Anal. Bioanal. Electrochem., Vol. 11, No.

8, 2019, 1070-1086

Analytical &
Bioanalytical
Electrochemistry
2019 by CEE
www.abechem.com

Full Paper
Stripping Square Wave Voltammetry for the
Determination of Uric Acid in Human Urine using
Iron(III) Doped Zeolite Graphite Powder Composite
Modified Glassy Carbon Electrode
Tadesse Gebregiyorgis,1,2,3 MeridTessema,2 Shimelis Admassie,2 Gebremariam
Birhanu1,4 and Ayenew Ashenef1,*
1
Department of Pharmaceutical Chemistry and Pharmacognosy , School of Pharmacy, College of
Health sciences, Addis Ababa University, P.o.Box. 1176, Addis Ababa, Ethiopia
2
Department of Chemistry, College of Natural Sciences, Addis Ababa University, P.o.Box. 1176, Addis
Ababa, Ethiopia
3
Department of Food Science and Nutrition, Ethiopian Public Health Institute, Addis Ababa, Ethiopia
4
Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences,
International Campus (TUMS-IC), Tehran, Iran
*Corresponding Author, Tel.:+251-118697101; Fax:+251-111558566
E-Mail: ayenew.ashenef@aau.edu.et

Received: 26 May 2018 / Received in revised form: 15 July 2019 /

Accepted: 31 July 2019 / Published online: 31 August 2019

Abstract- A novel stripping square wave voltammetric method has been developed for the
determination of uric acid (UA) in untreated human urine. In this study, we introduced a new
electrode based on iron(III) doped zeolite graphite powder composite (GPC) modified glassy
carbon electrode (GCE). Iron(III) loaded in zeolite matrix can increase anodic peak current by
adsorption of uric acid compound on the electrode surface. The results indicated that Fe+3
Y/GPC modified GCE facilitate the determination of uric acid with good sensitivity and
reproducibility. The optimum conditions selected for the determination of uric acid using
square wave voltammetry were: pulse amplitude 40 mV, Scan rate100 mV/s, accumulation
time 30 s, and accumulation potential +100 mV, pH of supporting electrolyte (pH)=7. Under
these optimum conditions, the oxidation peak current of UA increased linearly with
concentration in the range of 2.0×10−6 to 8.0×10-5 mol L−1 with linear equation, correlation
coefficient (R2) and detection limit (based on the formula LoD=3δ/m) of Ipa (µA)=0.1952–
0.3252 C (µM), 0.99903 and 2.32×10-7 mol L-1, respectively.

Keywords- Stripping square wave voltammetry, Uric acid, Zeolite, Modified glassy carbon
electrode, Urine, Graphite powder
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1071

1. INTRODUCTION

Zeolites are hydrated crystalline alumino-silicate minerals, natural or synthetic, with the
general formulation (Cn+)x[(AlO-2)nx(SiO2)y].m(H2O) and are widely used as adsorbents, ion
exchangers, or catalysts [1, 2]. The first report that utilises zeolites for electrochemical
purposes, dated 1939, has been described by Marshall and involves the potentiometric response
of zeolite-containing membrane electrodes to various mono- and divalent cations [3]. This
work and subsequent investigations by considered the inorganic membranes as polyelectrolytes
with mobile cations, allowing the characterization of cation activities similar to the way that
the glass membrane electrode is used for determining hydrogen ion activities (pH). Barrer and
James [4] in 1960, had worked on providing a more quantitative treatment of the zeolite
membrane potential and discussed its relation to the selectivity with respect to cation mixtures
in solution. Theses potentiometric applications exploit the solution-like ionic conduction of
zeolites [5,6].
Investigations concerning the zeolite-electrochemistry intersection began in the 1980s with
considerable research on ZMEs since 1988. Preliminary works performed in this initial mid
80’s period suggested most of the application types as well as electrode configurations on these
materials. Some milestones are: Pereira-Ramos et al. [7] prepared zeolite-supported metal
catalyst using electrochemical techniques. By means of a pressed composite electrode made
of graphite and silver-exchange mordenite particles, they were able to produce
electrochemically some clusters of metallic silver within the mordenite particles, and
crystallities or dendritic deposits on the graphite particles. One year later, Murray et al. [8]
grew electrogenerated coatings comprising zeolites; this was achieved by continuous potential
cycling at a rotated disk electrode (Pt or C) in an organic solvent containing fine zeolite
particles in suspension and an appropriate soluble electro-reactant. Concurrent and competing
with this approach it is the evaporative deposition of a zeolite-polystyrene composite layer on
solid electrode surfaces, which is carried out with suspensions of powdered zeolite in polymer
solutions, as first described by de Vismes et al. [9]. They have also incorporated some metal
porphyrins into the ZME and explored their electrocatalytic properties; in this case, the zeolite
is thought to enhance the chemical stability of the catalyst. Hernandez et al. [10] described the
first zeolite-modified carbon paste electrode, by mixing graphite particles with a natural zeolite
from the Canary Island and a mineral oil binder, they applied it to the voltammetric analysis of
HgII after chemical accumulation by ion exchange within the zeolite. Finally, the group of Shaw
[11] initiated what has become one of the largest challenges of ZME electrochemistry: (a) to
determine and, if possible, control the factors affecting the behavior of ZMEs; and (b) to
understand the origin of the electrochemical response of ZMEs by proposing mechanistic
models for charge transfer.
At the end of the 1980s, the two main methods for preparing ZMEs were zeolite overlayers
on solid electrodes and zeolite dispersions into a composite electrode material. Subsequent
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1072

