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Full Paper

Voltammetric Determination of Uric Acid at a Fullerene-C60-

Modified Glassy Carbon Electrode
Rajendra N. Goyal,* Vinod K. Gupta, Aditi Sangal, Neeta Bachheti
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee-247 667, India
*e-mail: rngcyfcy@iitr.ernet.in

Received: June 2, 2005

Accepted: August 3, 2005

Abstract
Glassy carbon electrode modified by microcrystals of fullerene-C60 mediates the voltammetric determination of uric
acid (UA) in the presence of ascorbic acid (AA). Interference of AA was overcome owing to the ability of pretreated
fullerene-C60-modified glassy carbon electrode. Based on its strong catalytic function towards the oxidation of UA and
AA, the overlapping voltammetric response of uric acid and ascorbic acid is resolved into two well-defined
voltammetric peaks with lowered oxidation potential and enhanced oxidation currents under conditions of both linear
sweep voltammetry (LSV) and Osteryoung square-wave voltammetry (OSWV). At pH 7.2, a linear calibration graph
is obtained for UA in linear sweep voltammetry over the range from 0.5 mM to 700 mM with a correlation coefficient
of 0.9904 and a sensitivity of 0.0215 mA mM
1 . The detection limit (3s) is 0.2 mM for standard solution. AA in less than
four fold excess does not interfere. The sensitivity and detection limit in OSWV were found as 0.0255 mA mM
1 and
0.12 mM, for standard solution respectively. The presence of physiologically common interferents (i.e. adenine,
hypoxanthine and xanthine) negligibly affects the response of UA. The fullerene-C60-modified electrode exhibited a
stable, selective and sensitive response to uric acid in the presence of interferents.

Keywords: Uric acid, Ascorbic acid, C60, Modified electrode, Interferents

DOI: 10.1002/elan.200503353

1. Introduction cardiovascular disease [10, 11]. In patients having conges-

Uric acid (2,6,8-trihydroxypurine) (UA) is a relatively water
insoluble nitrogenous end product of purine nucleotide
catabolism in human system [1]. It is found distributed in
biological fluids of human, mainly in serum, blood and urine
and is largely excreted from the human system by kidneys [2].
In a healthy human being, the normal concentration ranges of
UA in human serum, human blood and urine are 240 –
520 mM, 120 –450 mM and 1.2 –4.4 mM, respectively [3, 4].
Change in UA concentration leads to numerous diseases and
physiological disorders. An overproduction of UA occurs
when there is excessive breakdown of cells that contain
purines or an inability of the kidneys to excrete UA. Greater
than normal levels of UA may be indicated in the diseases such
as diabetes, gout, hyperuricemia, Lesch-Nyhan syndrome, and
renal failure [5, 6]. Low uric acid levels may be associated with
a molybdenum deficiency, copper toxicity, and a worsening of
multiple sclerosis. An association between low serum uric acid
and hypersensitivity problems has been noted. Abnormally
low uric acid levels may indicate that the patient suffers from
FanconiBs disease and WilsonBs disease [7].
Plasma UA level has been considered as one of the
predictors of blood pressure elevation and obesity [8]. UA
can independently predict mortality in patients with coro-
nary artery disease [9]. Many prospective studies in hyper-
tensive patients show a relationship between serum UA and
tive heart failure, serum UA is the most powerful predictor
of survival as well as hospitalization [12]. UA is also
employed as a clinical marker for renal dysfunction. UA
level is higher in people at high risk of diabetes mellitus with
abnormal glucose tolerance [13]. Besides such detrimental
actions, UA also has beneficial functions. It is an important
antioxidant in human adult plasma and is involved in many
pathological changes [14, 15]. Thus, UA concentration
determination is of paramount importance, due to its crucial
role in health assessment and monitoring.
Various methods such as enzymatic, colorimetric, electro-
chemical, spectrophotometric and HPLC have been report-
ed for the analysis of UA in the human body fluids [16 – 19].
The electrochemical techniques have been found more
selective, less costly and less time-consuming and being
claimed useful over other methods for the determination of
uric acid. However, a major problem has been found due to
the presence of high concentration of AA in human body
fluids, which oxidizes at a similar potential as UA and higher
potentials are required for their direct electrooxidation at
carbon-based electrodes. The ability to determine UA and
AA selectively has been a major goal of electroanalytical
research. Voltammetric methods based on Nafion-coated
carbon paste electrode, Nafion coated GCE and b-cyclo-
dextrin modified electrode incorporating carbon nanotubes
have been reported for the determination of uric acid that

