b) GC/HDA/GO
and (c) GC/HDA/ERGO electrode in 0.2 M PB solution (pH 7.2) at a scan rate of
50 mV s−1 . (d) CVs of GC/HDA/ERGO electrode in 0.2 M PB solution in the absence of
XN.
c: solid line). Interestingly, ERGO modified GCE effectively pre-
vents the surface fouling effect caused by the oxidation products
of UA (curve c: dotted line). The enhancement of oxidation current
might be due to ␲–␲ interaction between UA and ERGO film on
GCE.
Fig. 2 shows the oxidation of XN at bare GC, GC/HDA/GO and
GC/HDA/ERGO electrodes. Bare GCE oxidized it at 0.79 V (curve a:
solid line) and for further potential cycles, the oxidation current was
decreased and the oxidation potential was shifted to more positive
potential (curve a: dotted line). For GO modified GCE, broad oxi-
dation wave was observed at 0.87 V with enhanced peak current
compared to bare GCE (curve b: solid line) and the oxidation peak
was shifted to more positive potential for further potential cycles
with decrease in peak current (curve b: dotted line). At ERGO mod-
ified electrode, the oxidation of XN was observed at 0.73 V with
enhanced peak current compared to bare and GO modified GCEs
(curve c: solid line). Similar to UA oxidation, the oxidation peak of
XN was also stable at ERGO electrode with subsequent potential
cycles (curve c: dotted line). Fig. S3 shows the oxidation of HXN
at bare GCE, GO/HDA/GO and GC/HDA/ERGO electrodes. Bare GCE
oxidized it at 1.20 V and the oxidation was not stable at bare GCE
(curve a). Slight enhancement of oxidation current was observed for
HXN at GO modified electrode but oxidation peak was not stable
at GO modified electrodes (curve b). In the case of ERGO modified
electrode, the oxidation peak was shifted to less positive potential
and the oxidation current was increased in contrast to bare and
GO modified GCEs (curve c). On subsequent potential cycles, slight
decrease in oxidation peak current was observed. In the case of CAF
oxidation, bare GCE oxidize it at 1.47 V (Fig. S4: curve a) whereas
an increase in the oxidation current was obtained at GO modified
electrode (curve b). Compared to bare and GO modified GCEs, ERGO
modified electrode greatly enhanced the oxidation current on CAF
(curve c). Compared to bare GCE, GO modified electrode enhanced
the oxidation currents of all the four purine derivatives. But, the
oxidation potentials were not stable for further potential cycles.
Hence, GO modified GCE is not suitable for the determination of
purine derivatives. In the case of ERGO modified electrode, not only
the oxidation currents of four purine derivatives increased but also
their oxidation potentials were shifted to less positive potentials.
Further, it effectively prevented the surface fouling caused by the
oxidation products of the purine derivatives. Hence, ERGO modi-
fied GCE is highly suitable for the determination of UA, XN, HXN
and CAF.
Please cite this article in press as: M.A. Raj, S.A. John, Simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine
in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode, Anal. Chim. Acta (2013),
http://dx.doi.org/10.1016/j.aca.2013.02.017
Fig. 3. DPVs obtained for the oxidation of 0.5 mM of each UA, XN, HXN and CAF at
(a) bare GC and (b) GC/HDA/ERGO electrode in 0.2 M PB solution (pH 7.2). Solid line:
1st cycle and dotted line: 8th cycle.
3.4. Electrochemical behavior of purine derivatives in a mixture
at bare GCE and GC/HDA/ERGO films
Fig. 3 shows the differential pulse voltammograms (DPVs)
obtained for 0.5 mM each UA, XN, HXN and CAF in a mixture at
bare GC and GC/HDA/ERGO electrodes in 0.2 M PB solution. In the
first cycle, bare GCE shows the oxidations of UA, XN, HXN and CAF at
0.32 V, 0.71 V, 1.10 V and 1.42 V, respectively (curve a: solid line).
In the subsequent cycles, the oxidation peak potentials of them
were shifted to more positive potentials with significant decrease
in oxidation peak currents (curve a: dotted line). This clearly shows
that bare GCE was not suitable for the determination of four purine
derivatives in a mixture. In the case of ERGO modified electrode,
increase in oxidation peak currents and shifting of peak poten-
tials to less positive potential were observed for UA, XN, HXN and
CAF (curve b: solid line). The voltammetric signals of them remain
unchanged even after 8 subsequent potential cycles (curve b: dot-
ted line). This shows that the ERGO modified electrode effectively
prevents the surface fouling effect caused by the oxidation prod-
ucts of the four structurally similar purine derivatives in a mixture.