efforts were often directed to optimizing these generic procedures to get longer durability and
better electrochemical perspectives, rather than evaluating totally new directions (e.g., in situ
grown zeolite films on conducting substrates like gold or mercury [12]. The main applications
predicted during this startup period, including electrocatalysis and several aspects of
electroanalysis and sensors, were largely developed during the last decade of the 20th century.
One should also mention that the field of energy storage that exploits the adsorbent properties
of zeolites was still growing in the 1980s, especially prompted by Coetzer [13], and the use of
zeolites as solid electrolytes in batteries remains common [14].
Uric acid (UA) is purine metabolism product which is excreted through kidney. Normal
levels UA in serum ranges from 240–520 mM and excretion through the urine is in the range
250-750 mg/L over a day for a healthy human being [15].
Abnormal levels of UA in urine and serum are associated with several diseases such as
gout, hyperuricemia, pneumonia and Lesch–Nyhan syndrome. Hence determining the amount
of uric acid in humans help in the diagnosis of many diseases associated with purine
metabolism abnormalities [16-18].
Uric acid is aromatic because of the purine functional group with a molecular formula
C5H4N4O3 and a molecular mass of 168 g/mol, it is white crystalline in appearance, slightly
soluble in water and has a density of 1.87 g/mL [17].
There are many recent reports dealing with rapid and simple analytical methods for UA
determination [15-19]. A range of techniques which include chromatography, [20-24]
electrophoresis, [25, 26] mass spectroscopy, [27] enzymatic and colorimetric methods [28, 30]
have been reported and widely used for qualitative and quantitative detection of uric acid.
However these techniques had many drawbacks requiring centralized laboratories,
extensive labor and many analytical resources, and lengthy time. But electrochemical methods
can be advantageous by being selective, sensitive, lower cost and being rapid. Moreover
electrochemical sensors and biosensors have a ready capacity for miniaturization, and direct
electronic readout [31-34].
Electrochemical detection of UA can be inexpensive, rapid and can be deployed in the
laboratory as well as in the field. For such purposes previously many electrochemical sensors
and biosensors, For example Nafion-coated carbon paste electrode [31], Poly(N,N-
dimethylaniline) film-coated GC electrode [32], Glassy carbon electrode coated with paste of
multi walled carbon nanotubes and ionic liquids [33], Cysteine modified glassy carbon
electrode [35], Carbon-coated iron nanoparticle modified by glassy carbon electrode [36],
Iron(III) doped zeolite modified with carbon paste electrode [37], poly(bromocresol purple)
modified glassy carbon electrode [38], tiron modified glass carbon electrode [39], nanozeolite
modified multiwall carbon nanotube [40] and more had been developed and deployed for the
determination of UA.
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1073

Spectrophotometeric method is the standard official method utilized for the determination
of uric acid in clinical laboratories. This method is based on the principle of uric acid oxidation
by uricase to produce allantoin and hydrogen peroxide. The hydrogen peroxide product again
reacts with 4-aminoantipyrine and 3, 5-dichloro-2-hydroxybenzene sulfonate using peroxidase
at catalyst to produce a colored product that is determined by colorimetry in its linearity range
[41].
In our present investigation, iron(III) doped zeolite/graphite powder composite
(Fe+3Y/GPC) modified GCE was prepared. The electrochemical properties of this material
were studied and deployed for the analysis of uric acid in urine.

