Colloids and Surfaces B: Biointerfaces 101 (2013) 91–96

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Colloids and Surfaces B: Biointerfaces

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Poly(Patton and Reeder’s reagent) modified carbon paste electrode for the
sensitive detection of acetaminophen in biological fluid and pharmaceutical
formulations
Tony Thomas a , Ronald J. Mascarenhas a,∗ , Frederika Cotta b , Kalyani Sri Guha b , B.E. Kumara Swamy c ,
Praveen Martis d , Zineb Mekhalif d
a
Electrochemistry Research Group, Department of Chemistry, St. Joseph’s College, Lalbagh Road, Bangalore 560 027, Karnataka, India
b
Department of Chemistry, St. Joseph’s College, Lalbagh Road, Bangalore 560 027, Karnataka, India
c
Department of Post Graduate Studies and Research in Industrial Chemistry, Jnana Sahyadri, Kuvempu University, Shankaraghatta 577 451, Shimoga, Karnataka, India
d
Laboratoire de Chimie et d’Electrochimie des Surface, Faculteı̌s Universitaires Notre-Dame de la Paix, 61 Rue de Bruxelles, B-5000 Namur, Belgium

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

Article history: An electrochemical sensor for sensitive detection of acetaminophen (AAP) was developed by electropoly-
Received 4 June 2012 merizing Patton and Reeder’s reagent at carbon paste electrode (CPE). Modification improves the redox
Accepted 14 June 2012 kinetics of AAP with increased current sensitivity. A similar modification at multiwall carbon nanotube
Available online 28 June 2012
(MWCNT) modified CPE did not result in an impressive charge transfer. Electrochemical impedance spec-
troscopy (EIS) of the bare and modified electrodes investigated imply a least charge transfer resistance
Keywords:
at Patton and Reeder’s reagent modified carbon paste electrode (MCPE/PR) as compared to bare CPE and
Acetaminophen
MWCNT modified electrode. Differential pulse voltammetric (DPV) study at MCPE/PR electrode did not
Electropolymerization
Patton and Reeder’s reagent
suffer any interference from its hydrolytic degradation product 4-aminophenol (4-AP) even in 1000-
Carbon paste electrode fold excess of its concentration and enables its detection simultaneously. A linear dynamic range of
Multiwall carbon nanotube 0.7–100 ␮M with detection limit (S/N = 3) of 0.53 ␮M was obtained for AAP. This modified electrode is
4-Aminophenol easy to prepare, cheap, and having good reproducibility and stability. The analytical performance of the
modified electrode is assessed by successfully applying it for the estimation of acetaminophen in different
pharmaceutical samples and spiked biological fluid.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction
Acetaminophen (Paracetamol, N-Acetyl-p-aminophenol or
AAP) is the most widely used analgesic and antipyretic drug
in the world. When it is used as an analgesic drug it reduces
mild–moderate pain associated with headache, toothache, back-
ache, migraine, muscular aches and postoperative pain [1,2]. As
an antipyretic agent it reduces fevers of viral and bacterial origin
[3]. AAP controls the pain and fever by inhibiting the production of
prostaglandins synthesis in central nervous system and sedating
hypothalamic heat regulating center [4]. This non-carcinogenic
drug is also useful in osteoarthritis therapy, protects hardening
of arteries and ovarian cancer [5]. Apart from being used as
antipyretic and analgesic drug, it is also useful in the treatment
of cold, cough and asthma. Hence, AAP is the major ingredient in
cold and influenza medications [6,7]. It is having actions similar
∗ Corresponding author. Tel.: +91 9448756584; fax: +91 8022245831.
E-mail address: ronaldmasc2311@yahoo.co.in (R.J. Mascarenhas).
to aspirin and is suitable alternative drug for patients who are
sensitive to aspirin [8]. AAP does not cause any addiction even
in people who use it frequently [9]. As a weak acid with pKa of
9.5, it rapidly gets absorbed and distributed soon after its oral
administration and the metabolites are easily excreted through
urine [10]. Generally, AAP does not exhibit any harmful side effects
at therapeutic doses. However, hypersensitivity or overdose of
acetaminophen produces toxic metabolites and lead to severe
kidney and liver damage [11]. Overdose can also cause skin
rashes and pancreatitis [12]. Moreover, the hydrolytic degradation
product of AAP, 4-aminophenol (4-AP) can cause teratogenic
effect and nephrotoxicity. Sometimes 4-AP observed in tablet
as synthetic intermediate [13,14]. 4-AP is known to interfere in
the electrochemical detection of AAP and therefore, not only its
detection is rendered important but also its interference in the
detection of AAP should be eliminated. Maintaining the exact
content of AAP in pharmaceuticals is of utmost importance and
has significant impact on public health. Therefore, it is necessary
to develop simple, fast, sensitive and accurate method for the
determination of AAP for biomedical, diagnostic and industrial
applications.