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offered good selectivity with a low detection limit (0.2 mM)
[20 – 22]. However, these modified electrodes involved a
complex preparation procedure of the electrode and the
surface of the electrode is to be renewed each time. The
extent of renewal depends on applied potential and pH of
the cleaning solution. Few other modified electrodes [23, 24,
32] reported in literature for the determination of uric acid
also had the same complication.
Cai and co-workers reported an improved voltammetric
method using electrochemically pretreated carbon-paste
electrode in basic electrolyte media [25]. However, the
electrode material was found to swell after 30 measurements.
At several other electrodes modified with films of
complexes, nanodeposites the lowest detection limit report-
ed for uric acid is 0.2 mM [26 – 28].
Since the discovery of the new allotrope of carbon, the
fullerene, investigations have been initiated to explore
several possible fields of their applications. The reductive
electrochemistry of fullerene-C60-films has been studied
extensively. Szucs et al. have established that fullerene films
become conducting upon reduction which implies that it can
be used as an electrode material [29]. It was observed that
reduced fullerene films could be used as electrocatalysts. The
electrocatalytic behavior of fullerene film electrodes is quite
different from other type of electrodes since reduced full-
erene film on glassy carbon substrates show microelectrode
characteristics. Fullerene modified electrodes have recently
been widely used for catalytic and sensor applications, and to
study the electrochemistry of biomolecules. The present
paper describes simple voltammetric techniques for the
sensitive and selective determination of UA at fullerene-C60
modified glassy carbon electrode at physiological pH i.e. at
pH 7.2. The electrode not only has a strong catalytic function
towards the oxidation of UA and AA, but also resolves the
overlapping voltammetric response of the two compounds
into two well-defined voltammetric peaks. The voltammetric
response of UA in the presence of other biologically common
interferents has also been studied.
2. Experimental
2.1. Reagents
C60 was obtained from Aldrich, USA (purity 98%). UA,
AA, adenine, xanthine and hypoxanthine were purchased
from Sigma, USA. All these compounds were used without
further purification. Phosphate buffer solution (PBS) (m ¼
0.1 M) prepared according to the method of Christian and
Purdy [30] was used as supporting electrolyte. All other
reagents used were of analytical grade. All solutions were
prepared in double distilled water.
2.2. Instrumentation
The voltammetric experiments were performed on BAS
(Bioanalytical Systems, West Lafayette, IN, USA) CV-50W
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R. N. Goyal et al.
Voltammetric analyzer. The electrochemical measurements
were carried out in a single-compartment three-electrode
glass cell with a 3 mm diameter glassy carbon electrode
(GCE) as the working electrode, a platinum wire as counter
electrode and Ag/AgCl electrode as reference (Model MF-
2052 RB-5B). All experiments were carried out at an
ambient temperature of 25  2 8C.
2.3. Procedures
For recording voltammograms, stock solutions of UA
(2 mM) and AA (2 mM) were prepared in doubly distilled
water. 2 mL of the stock solution was added to 2 mL of PBS
(0.1 M, pH 7.2). Osteryoung square-wave voltammetry
employed the following parameters: step height, 4 mV;
square-wave amplitude, 25 mV; frequency, 5 Hz; quiet time,
2s and sensitivity, 100 mA.
2.4. Preparation of Fullerene-C60-Modified Electrode
Prior to electrode modification, the GCE surface was
cleaned by polishing with alumina on microcloth pads
(BAS, USA) and then zinc oxide (Aldrich) to a mirror-like
finish. It was then dipped in a beaker containing 0.2 M H3
PO4 solution to remove the adhered powder, rinsed with
distilled water and dried in an oven at temperature 45 8C for
2 – 3 minutes. Stock solution of C60 was prepared by
dissolving in CH2Cl2 (150 mM). A known volume (40 mL)
of this solution was adsorbed onto the surface of the clean
and dried GCE using a microsyringe and dried in a stream of
hot air (45 8C). The most important point is the rapid
evaporation of dichloromethane providing a fine and even
dispersion of the deposited particles, thus, avoiding the
formation of large crystalline particles. The C60 film formed
was then reduced in 1 M KOH in the potential range 0.0 V to

1.5 V at 10 mV/s. PBS (50 mM) of pH 7.2 was taken in
another cell and the electrode surface was equilibrated in it
by cyclic scanning in the potential range of 550 mV to