2. MATERIALS AND METHODS

The following analytical grade chemicals and reagents was used in the study: Ferric
chloride (Research Chemicals Ltd, England), Zeolite Y, Hydrochloric acid (BDH, England),
Polystyrene, Dichloroethane (Riedel-de Haen, Germany), Tetrahydrofuran (BDH, England),
Uric acid (Research-lab Fine ChemInustries, India), Graphite powder, Sodium nitrate (Riedel-
de Haen, Germany), dipotassium hydrogen phosphate (Wagtech international Ltd, UK),
Potassium dihydrogen phosphate (Riedel-deHaen, Germany).

2.1. Supporting electrolyte Preparation

Electrolytes of phosphate buffers (KH2PO4 –K2HPO4) in the pH range 5-9 were prepared
from 0.1 M KH2PO4 and 0.1 M K2HPO4 in distilled water. The pHs were adjusted by adding
drops of concentrated H3PO4 and KOH. Experiments were carried out at room temperature.
Deionized water was employed to prepare the solutions [31].

2.2. Electrode preparation

Four types of electrodes were used as working electrodes. These are bare GCE, GCE
modified with graphite powder, GCE modified with un-doped zeolite and graphite powder
composite, GCE modified with Fe3+ doped-zeolite and graphite powder composite. The last
three electrodes contain polished GCE, graphite powder, di-chloroethane, tetrahydrofuran and
polystyrene in common.
The iron(III) doped zeolite was prepared per the previous method [42]. Briefly, 1 g of
zeolite was lightly ground and immersed in 250 mL of 0.01 M FeCl3 solution. It was stirred for
48 h. The Fe3+ doped zeolite was carefully washed by HCl solution (pH of 2.0) to remove
occluded material and surface-adherent salt. Then, it was re-washed with distilled water to
remove chloride ion. At last the material prepared was dried at room temperature.
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1074

The preparation of the iron(III) doped zeolite/ graphite powder composite modified GCE
involves lightly grinding a mixture of 50 mg of the iron(III) doped zeolite and an equal amount
of graphite powder. Then the composite was dispersed in a mixture of 0.2 mL of
tetrahydrofuran, 0.3 mL of dichloroethane and 10 mg of polystyrene. It was sonicated for 30
minutes after through hand-mixing. 10 µL of the mixture was casted on the surface of a
well-polished GCE. It was dried at room temperature for about 30 min. Similarly, the other
modified electrodes were made following the same procedure but omitting the iron(III)
exchanging step or zeolite addition step. After this process, the modified electrode was
immersed in pH 7.0 PBS until use.
To compare the responses of bare GCE, GCE modified with undoped zeolite/ Graphite
powder composite, GCE modified with iron(III) doped zeolite/Graphite powder composite and
GCE modified with graphite powder for 1 mM uric acid in 0.5 mol L-1 NaNO3 (pH 7.0 PBS)
have been studied.
In order to evaluate the analytical performance of the method a series of concentration of
uric acid ranging from 2 to 800 µM were prepared from a 1 mM uric acid stock solution

3. RESULT AND DISCUSSION

3.1. Electrochemical characterization of Fe3+ Y/GPC modified GCE

As shown above Fig. 1A depicts the effect of potential scan rate on the CV of Fe3+ Y/GPC
modified GCE in the range of 20-400 mV s-1 scan rate. A pair of redox peaks was obtained
which are characteristic of Fe3+/Fe2+ indicating the incorporation of Fe3+ in to the cavities of
zeolite [35]. The relationship between oxidative and reductive peak currents with scan rates
was found linear as shown below.