0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2012.06.020
92 T. Thomas et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 91–96
As discussed elsewhere [15] many methods have been utilized
for the determination of AAP: such as chromatography, spectropho-
tometry, titrimetry, spectrofluorometry, capillary electrophoresis,
electroanalytical methods and chemiluminescence. Most of these
methods involve tedious sample pretreatment prior to determi-
nation and are time consuming and expensive [16]. Being an
electro-active compound, different electroanalytical methods are
also used for assay of AAP in physiological and pharmaceutical
samples. In addition, electrochemical methods carry certain advan-
tages such as quick response, high sensitivity and selectivity, cost
effective, amenability to miniaturization, simplicity, low power
requirements and have wide dynamic range [17,18]. However,
at traditional working electrodes AAP exhibit poor voltammetric
response due to sluggish electrode kinetics [19]. Another prob-
lem commonly encountered at bare electrode is that they get
poisoned by several species and decrease sensitivity and repro-
ducibility. Hence, considerable efforts have been devoted to modify
the electrode for enhancing its voltammetric response and analyt-
ical performance.
Electropolymerization is a common method to modify CPE
because of its simplicity, strong adherence of polymer film to
electrode surface, broad potential window, chemical stability, abil-
ity to provide larger surface area by forming homogeneous film,
promote electron transfer rates and good sensitivity [20]. Elec-
tropolymerization of numerous molecules on different electrodes
has been used for the selective detection of AAP [21–28]. CPE
has been used as working electrode for many biosensor appli-
cations due to their simplicity in preparation, easy renewability
of surface, low background current, cheap and above all, its
biocompatibility [29]. Patton and Reeder’s reagent (2-hydroxy-1-
(2-hydroxy-4-sulfo-1-napthylazo)-3-naphthoic acid) is a blue dye
used in complexometric estimation of calcium.
In continuation of our effort to use CPE and modified CPE (MCPE)
for different applications [30–33], in the present work our objective
was to develop a modified electrode which is free from its interfer-
ing hydrolytic degradation product 4-AP and which can be used for
the estimation of AAP in pharmaceuticals and biological fluid like
blood serum. Owing to its many documented advantages and abil-
ity to display some unusual properties, the CNT electrodes are being
subject of intense research for the design of modified electrodes.
This prompted us to explore the possibilities of obtaining improved
voltammetric response using materials such as CNT. Hence, we
tried to fabricate the electrode using MWCNT and subjected it to
similar modification in order to verify whether it would demon-
strate unusual properties in this case too. The modification involves
electropolymerization of Patton and Reeder’s reagent both at CPE
and MWCNT modified CPE (MCPE/MWCNT). The electropolymer-
ization at CPE electrode shows good sensitivity and reproducibility
and allows accurate determination of AAP in presence of 4-AP.
2. Materials and methods
2.1. Reagents and chemicals
Acetaminophen (Micro Labs Ltd), CH3 COONa·3H2 O, CH3 COOH,
NaOH pellets, KH2 PO4 , H3 PO4 , silicon oil (all from Merck) Patton
and Reeder’s reagent and 4-aminophenol (Loba Chemie) were of
analytical grade and used as received. All aqueous solutions were
prepared with ultra pure water (>18.2 M cm) from Milli-Q Plus
system (Millipore). Stock solutions of AAP and 4-AP were prepared
in water and 0.01 M NaOH respectively. Acetate buffer solutions
(ABS) were prepared from CH3 COONa·3H2 O & CH3 COOH and pH
was adjusted using CH3 COOH or NaOH. Phosphate buffer solu-
tion was prepared from KH2 PO4 and pH was adjusted using H3 PO4
or NaOH. Graphite powder was obtained from Graphite India Ltd.
The thin MWCNTs obtained from Nanocyl SA, were synthesized by
decomposition of ethylene using the combustion chemical vapor
deposition method. The MWCNTs have an average diameter of
10 nm and length of several (0.1–10) micrometers.