Thus, the present modified electrode can be used to simultaneously
determine them in a mixture. The stability and reproducibility of
GC/HDA/ERGO electrode was examined by series of 20 successive
DPV measurements for 0.5 mM each UA, XN, HXN and CAF. The rel-
ative standard deviation was found to be 2.4%, indicating that the
modified electrode showed good stability and repeatability within
a confidence level of 97.4%. The above results showed the higher
stability of the present modified electrode.
3.5. Simultaneous determination of UA, XN, HXN and CAF at
ERGO modified electrode
Fig. 4 shows the DPVs obtained for UA, XN, HXN and CAF in a
mixture at GC/HDA/ERGO electrode in 0.2 M PB solution (pH 7.2).
A well defined oxidation peaks were observed for 20 ␮M UA, each
10 ␮M XN and HXN and 20 ␮M CAF at 0.26 V, 0.63 V, 0.97 V and
1.33 V, respectively. When the concentrations of UA and CAF were
increased from 20 ␮M to 120 ␮M and XN and HXN were increased
from 10 ␮M to 60 ␮M, the peak currents of the respective ana-
lytes were increased linearly. The plots of concentrations of UA,
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XN, HXN and CAF versus their respective oxidation peak currents
were linear with correlation coefficients of 0.9912, 0.9934, 0.9920,
and 0.9962, respectively (Fig. S5). The oxidation potentials of UA
and CAF were not shifted on each addition whereas the oxidation
potentials of XN and HXN were slightly shifted toward more pos-
itive potentials. These results suggested that the ERGO modified
GCE was highly suitable for the simultaneous determination of the
four purine derivatives.

3.6. Selective determination at ERGO modified electrode

The other objective of the present investigation is to determine

Fig. 4. DPVs obtained for the simultaneous increment of 20 ␮M of each UA and
CAF and 10 ␮M each XN and HXN at GC/HDA/ERGO electrode in 0.2 M PB solution
(pH 7.2).
one purine derivative selectively in the presence of low concentra-
tions of other three purine derivatives. Fig. 5A shows the selective
determination of UA in the presence of low concentrations of XN,
HXN and CAF. A clear voltammetric signal was observed at 0.26 V
for the addition of 5 ␮M UA in the presence of each 25 ␮M of XN and
HXN and 50 ␮M CAF. For each addition of UA, the current increases
linearly in the range of 5–1000 ␮M with a correlation coefficient of
0.9934 (Fig. S6A). The peak currents and oxidation potentials cor-
respond to XN, HXN and CAF were not changed for each addition of
UA. Fig. 5B shows the selective determination XN in the presence
of each 25 ␮M UA and HXN and 50 ␮M CAF. The oxidation of XN
was observed at 0.64 V in the presence UA, HXN and CAF. For each

Fig. 5. DPVs obtained for the increment of (A) (a) 5 ␮M, (b) 5 ␮M, (c) 10 ␮M, (d) 10 ␮M, (e) 10 ␮M, (f) 20 ␮M, (g) 20 ␮M, (h) 20 ␮M, (i) 50 ␮M, (j) 50 ␮M, (k) 100 ␮M, (l)
200 ␮M and (m) 500 ␮M UA in the presence of each 25 ␮M XN and HXN and 50 ␮M CAF, (B) (a) 5 ␮M, (b) 5 ␮M, (c) 10 ␮M, (d) 10 ␮M, (e) 10 ␮M, (f) 20 ␮M, (g) 20 ␮M, (h)
20 ␮M, (i) 50 ␮M, (j) 50 ␮M, (k) 50 ␮M and (l) 50 ␮M XN in the presence of each 25 ␮M UA and HXN and 50 ␮M CAF, (C) DPVs obtained for the increment of (a) 5 ␮M, (b)
5 ␮M, (c) 10 ␮M, (d) 10 ␮M, (e) 10 ␮M, (f) 20 ␮M, (g) 20 ␮M, (h) 20 ␮M, (i) 50 ␮M, (j) 50 ␮M and (k) 100 ␮M HXN in the presence of each 25 ␮M of UA and XN and 50 ␮M
CAF and (D) (a) 10 ␮M, (b) 10 ␮M, (c) 20 ␮M, (d) 20 ␮M, (e) 20 ␮M, (f) 20 ␮M, (g) 50 ␮M, (h) 50 ␮M, (i) 100 ␮M and (j) 200 ␮M CAF in the presence of each 25 ␮M UA, XN and
CAF at GC/HDA/ERGO electrode in 0.2 M PB solution (pH 7.2).