Ipa(µA)=31.2375+0.99295ν (mV s-1) (R2=0.9999) and

Ipc(µA)=13.99468+0.66934ν (mV s-1) (R2=0.9947), respectively

This linear relation of the peak current and potential scan rate indicates redox processes for
surface-confined species [36]. Thus, we can conclude that the iron(III) is undergoing electrode
surface redox processes.
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1075

350
300
250
200
150
100
50

Current / A
0 20
-50
-100
-150
-200
-250 400
-300
-350
-400
-450
-500
-0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Potential / V

450
Ipa
400

350

300
ipc
250
Ip / A

200

150

100

50

0
0 50 100 150 200 250 300 350 400 450

Scan rate (mV s-1)

Fig. 1. (A) Cyclic voltammogram of stabilized Fe3+ Y/GPC modified GCE in 0.5 mol L-1
NaNO3 (PBS of pH 7) at different scan rates (from inner to outer waves: 20, 40, 60, 80, 100,
200 and 400 mV s-1); (B) Peak current (Ip) vs. Scan rate (ν)

3.2. Oxidation of uric acid electrochemically at different electrodes

The responses of the modified electrodes for 1 mM UA in 0.5 mol L-1 NaNO3 (pH 7.0 PBS)
have been investigated.
Fig. 2 above depicts the cyclic voltammograms of 1 mM of UA in o.5 M NaNO3 (pH 7.0
PBS) at different electrodes. UA undergoes an irreversible oxidation at both the bare and
graphite powder modified GCEs. The current response and highest potential of UA at the bare
(Fig. 2A(c)) and graphite powder modified GCE (GPMGCE) (Fig. 2A(d)) are comparable. A
fourfold increment of the highest current and a potential change of about 300 mV to a lower
positive potential (Fig. 2A(f)) have been observed at the Fe3+Y/GPC modified GCE indicating
the catalytic power of the Fe3+doped zeolite modified GCE which lowers the over potential
thereby assisting the electron exchanging process between the electrode surface and the
analyte.
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1076

30

20

a-b
10

Current / A
c
-10
d
-20

-30
f
-40

-50 e

-60

-400 -200 0 200 400 600 800 1000 1200 1400

Potential (mV)

(A)

40

20

0 c
Current / A

-20

-40 b
d a

-60

-80
-400 -200 0 200 400 600 800 1000 1200 1400

Potential (mV)

(B)
Fig. 2. (A) Cyclic voltammograms of (A) bare GCE (a and c), GPMGCE (b and d) and Fe3+
Y/GPC modified GCE (e and f) in 0.5 mol L-1 NaNO3 (pH 7.0 PBS) containing no UA (a, b
and e) and in 0.5 mol L-1 NaNO3 (pH 7.0 PBS) containing 1 mM UA (c, d and f); (B) Cyclic
voltammograms of Undoped zeolite/GPC modified GCE (a and b) and Fe3+ Y/GPC modified
GCE (c and d) in 0.5 M NaNO3 (pH 7.0 PBS) in the absence (a and c) presence (b and d) of 1
mM UA scan rate: 100 mV s-1

Comparison has also been made between the current responses of the undoped and Fe3+
doped zeolite by recording their cyclic voltammograms (Fig. 2B) for 1 mM UA in 0.5 M
NaNO3 (pH 7.0 PBS). It can be concluded that the potential shift and current enhancement
observed (Fig. 2A) is not due to the zeolite but due to the presence of the Fe3+ in the cavities
of zeolite.
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1077

3.3. Effect of scan rate on the peak potential and peak current of UA at Fe3+ Y/GPC
modified GCE

The dependence of the peak current of UA on the potential sweep rate has been
investigated. The oxidation current for 1 mM UA increased linearly with potential scan rate (ν)
in the range 20 to 1000 mV s-1 (Fig. 3B) with R2 of 0.9980, telling an electron transfer
controlled reaction process. The shift of the peak potential to a more positive direction
proportionally with potential scan rate observed from Fig. 3A above is an affirmation for the
inverse relation of the oxidation reaction of UA at the modified GCE [35].