2.2. Apparatus
All electrochemical experiments were performed using
ChemiLink model EA-201 Electro Analyzer. A conventional three
electrode system was used for all electrochemical experiments,
which comprise a bare or modified CPE or modified MWCNT as
working electrode, a platinum wire as auxiliary electrode and all
potentials were measured and applied using saturated calomel
electrode (SCE) as a reference electrode. Lugin capillary whose tip
was set approximately at a distance of 1 mm from the surface of
the working electrode–bare and modified CPE in order to mini-
mize error due to IR drop in the electrolyte. The electrochemical
experiments were performed in quiescent solution and voltam-
metric curves were recorded at room temperature (∼300 K). The
structural morphology of the electrodes was studied using a JEOL
JSM-848 scanning electron microscope (SEM). Electrochemical
impedance spectroscopy (EIS) was performed using VersaSTAT 3.
2.3. Generation of oxygen functionalities on MWCNTs
Since the oxygen functionalities on the surface of MWCNT
improve their electrochemical properties, the same were gener-
ated by treating them with a mixture of concentrated H2 SO4 and
HNO3 (molar ratio 3:1). In a typical experiment 75 ml of conc. H2 SO4
(97%) and 25 ml of conc. HNO3 (65%) were mixed and added to 1 g
of MWCNTs in a round-bottomed flask and heated under constant
agitation at 50 ◦ C for 8 h. After having allowed it to cool down to
room temperature an equal quantity of deionized water is added,
filtered and the residue was washed several times with deionized
water until neutral pH was attained. The residue was then filtered
and freeze-dried [34].
2.4. Preparation of bare and poly(Patton and Reeder’s reagent)
modified carbon paste electrode
After having optimized the ratio of graphite powder to binder,
CPE was prepared by thoroughly hand mixing the graphite powder
and silicone oil at a ratio 70:30 (w/w) in an agate mortar using a
pestle to obtain a homogeneous paste. A portion of the resulting
homogeneous paste was packed into the cave of the Teflon tube.
A copper wire fixed to a graphite rod and inserted into the Teflon
tube served to establish electrical contact with the external circuit.
Similarly, MWCNT modified CPE, (MCPE/MWCNT) was prepared by
bulk modification method since the modifier did not form a stable
film at MWCNT paste electrode. Extensively studied potassium fer-
ricyanide redox system was used to study and optimize the ratio
of MWCNT and graphite powder (details will be provided if asked)
till satisfactory results were obtained. The MWCNT bulk modified
CPE was prepared by mixing carbon paste with MWCNT at a ratio
of 95:5 (w/w).
Electropolymerization of Patton and Reeder’s reagent at CPE and
MCPE/MWCNT was optimized by carrying out the experiments in
0.1 M phosphate buffer solution (pH = 8.0) containing 1 × 10−5 M
Patton and Reeder’s reagent. Satisfactory polymer growth was
achieved by applying potential between −300 mV and 1000 mV at
a scan rate of 50 mV s−1 continuously for 10 cycles using cyclic
voltammetry as shown in Fig. S1 (supplementary information).
After polymerization, the electrode was thoroughly washed with
distilled water and used for further electrochemical analysis. The
electrochemical inertness of the prepared electrode was illustrated
by the absence of cyclic voltammetric peaks in 0.1 M acetate buffer
 T. Thomas et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 91–96
Fig. 1. SEM images of (a) CPE and (b) MCPE/PR.
of pH 5.0 (blank) in the potential window applied for the detection
of the analyte. The resulted electrodes were denoted by MCPE/PR
and MCPE/MWCNT/PR respectively. Surface morphology of both
CPE and MCPE/PR has been studied by recording SEM images as
shown in Fig. 1a and b. Smooth surface of MCPE/PR compared to
CPE indicates uniform coating of poly(Patton and Reeder’s reagent)
during its electropolymerization [35].
3. Results and discussion
3.1. Electropolymerization of Patton and Reeder’s reagent at CPE
and MCPE/MWCNT
Fig. S1 (supplementary information) shows the consecutive
cyclic voltammogram of 1 × 10−5 M Patton and Reeder’s reagent in
0.1 M phosphate buffer of pH 8.0 at CPE. A pair of redox peaks was
observed. A cathodic peak (Pc1) was observed at 220 mV and anodic
peak (Pa1) was observed at 270 mV. The mechanism of polymer-
ization of Patton and Reeder’s reagent has been already reported
[36]. The increase in peak currents on successive scans indicates
the growth of conducting polymeric film at CPE and MCPE/MWCNT
[37]. An approximate estimation of surface coverage of MCPE/PR is
calculated using the method described elsewhere [38] and it was
found to be 2.27 × 10−7 mol cm−2 .
3.2. Characterization by electrochemical impedance spectroscopy
EIS is an effective tool to investigate the electrical properties of
electrode/electrolyte interface. EIS spectra of four different elec-
trodes are shown in the form of Nyquist plot in Fig. 2. EIS data
Fig. 2. Nyquist plots of 5 × 10−4 M AAP in 0.1 M acetate buffer of pH 5.0 using dif-
ferent electrodes at their oxidation peak potential.