Please cite this article in press as: M.A. Raj, S.A. John, Simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine
in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode, Anal. Chim. Acta (2013),
http://dx.doi.org/10.1016/j.aca.2013.02.017
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Fig. 6. (A) DPVs obtained for (a) human blood serum and (b) after the addition of 10 ␮M UA, 5 ␮M XN, 10 ␮M HXN and 30 ␮M CAF at GC/HDA/ERGO electrode in 0.2 M PB
solution. (B) DPVs obtained for (a) human urine and (b) after the addition of each 25 ␮M UA, each 10 ␮M XN and HXN and 30 ␮M CAF at GC/HDA/ERGO electrode in 0.2 M PB
solution.
addition of XN, the oxidation peak current was increased linearly
in the range of 5–300 ␮M with a correlation coefficient of 0.9946
(Fig. S6B). The peak potential and the peak currents of other purine
derivatives were not affected significantly with the increment of XN
concentration suggesting that the present modified electrode was
suitable for the selective determination of XN. Fig. 5C shows the
selective determination of HXN in presence of each 25 ␮M UA and
XN and 50 ␮M CAF. The oxidation potential of HXN was observed
at 0.97 V and for each addition the current increases linearly. The
concentration of HXN was increased from 5 ␮M to 300 ␮M and the
plot of concentration of HXN versus peak current was linear with a
correlation coefficient of 0.9878 (Fig. S6C). The peak currents of UA,
XN and CAF was not shifted suggesting that selective determination
of HXN was possible in the presence of very low concentrations of
UA, XN and CAF. Fig. 5D shows the selective determination of CAF
in the presence of low concentrations of UA, XN and HXN. The con-
centration of CAF was increased from 10 ␮M to 500 ␮M and the
current increases linearly with a correlation coefficient of 0.9889
(Fig. S6D). These results revealed that ERGO modified electrode
was highly suitable for the selective determination of one purine
derivative in a wide concentration range in the presence of very
low concentrations of other purine derivatives.
Ascorbic acid (AA) usually coexists in higher concentration along
with the purine derivatives in body fluids. For adults, AA doses of
100–2000 mg day−1 are required for eliminating vitamin C defi-
ciency and hence the level of AA is usually higher in body fluids
[51]. Hence, the determination of purine derivatives in the pres-
ence of wide concentration range of AA is very important. Fig. S7
shows the DPVs obtained for the increment of AA in the presence of
each 25 ␮M of UA, XN and HXN and 50 ␮M CAF. When the concen-
tration of AA was increased from 25 ␮M to 750 ␮M, the oxidation
peak current corresponds to AA oxidation current increased with-
out affecting the oxidation peak potential and peak currents of UA,
XN, HXN and CAF. The plot of concentration of AA versus the oxida-
tion peak current was linear with a correlation coefficient of 0.9990.