200
150
100
50
0 20 mV/s
-50
Current / A

-100
-150
-200
-250
-300
-350
-400 1000 mV/s
-450
-500

-400 -200 0 200 400 600 800

Potential (mV)

(A)

500

400

300
Current (uA)

200 -1
Ipa () =  + (mV s )
2
R = 0.9980

100

0
0 200 400 600 800 1000

Scan rate (mVs-1)

(B)
Fig. 3. (A) Cyclic voltammograms of 1 mM UA in 0.5 M NaNO3 (pH 7.0 PBS) at different
scan rates (20, 40, 60, 80, 100, 200, 400, 600, 800 and 1000 mV/s); (B) Graph of peak current
versus potential scan rate
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1078

(A)

80

75
Oxidative peak current /A

70

65

60

55

50

5 6 7 8 9 10

pH

(B)

500

480

460
Epa (mV) = 702.6 - 42.6pH and
2
440 R = 0.99069

420
Epa / mV

400

380

360

340

320

300
5 6 7 8 9

pH

(C)
Fig. 4. (A) Cyclic voltammograms of 1 mM UA in 0.5 M NaNO3 (a to e): pH=5, pH=6, pH=7,
pH=8, pH=9 at Fe3+ Y/GPC modified GCE, scan rate: 100 mV s-1; (B) Variation of the peak
current of 1 mM UA with the pH of the supporting electrolyte; (C) Plot of peak potential versus
the pH of the supporting electrolyte
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1079

3.4. Effect of the pH change of the supporting electrolyte

The influence of the pH of the supporting electrolyte on the peak potential and peak current
for 1 mM UA at the Fe3+ Y/GPC modified GCE has been investigated. As seen in Fig. 4A both
the peak current and peak potential changed with variation with pH of the solution.
As shown above in Fig. 4B the peak current increased significantly with pH till its peak
value at pH of 7.0 and then decreased above pH 7.0. The trend could be explained taking two
consequences of increasing the pH in to account. Firstly; with raising the pH of the solution,
the uric acid (pKa=5.8) becomes more negative and exerts stronger attractive force towards the
Fe3+ doped zeolite surface of the GCE resulting a rise in peak current with pH [36]. On the
contrary, the extent the surface of the modified GCE is rich in the Fe3+ ions which catalyze the
oxidation of UA gets lower. This is in agreement with the decreasing trend of the peak current
with increasing the solution pH above 7.0, the optimal physiological pH taken.
There was also linear shift of the peak potential to a more negative direction with increasing
the pH of the supporting electrolyte from pH 5.0 to pH 9.0 with an equation of
Epa (mV)=702.6–42.6 pH and R2=0.99069 respectively showing the involvement of protons
in the oxidation of UA. A slope of 42.6 mV is a confirmatory of equal number of electrons and
protons were participated in the oxidation of UA at Fe3+Y /GPC modified GCE [36]. As a result
of this, the most possible mechanism of reaction for the oxidation of UA at Fe3+ Y/GPC
modified GCE is shown below.

2Fe3+ + 2e- 2Fe2+

O H
H -
O N
N -2e NH2
HN O +
O 2H2O -2H+ CO2
O N N
O N N H H
H H

3.5. Square wave voltammetry of UA

The SW voltammograms of 1 mM UA in 0.5 M NaNO3 (pH 7.0 PBS) at bare GCE and the
iron(III) doped-zeolite graphite powder composite modified GCE are shown in Fig. 5. These
voltammograms showed clearly the catalytic role of the modifier by lowering the over potential
and facilitating the electron exchange process at the surface. Hence, square wave voltammetry
method can be used for UA concentration determination in real samples.
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1080

a
0

Anodic current / A
-20

-40
b

-60

-80

-400 -200 0 200 400 600 800 1000 1200 1400

Potential (mV)

Fig. 5. Square wave voltammograms of 1 mM UA in 0.5 M NaNO3 (pH 7.0 PBS) at bare and
Fe3+ Y/GPC modified GCE

3.6. Effect of accumulation potential (Eacc)

The effect of accumulation potential (Eacc) on the SWV peak current at various
accumulation potentials between -50 mV and 200 mV was studied. The maximum peak current
was obtained at an accumulation potential (Eacc) of +100 mV as shown in Fig. 6. Therefore, the
accumulation potential (Eacc) of +100 mV was chosen as the maximum potential.