93
were recorded in the range of ac frequency varying from 0.1 Hz
to 100 kHz with an applied potential corresponding to Epa of AAP
in 0.1 M acetate buffer of pH 5.0. The charge transfer resistance
(Rct ) reflects the electron transfer kinetics of AAP at different elec-
trode/electrolyte interface. Rct values at different electrodes were
obtained by fitting the obtained impedance data to appropriate
circuit. Fig. S2 (supplementary information) represents the equiva-
lent circuit used for the fitting of impedance spectra. The Rct values
for CPE, MCPE/CNT, MCPE/PR and MCPE/MWCNT/PR are 4.36 × 104 ,
14.0 × 104 , 1.46 × 103 and 2.32 × 103  respectively. The presence
of polymer at electrode surface significantly reduces the value of
Rct . The least Rct value was obtained for MCPE/PR which makes this
particular electrode a good electrochemical sensor for AAP.
3.3. Electrochemical behavior of acetaminophen at MCPE/PR
The fabricated electrode was employed in order to investigate
the electrochemical behavior of AAP by cyclic voltammetric tech-
nique. Cyclic voltammograms obtained for 5 × 10−4 M AAP in 0.1 M
acetate buffer of pH 5.0 at CPE (dashed line) and MCPE/PR (solid
line) are shown in Fig. 3. At both electrodes, AAP shows quasi-
reversible behavior. Anodic peak potential (Epa) and cathodic peak
potential (Epc) of AAP at CPE were observed at 454.8 ± 4.6 mV
and 390.0 ± 1.8 mV while the same at MCPE/PR were observed at
462.5 ± 2.3 mV and 413.7 ± 3.7 mV. The small difference in anodic
and cathodic peak potentials (Ep) of AAP at MCPE/PR indicates
the better redox kinetics at MCPE/PR [39]. The anodic peak cur-
rent (Ipa) and cathodic peak current (Ipc) of AAP at CPE were
Fig. 3. Cyclic voltammograms of 5 × 10−4 M AAP in 0.1 M acetate buffer of pH 5.0
at CPE (dashed line), MCPE/MWCNT (short dashed line), MCPE/MWCNT/PR (dotted
line), MCPE/PR (solid line) and blank (dash dot line) at MCPE/PR. Scan rate: 50 mV s−1 .
94 T. Thomas et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 91–96
Fig. 4. (a) DPVs of various concentrations of AAP at MCPE/PR in 0.1 M acetate buffer
solution of pH 5.0. AAP concentration (in ␮M): 100, 70, 40, 10, 8, 6, 4, 2, 0.9 and 0.7
(a–j). Scan rate: 5 mV s−1 and pulse amplitude: 100 mV. (b) Calibration plot of Ipa
versus concentration of AAP.
Fig. 5. (a) DPVs of solutions containing 100 ␮M 4-AP and various AAP concentra-
tions; 100, 70, 40, 10, 7, 4, 2, 0.9 and 0.7 ␮M (a–i) at MCPE/PR. Scan rate: 5 mV s−1
and pulse amplitude: 100 mV. (b) Calibration plot of Ipa versus concentration of AAP
in binary mixture.
measured to be −17.84 ± 0.93 ␮A and 11.94 ± 1.14 ␮A whereas the
same at MCPE/PR were −35.86 ± 3.39 ␮A and −21.34 ± 2.09 ␮A. The
peak currents of AAP were doubled at modified electrode which is
attributed to the increase in active surface area of the electrode on
modification [40]. There is a possibility of hydrogen bond forma-
tion between polymer at electrode surface and hydroxyl group of
AAP molecule. This may also contribute to better charge transfer
kinetics of AAP at MCPE/PR.
When the concentration of AAP was varied from 1 × 10−4 M
to 4 × 10−3 M, Ipa increased linearly with a coefficient of
determination, R2 = 0.9944 at MCPE/PR as shown in Fig. S3 (sup-
plementary information). The corresponding linear regression
equation for Fig. 5 is Ipa (␮A) = −15.73–32.84 CAAP (mM). The effect
of scan rate on peak currents of AAP was investigated taking
5 × 10−4 M AAP in acetate buffer at pH 5.0. Ipa is proportional to
the scan rate in the range of 10–300 mV s−1 (Fig. S4, supplemen-
tary information) with a coefficient of determination, R2 = 0.9962
which suggests that redox reaction of AAP is adsorption controlled
[41]. The resulted linear regression equation for above plot is Ipa
(␮A) = −15.57–0.4345 ␯ (mV s−1 ). Epa shifts in positive direction
with increase in scan rate and concentration of AAP and corre-
sponding negative shift was observed for Epc. Scheme 1 represents
the electrochemical oxidation of AAP.