50 mV (vs. Ag/AgCl) at a scan rate of 20 mV/s for
20 minutes under a nitrogen atmosphere [31]. The film
changed color upon reduction i.e. the initially brownish
yellow film appears to become brownish red. This very easy
technique allowed us to form a compact layer of reduced C60
on glassy carbon substrate. The fullerene-C60-modified
electrode was then ready for use.
3. Results and Discussion
3.1. Electrochemical Behavior of Uric Acid
It has been established that fullerene-C60 becomes conduc-
tive upon reduction and doping with alkali metal ions, the
most active surface being produced with Kþ. These reduced
fullerene-C60 films have been suggested to have special
G 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Voltammetric Determination of Uric Acid
Fig. 1. Linear sweep voltammograms for (a) 0.5 mM Uric acid at pH 7.2 at bare (- - -) and fullerene-C60-modified (–) glassy carbon
electrode (b) PBS (pH 7.2) at fullerene-C60-modified glassy carbon electrode ( ·· · ·). Sweep rate 100 mV/s.
Fig. 2. Dependence of peak current on Uric acid concentration
at pH 7.2.Sweep rate 100 mV/s. (~) for linear sweep and (&) for
OSWV.
electrocatalytic properties [29]. It is also likely that the more
conducting KxC60 species, which is formed on reduction,
helps in the electron transfer at the interface.
It was observed that modification of electrode surface by a
thin film of fullerene-C60 remarkably improves the reactivity
of GCE towards UA and AA oxidation. Figure 1 illustrates
the voltammograms of UA at a bare GCE and fullerene-C60-
modified electrode at pH 7.2. At the bare GCE, UA
oxidation takes place at 490 mV, which is in close agreement
with a previous report [32]. While at fullerene-C60-modified
electrode, the electrooxidation of UA occurred at 350 mV
with an enhancement in peak current. This shift of peak
potential (ca.140 mV) to less positive potentials with
increased peak current indicates that the exchange current
increases as a result of a lower overpotential for the reaction
in the presence of fullerene-C60.
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The dependence of peak current function (with correction
of background current) on the concentration of UA at
pH 7.2 was linear in the range 0.5 mM to 0.7 mM (Fig. 2)
having a correlation coefficient of 0.9904 and can be
expressed by the equation
ip (mA) ¼ 0.0215 C where C is in mM.
The detection limit (3s) is 0.2 mM. The error in the
calculation of detection limit was estimated to be 1.45%.
At concentrations greater than 0.7 mM of UA the peak
current became constant which indicates adsorption of UA
at the surface of the modified electrode at higher concen-
trations. Thus, by using fullerene-C60-modified GCE, UA
can be detected in a solution as low as 0.5 mM; however, it is
not possible to detect concentrations greater than 0.7 mM.
3.2. Oxidation of Ascorbic Acid
One of the major problems frequently encountered in the
electrochemical detection of biomolecules is the serious
interference due to ascorbic acid. The oxidation of AA in
linear sweep voltammetry at bare GCE occurred at 440 mV
and the peak was broad. In contrast, at the fullerene-C60-
modified electrode, the oxidation current increased greatly
and the peak potential shifted negatively to 200 mV. Thus,
an increment in the current response with a negative shift
(ca.240 mV) of the oxidation potential at the fullerene-C60-
modified electrode was obtained for AA. The enhanced
current responses and lowered oxidation peak potentials for
AA clearly indicates the strong catalytic action of fullerene-
C60-modified electrode. Since AA is more hydrophilic and
more soluble in water than UA, this may account for higher
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catalytic action of the modified electrode towards AA
oxidation.
3.3. Simultaneous Detection of Uric acid and Ascorbic
Acid
It was observed in the linear sweep voltammetric experi-
ments that oxidation potential of AA showed a large
negative shift (ca.240 mV) with an enhanced current
response at fullerene-C60-modified electrode. This observa-
tion is explored to detect simultaneously AA and UA using
fullerene-C60-modified electrode in the present studies.
Figure 3 shows the linear sweep voltammograms of AA
(1.0 mM) and UA (1.0 mM) both coexisting at pH 7.2, at the
bare and fullerene-C60-modified electrode. The bare elec-
trode could not separate the voltammetric signals of UA and
AA, and gave a single broad anodic peak at a potential
different from the oxidation potentials of AA and UA. Thus,
the peak potentials for UA and AA were indistinguishable
and it was impossible to deduce any conclusive information
from the broad voltammetric peak. On the other hand, the
presence of the fullerene-C60-film at the electrode surface
resolved the voltammetric response into two well-defined
peaks corresponding to almost the same potential as those
obtained for the individual oxidation of UA and AA. The
separation between the two peak potentials was  150 mV,
which was large enough for the simultaneous determination
of UA and AA in a mixture.
Fig. 3. Linear sweep voltammograms for solutions containing both uric acid (1.00 mM) and ascorbic acid (1.00 mM) at pH 7.2 at bare
(- - -) and fullerene-C60-modified (–) glassy carbon electrode. Sweep rate 100 mV/s.
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R. N. Goyal et al.
The response of UA and AA, coexisting in a solution, was
further studied by Osteryoung square-wave voltammetry
(OSWV), which has been claimed as a more sensitive
technique. Figure 4 shows the typical OSWVs obtained at
fullerene-C60-modified GCE at pH 7.2 for varying concen-
trations of UA keeping the concentration of AA constant.
Two well-defined oxidation peaks for UA and AA were
observed at 350 mV and 200 mV, respectively. Figure 2
represents the relation between the oxidation peak current
and the concentration of UA (0.5 mM to 0.8 mM) obtained
for OSWV technique. The peak current (ip) increased
linearly with increase in UA concentration (C) having a
correlation coefficient of 0.9958. The linear dependence can
be expressed by the equation
ip (mA) ¼ 0.0255 C
where C is in mM and a sensitivity of 0.0255 mA mM
1 is
observed. The detection limit (3s) in OSWV was found as
0.12 mM. The error in the calculation of detection limit is
1.65%. Ascorbic acid oxidized at  150 mV less positive
potential than UA and the voltammetric peak of AA
remained almost unchanged during the oxidation of UA.
Thus, using fullerene-C60-modified GCE it is possible to
determine UA in the presence of AA with detection limit as
low as 0.12 mM. This method is superior to other methods
reported earlier because no prior treatment of the electrode
is needed. Also cleaning of the electrode surface after each
run is not required which is a prerequisite to many methods
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Voltammetric Determination of Uric Acid 2221