150

145

140
Current / A

135

130

125

120
-50 0 50 100 150 200

Potential / mV

Fig. 6. Effect of deposition potential on the SWV peak current for 1 mM UA in 0.5 M NaNO3
(pH 7.0 PBS) at Fe3+ Y/GPC modified GCE. Amplitude: 20 mV; deposition time: 10 s

3.7. Impact of accumulation time (tacc)

The influence of tacc on peak current was carried out using 1 mM UA. The responses
obtained are shown in Fig. 7. The change of adsorption time between 0 and 40 s at an adsorption
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1081

potential of 100 mV, indicated that the peak current rose directly with the accumulation time
(tacc) up to 30 s and then leveled off.
The increase of peak current directly with accumulation time (tacc) indicated that UA can
be accumulated at the zeolite cavities on the surface of the GCE. The leveling off in peak
current after 30 s indicates the saturation of the cavities. So the tacc of 30 s was selected as the
best accumulation time (tacc) for further studies.

140

130

120
Ipa / A

110

100

90
0 10 20 30 40

Deposition time / s

Fig. 7. Effect of deposition time on the oxidative peak current of 1 mM UA in 0.5 M NaNO3
(pH 7.0 PBS). Deposition potential: 100 mV

3.8. Analytical performance of the method

The optimum conditions selected for the analysis of uric acid using square wave
voltammetry were: pulse amplitude 40 mV, Scan rate100 mV/s, accumulation time 30 s, and
accumulation potential +100 mV. Under these optimum conditions, the oxidation peak current
of UA raised directly with concentration in the range of 2.0×10−6 to 8.0×10-5 mol L−1 with an
equation, R2 and detection limit (based on the formula LOD=3δ/m) of Ipa (µA)=0.1952-
0.3252C (µM), 0.99903 and 2.32×10-7 mol L-1 respectively.
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1082

(A)

Ipa (A) = 0.1952 - 0.3252C(M)

-5 2
R = 0.99903
Peak current / A

-10

-15

-20

-25

-30
0 20 40 60 80

UA concentartion / M

(B)
Fig. 8. (A) Square wave voltammograms of Fe3+ Y/GPC modified GCE for different
concentrations (2, 10, 30, 40, 50, 60, 80, 100, 200, 400, 600 and 800µM ) of UA in 0.5 M
NaNO3 (in pH 7.0 PBS) at scan rate, deposition time, deposition potential and pulse amplitude
of 100 mV s-1, 30 s, 100 mV and 40 mV; (B) graph of peak current versus concentration

Table 1. Comparison of the proposed electrode with other modified electrodes

Electrode Modifier LR (mol L-1)a LOD (mol L-1)b Ref.

Carbon paste Poly(N,N-dimethylaniline) 1.25-68.75 µ 1.2510-6 [32]
Glassy carbon Nafion 0-50 µ 2.510-7 [34]
Glassy carbon Cysteine 0.7-300 µ 3.710-7 [35]
Glassy carbon Carbon-coated 0.5-20 µ 1.510-7 [36]
Carbon paste Fe3+ doped zeolite 0.3-700 µ 0.810-7 [37]
Glassy carbon Poly(bromocresol purple) 0.5-120 µ 210-7 [38]
Glassy carbon Fe3+ doped zeolite/graphite 2-80 µ 2.3210-7 This work
a
Linear range, bLimit of detection
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1083

3.9. Analytical applications

For the purpose of evaluating the validity of the method proposed, the Fe3+ doped
zeolite/graphite powder composite modified GCE was used for the direct determination of uric
acid in urine samples collected from three different volunteers. The method was applied to
check concentrations of standard uric acid spiked in to urine solutions. The collected urine
samples were divided in to two parts; one part for analysis after suction filtration with 0.45 µm
filter paper and the other for direct analysis without filtration.
To fit into the linear range, the whole urine samples (filtered and unfiltered) were diluted
200 times [25] with 0.5 M NaNO3 (pH 7.0 PBS) to reduce the matrix effect of the human urine
samples. Thus, determination of the UA from three human urine samples were performed with
the diluted samples while recovery analysis of the standards (30, 50 and 70 µM) were from
solutions of the standard UA and the diluted human urine samples. To know the accuracy of
the results, three measurements have been carried for each sample. The results found are given
in Table 2.