Even in the presence of higher concentration of AA, the voltammet-
ric signals of UA, XN, HXN and CAF were clearly observed at 0.27 V,
0.63 V, 0.97 V and 1.31 V, respectively. This shows that the present
modified electrode is highly suitable for the selective determination
of purine derivatives in the presence of wide concentration range
of AA. Using DPV, we have calculated the detection limit for UA,
XN, HXN and CAF at GC/HDA/ERGO electrode. The detection lim-
its were found to be 8.8 × 10−8 M, 1.1 × 10−7 M, 3.2 × 10−7 M and
4.3 × 10−7 M for UA, XN, HXN and CAF, respectively. The obtained
Please cite this article in press as: M.A. Raj, S.A. John, Simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine
in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode, Anal. Chim. Acta (2013),
http://dx.doi.org/10.1016/j.aca.2013.02.017
lowest detection limits of purine derivatives at GC/HDA/ERGO elec-
trode were compared with the reported modified electrodes (Table
S1: Supplementary information). To the best of our knowledge, no
report is available in the literature for the simultaneous determina-
tion of four purine derivatives. When compared to reported papers,
the present method showed appreciable detection limits for UA,
XN, HXN and CAF (Table S1). Although the present modified elec-
trode was successfully used for the determination of four purine
derivatives, it is limited to the detection of UA, XN, HXN and CAF
in the absence of drugs such as dopamine, acetaminophen, sali-
cylic acid, acetylsalicylic acid and other purine derivatives including
adenine, and guanine.
3.7. Determination of purine derivatives in human blood serum
and urine samples
The practical application of the present modified electrode was
demonstrated by determining the concentration of purine deriva-
tives in human blood serum and urine samples. The serum samples
were collected from the clinical laboratory. The standard addition
technique was used for the determination of purine derivatives in
serum samples. The human blood serum samples were diluted to
10 times using 0.2 M PB solution (pH 7.2). DPV of serum sample
in PB solution shows four oxidation peaks at 0.35 V, 0.65 V and
0.98 V and the obtained peaks may be due to the oxidation of UA,
XN and HXN, respectively (curve a in Fig. 6A). To confirm this,
commercial samples of UA, XN, HXN and CAF were injected into
the serum solution and the oxidation peak currents at 0.35, 0.65
and 0.98 V were increased and an additional peak at 1.38 V was
appeared which might be due to the oxidation peak of CAF (curve
b in Fig. 6A). The present modified electrode shows good recov-
ery for the simultaneous determination of UA, XN, HXN and CAF
in human serum samples (Table S2). Similar recoveries were also
obtained in human urine samples diluted to 25 times by PB solution
(Fig. 6B). The results were summarized in Table S2. The proposed
method shows good recoveries for the spiked UA, XN, HXN and
CAF in human serum and urine samples. Thus, the present modified
electrode can be utilized for the determination of these analytes in
real samples.
4. Conclusions
In this paper, we have demonstrated the determination of struc-
turally similar purine derivatives using ERGO modified GCE. GO
 ARTICLE IN PRESS
G Model
ACA-232403; No. of Pages 7
M.A. Raj, S.A. John / Analytica Chimica Acta xxx (2013) xxx–xxx
was self-assembled on GC/HDA electrode and then electrochemi-
cally reduced at negative potentials to retain the aromatic lattice
of graphene. Raman spectral studies showed the increase in D/G
intensity ratio after the electrochemical reduction of GO suggest-
ing the successful attachment of GO followed by its electrochemical
reduction. Compared to bare and GO modified GCEs, ERGO modi-
fied electrode greatly enhanced the oxidation currents of UA, XN,
HXN and CAF and shifted their oxidation potentials toward less
positive potentials. The increase in the oxidation currents was due
to the ␲–␲ interaction between aromatic backbone of graphene
layer on GCE and the purine derivatives. Bare and GO modified
GCE failed to give stable oxidation peaks in a mixture of purine
derivatives whereas stable voltammetric peaks were observed
at ERGO modified electrode. The simultaneous determination of
four purine derivatives was successfully demonstrated using the
present modified electrode. Selective determination of one purine
derivative in the presence of low concentrations of other purine
derivatives was also successfully demonstrated using the present
modified electrode. Using DPV, detection limits were calculated
and it was found to be 8.8 × 10−8 M, 1.1 × 10−7 M, 3.2 × 10−7 M
and 4.3 × 10−7 M, respectively (S/N = 3). Further, the practical appli-
cation of the present modified electrode was demonstrated by
simultaneously determining the concentrations of UA, XN, HXN and
CAF in human blood serum and urine samples.
Acknowledgement
M. Amal Raj thanks the Council of Scientific and Industrial
Research (CSIR), New Delhi, for the award of Senior Research Fel-
lowship (09/715(0014)/2010-EMR-I).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.aca.2013.02.017.
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