surface after each run and studies are carried out at

physiological pH. The ease of preparation of the modified
electrode and its stability is also an advantage over the
reported methods.

3.4. Effect of Interferents

The voltammetric response to UA generally suffers from the
interference of ascorbic acid as well as xanthine, hypoxan-
thine and adenine existing in biological samples. Thus, prior
to the application of a fullerene-C60-modified electrode to
biological samples, effect of interferents on the response of
the specific analyte is checked. The specificity of the
fullerene-C60-modified electrode to 0.05 mM of UA in the
presence of xanthine, hypoxanthine and adenine was carried
out by carrying out OSWV experiments. The OSWV
response for the oxidation of UA (0.05 mM) was recorded
after addition of varying concentration of each interferent
(0.001 – 0.4 mM). The peak current response obtained in
case of adenine, hypoxanthine and xanthine is compiled in
Table 1. Xanthine and hypoxanthine affected the peak
current of UA at 6 and 8 fold-excess respectively. However,
adenine did not show any interference up to 8 fold-excess.
The interference effect of AA was investigated in further
detail. Table 2 shows the effect of varying concentrations of
Fig. 4. Osteryoung square-wave voltammograms of a mixture of AA on the OSWV response of 0.10 mM UA at the fullerene-
AA and UA at the fullerene-C60 modified glassy carbon electrode C60-modified electrode. The peak current for AA increased
at pH 7.2. Concentration of UA was changed keeping the
with increasing AA concentration. The peak potential and
concentration of AA constant, i.e., [AA] ¼ 0.10 mM, [UA]: a)
0.00, b) 0.10, c) 0.25, d) 0.30, e) 0.40, f) 0.50, g) 0.60, h) 0.70, i) peak current of UA remained almost constant in less than 4
0.80 mM. fold-excess of AA as shown in Figure 5. At and above 4-fold
excess of AA, a significant increase in peak current response
Table 1. Effect of interferents on the square-wave voltammetric with a shift in peak potential of UA to more positive
response of 0.05 mM UA at the fullerene-C60-modified glassy potential is observed which indicated that the four-fold
carbon electrode. excess of AA interferes in the determination of UA.
Interferents Concentration Change in
(mM ) peak current (mA )
Adenine 0.40 0.000 3.5. Determination of Uric Acid in Real Samples
Hypoxanthine 0.40 0.348
The linear sweep voltammetric method was applied to the
Xanthine 0.30 0.348
direct analysis of two human urine samples. To fit within the
linear range, the urine samples were diluted 500 times. The
dilution process greatly reduces matrix effect of real
[20 – 22, 33] where low detection limit (0.05 mM) is reported. samples. The results obtained are shown in Table 3. To
The regeneration of the electrode surface is dependent on ascertain the correctness of the results, the samples were
the potential scan and pH of the solution used. The present spiked with certain amounts of UA in about the same
method does not require renewal/regeneration of electrode concentration as found in the samples themselves. The