Table 2. Direct determination of UA in three filtered and unfiltered diluted human urine
samples

Urine sample Unfiltered urine Filtered

code Ipa* Conc. Standard Ipa* Conc. Standard
(µA) (µM) Deviation (µA) (µM) deviation
1 2.420 8.0418 0.305 1.817 6.1866 0.193
2 4.987 15.935 0.021 4.723 15.124 0.123
3 6.960 22.002 0.546 6.517 20.640 0.280
*Mean anodic peak current of Triplicate measurements

As shown in Table 2, the detected concentrations of uric acid in the three unfiltered and
filtered human urine samples are all in the uric acid concentration range for a normal person
which is 1.5-4.5 mM [41] with standard deviations less than unity. The lower concentration of
UA in the filtrated sample labeled code 1 could be ascribed to the possible mass loss during
the filtration in the raw urine. The similarity between the responses for the UA concentrations
detected in the filtrated and unfiltered urine sample indicated the selectivity of the method
developed. These results suggested the modified electrode for the direct analysis of UA in
human urine without any treatment as in most of the conventional techniques.
Compared to the official method of determining uric acid, the proposed modified electrode
does not need prior treatment of sample.
 Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1084

Table 3. Recovery of standard UA from three filtered and unfiltered human urine samples
Urine sample

Unfiltered urine sample Filtered urine sample

UA UA
SD**
present* added found SD** Recovery present* added Found Recovery
(µM) (µM) (µM) (%) (µM) (µM) (µM) (%)
30.0 38.198 0.500 100.52 30.0 38.218 0.210 106.77

1 8.0418 50.0 53.532 0.480 90.98 6.1866 50.0 53.665 0.707 94.96

70.0 75.990 0.414 97.07 70.0 72.033 0.916 94.07

30.0 44.276 0.534 94.47 30.0 45.362 0.430 100.79

2 15.935 50.0 64.356 0.386 96.84 15.124 50.0 63.515 0.340 96.78
70.0 82.724 0.448 95.41 70.0 81.904 0.207 95.40
30.0 50.416 0.282 94.71 30.0 50.723 0.295 100.28

3 22.002 50.0 68.979 0.140 93.95 20.640 50.0 69.624 0.889 97.97

70.0 86.506 0.668 92.15 70.0 87.716 0.210 95.82

* Mean concentration of UA detected in the 200 times diluted human urine as per the results in table 1
** Standard deviation of triplicate measurements

The developed method can be used for the analysis of uric acid spiked with human urine.
The determination results and recoveries are given in Table 3. It can be seen that recovery
varied from 90.98% to 106.77%, showing that the method developed is appropriate for analysis
of uric acid in human urine. The system exhibited excellent reproducibility, with relative
standard deviation less than 0.3, making it suitable for routine analysis.

4. CONCLUSION
In this study, a new electrode based on iron(III) doped zeolite graphite powder composite
modified glassy carbon electrode was developed. Iron(III) loaded in zeolite matrix can rose
anodic peak current by adsorption of uric acid on the electrode surface. The finding here
showed that, Fe3+ Y/GPC modified GCE enhance uric acid determination of with good
sensitivity and reproducibility. The electrode here can be used in analysis of uric acid in human
urine without any sample pretreatment and has no any time consuming extraction procedures
before the analysis, and is with satisfactory recovery. The fabrication protocol, reproducibility,
wide linear dynamic range, low detection limit, high sensitivity, makes this electrode
appropriate for routine analysis.
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1085