Table 2. Influence of AA addition on the OSWV response of 0.10 mM UA at the fullerene-C60-modified electrode.
Concentration of AA (mM ) Peak potential of UA (mV ) Peak current of UA (mA ) Peak current of AA (mA )
0.10 351.2 2.526 1.758
0.15 353.7 2.524 1.882
0.20 351.0 2.523 1.961
0.25 353.7 2.515 2.067
0.30 352.9 2.522 2.173
0.35 352.1 2.525 2.278
0.40 381.4 3.229 2.489

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Fig. 5. Osteryoung square-wave voltammograms of a mixture of AA and UA at the fullerene-C60-modified glassy carbon electrode at
pH 7.2. Concentration of AA was changed keeping the concentration of UA constant, i.e., [UA] ¼ 0.10 mM, [AA]: a) 0.10, b) 0.15, c)
0.20, d) 0.25, e) 0.30, f) 0.35, g) 0.40 mM.
Table 3. Determination of UA in urine samples with fullerene-
C60-modified electrode.
Urine1
Original value (mM ) 2.968
Spike (mM ) 3.000
Recovery (%) 95.74
Total value [a] (mM ) 1.484
[a] The total value was obtained by multiplying the detected value and the
dilution factor.
recovery rates of the spiked samples varied between 95.7%
and 101.2%.
3.6. Stability and Reproducibility of the Fullerene-C60-
Modified GCE
The procedure of electrode preparation being easy and rapid,
it is not so important for the electrode to be stable for a
prolonged time. However, the day-to-day stability of the
electrode was evaluated. As an indicator of its stability, the
performance of a particular fullerene-C60-modified electrode
over a period of 7 days with measurements of the oxidation of
peak current for 0.01 mM UA in 0.1 M phosphate buffer
solution (pH 7.2) was tracked each consecutive day. Our
linear sweep voltammetric experiment has demonstrated that
no apparent decrease in current responses occurred during
the first day, and 3% decrease was noted after 3 days. Since
the activity remains virtually constant during this period of
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R. N. Goyal et al.
time, the result indicates good stability of the electrode. The
response time of the electrode was very fast, hence, all the
Urine2 measurements were done immediately after the fullerene-
C60-modified electrode was immersed into the test samples.
2.381 The reproducibility of fullerene-C60-modified electrode
2.400
was also investigated. Repetitive measurements were car-
101.26
1.191 ried out in 0.01 mM UA solution. The results of five
successive measurements show a relative standard deviation
of 1.76%. This also demonstrates the intraday stability of
fullerene-C60-modified electrode. To check the reproduci-
bility of the results further, three different glassy carbon
electrodes of almost the same area were modified with same
volume of fullerene-C60 solution in dichloromethane and
their response towards the oxidation of 0.01 mM UA was
tested. The current obtained in five repeated measurements
of the three independent electrodes showed a relative
standard deviation of  1.43 %, confirming that the results
are reproducible. Thus, the electrode is very stable and good
reproducibility is observed.
4. Conclusions
The simultaneous oxidation of UA and AA poses a very
strong challenge because of their very similar oxidation
peak potentials. The present study demonstrates the use of
fullerene-C60-modified electrode, which successfully sepa-
rated the peaks of UA and AA present in the mixture,
indistinguishable at the bare GCE. A linear relationship
between UA concentration and current response was
obtained in the concentration range of 0.5 – 700 mM with
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Voltammetric Determination of Uric Acid
excellent reproducibility of the current. Since a better peak
current response is obtained, for UA and AA coexisting in a
solution, on application of OSWV method, it offers an
improved sensitivity in electrochemical signal and thus is
better used in the determination. The fullerene-C60-modi-
fied electrode shows a stable and reproducible response
without any influence of interferents commonly existing in
biological fluids. Hence, an excellent approach towards the
development of a novel fullerene-C60-modified electrode
has been presented, offering a possibility for extending this
technique to the routine analysis of UA in clinical samples.
The result shows that the method may be especially valuable
in sensor fabrication.
5. Acknowledgements
The authors (A. S.) and (N. B.) are thankful to the Council of
Scientific and Industrial Research, Delhi for awarding
Senior and Junior Research Fellowship (Grant No. 8810 –
13 – 414), respectively.
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