REFERENCES

[1] N. D. Zaharri, N. Othman, and Z. A. Mohad Ishak, Advances in Mater. Sci. Eng. (2013)
1.
[2] L. M. Muresan, Pure Appl. Chem. 83 (2011) 325.
[3] S. K. George, M. T. Dipu, U. R. Mehra, and P. Sing, J. Chromatgr. B 832 (2006) 134.
[4] R. M. Barrer, and S. D. James, J. Phys. Chem. 64 (1960) 417.
[5] D. C. Freeman , Us Patent Number 3, 186 (1965) 875, Br Patent No. 999 (1965) 948.
[6] N. Petranovic, and M. V. Susic, Zeolites 3 (1983) 271.
[7] J. P. Pereira-Ramos, R. Messina, and J. Perichon, J. Electroanal. Chem. 146 (1983) 157.
[8] C. G. Murry, R. J. Nowak, and D. R. Rolison, J. Electroanal. Chem. 164 (1984) 205.
[9] B. de Vismes, F. Bedioui, J. Devynck, and C. Bied-Charreton, J. Electroanal. Chem. 187
(1985) 197.
[10] P. Hernandez, E. Alda, L. Hernandez, and Fresenius, J. Anal. Chem. 327 (1987) 676.
[11] H. A. Gemborys, and B. R. Shaw, J. Electroanal. Chem. 208 (1986) 95.
[12] S. Mintova, V. Valtchev, V. Engstrom, and B. J. Schoeman, J. Sterte. Micropor. Mater.
[13] J. Coetzer, ElectrochimActa. 23 (1978) 787.
[14] K. Mori, K. Kagohara, and H. Yamashita, J. Phys. Chem. 112 (2008) 2593.
[15] M.D. Baker, C. Senaratne, Anal Chem. 64 (1992) 697.
[16] R. T. Kachoosangi, C.E. Banks, R.G. Compton, Electroanal. 18 (2006) 741.
[17] R.N. Adams, Electrochemistry at Solid Electrodes, New York: Marcel Dekker (1969).
[18] K. Kalcher, J.M. Kauffmann, J. Wang, I. Svancara, K. Vytras, C. Neuhold, Z. Yang,
Electroanl. 7 (1995) 5.
[19] L. Fernandez, and H. Carrero, Electrochim. Acta 50 (2005) 1233.
[20] N. Wijemanne, P. Soyse, and S. Wijesundara, and H. Perera, IJAC (2018) 1.
[21] C. Kevin, and H. Church, J. Anal, Bioanal. Sep. Tech. 2 (2017) 1.
[22] X. Dong, IJPSR 8 (2017) 925.
[23] W. Shaoshi, Z. Zhang, X. Chen, J. Liu, H. Yu, L. Han, L. Jin, Y, Zhang, and T. Wang,
Acta Chromatogr. (2018) 1.
[24] J. Markelj, T. Zupancic, and B. Pihlar, Acta Chim. Slov. 63 (2016) 8.
[25] H. L. Lee, and S. C. Chen, Talanta, 64 (2004) 750.
[26] Y. Guan, T. Wu, and J. Ye, J. Chromatgr. B 821 (2005) 229.
[27] J. PerellPo, P. Sanchis, F. Grases, J. Chromatgr. B 824 (2005) 175.
[28] M. J. Stankov, P. Djurdjevic, and D. Stankov, J. Serb. Chem. Soc. 68 (2003) 691.
[29] R. K. Bera, A. Anoop, and C. R. Raj., Chem. Commun. 47 (2011) 11498.
[30] I. I. Toshihiko, S. Nobuhiko, Biosens. Bioelectron. 10 (1995) 435.
[31] W. Sroysee, S. Chairam, M. Amatatongchai, P. Jarujamrus, S. Tamuang, S. Pimmongkol,
L. Chaicharoenwimolkul, and E. Somsook, J. Saudi Chem. Soci. 22 (2018) 173.
[32] P. R. Roy, T. Okajima, and T. Ohsaka, J. Electroanal. Chem. 561 (2004) 75.
Anal. Bioanal. Electrochem., Vol. 11, No. 8, 2019, 1070-1086 1086

[33] Q. Yan, F. Zhao, G. Li, and B. Zeng, Electroanalysis 11 (2006) 1075.

[34] Z. J. M. Zen, C. T. Hsu, Talanta. 46 (1998) 1363.
[35] Z. Yan, J. R. Zhang, H. Q. Fang, Anal. Lett. 32 (1999) 223.
[36] S. Wang, Qiao Xu, Guodong Liu, Electroanal. 20 (2008) 1116.
[37] A. Babaei, M. Zendehdel, B. Khalizadeh, A. Taheri, Coll. Surf. B: Biointerfaces. 66
(2008) 226.
[38] Y. Wang, Li-li Tong, Sens. Act. B, 150 (2010) 43.
[39] M. Noroozifara,, M. Khorasani -Motlaghb, R. Akbaria and M. B. Parizia, Anal. Bioanal.
Chem. Res. 1 (2014) 62.
[40] A. A. Ensafi, M. Taei, T. Khayamian, Int. J. Electrochem. Sci. 5 (2010) 116.
[41] R. W. E. Watts, Ann. Clin. Biochem. 11 (1974) 103.
[42] P. Proti, Introduction to Modern voltammetric and polarographic Analysis Techniques,
Amel electrochemistry, 4th edition. New York (2001).

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