Scheme 1. Electrochemical oxidation of AAP.
Reproducibility of the modified electrode being an important
issue, the reproducibility at MCPE/PR was verified by fabricating six
similar electrodes and conducting cyclic voltammetric experiments
under similar conditions at pH 5.0 using 5 × 10−3 M solution of AAP.
The results obtained showed excellent reproducibility at MCPE/PR
with a relative standard deviation (RSD) of 2.72%.
3.4. Effect of MWCNT in CPE matrix for the electrochemical
behavior of AAP
The electrochemical behavior of AAP at MCPE/MWCNT/PR was
investigated using cyclic voltammetry. Fig. 3 shows the cyclic
voltammograms of 5 × 10−4 M AAP in 0.1 M acetate buffer of
pH 5.0 at MCPE/CNT (short dashed line) and MCPE/MWCNT/PR
(dotted line). AAP shows quasi reversible behavior at both the
electrodes. Epa and Epc of AAP at MCPE/CNT were observed at
474.0 ± 3.7 mV and 398.0 ± 3.3 mV while at MCPE/MWCNT/PR, the
same were observed at 490 ± 3.5 mV and 426 ± 3.8 mV respectively.
The Epa of AAP at MCPE/MWCNT and MCPE/MWCNT/PR is shifted
to more positive side compared to CPE and MCPE/PR. The differ-
ence in anodic and cathodic peak potentials (Ep) of AAP was also
increased to a large extent. This indicates the poor redox kinetics
of AAP at MCPE/MWCNT and MCPE/MWCNT/PR. The Ipa and Ipc of
AAP at MCPE/MWCNT/PR were −24.8 ± 2.3 ␮A and 15.9 ± 1.4 ␮A.
The same at MCPE/MWCNT were −22.2 ± 3.1 ␮A and 12.7 ± 1.9 ␮A.
Even though our experimental results have shown a slight
increase in response currents at MCPE/MWCNT/PR as compared
to CPE, the increase was not significant as compared to that at
MCPE/PR. In order to rule out thickness of polymeric film play-
ing any significant role in the voltammetric behavior of AAP at
the MWCNT modified electrode, we studied the voltammograms
by gradually varying the number of cycles in the electropoly-
merization process. The voltammetric response did not show any
significant improvement at MCPE/MWCNT/PR even after control-
ling the thickness of the polymer film. The less current sensitivity
may be associated with large capacitance current resulted, and the
sluggish kinetics of AAP at MCPE/MWCNT/PR could be due to the
resistance at the junction of the polymeric film and MCPE/MWCNT.
Probably due to this, the anodic peak potential of AAP shifts
toward more positive side. The results were not impressive at
MCPE/MWCNT/PR. This implies that a better support material for
electro polymerization of this film is CPE. On account of its bet-
ter charge transfer kinetics and cost effectiveness, MCPE/PR gets a
preference over MCPE/MWCNT/PR as a working electrode in our
study of the voltammetric behavior of AAP.
3.5. Effect of pH
It has been reported in literature that pH of the supporting
electrolyte plays a major role in the electrochemical oxidation of
AAP at different modified electrodes. The redox behavior of AAP
with pH provides significant information on mechanisms by which
it undergoes electrochemical reaction at MCPE/PR. Hence, influ-
ence of pH on Epa and Ipa of AAP has been investigated over
pH range 3.5–6.0 in 0.1 M acetate buffer solution using cyclic
voltammetry. The plot of Ipa vs. pH is shown in Fig. S5a (supplemen-
tary information). MCPE/PR shows maximum current sensitivity at
pH 5.0 with well defined redox peaks. Therefore, pH 5.0 has been
maintained throughout our studies. A negative shift in Epa was
observed with increase in pH as shown in Fig. S5b (supplemen-
tary information), which is described by linear regression equation
Epa (mV) = 766.6–59.03 pH, with regression coefficient 0.9857. The
slope of Epa vs. pH plot is −59.03 mV pH−1 . This indicates that equal
number of protons and electrons are involved in redox reaction of
AAP and is likely to be two as reported in literature [42].
 T. Thomas et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 91–96
Table 1
Comparison of MCPE/PR with other working electrodes.
Electrode Technique
GCE/f-MWCNT DPV
BDDE DPV
Nafion/ROPCME/GCE SWV
SPE/PEDOT DPV
Cu(II)-DPM/DDT/Au OSWV
C60 /GCE DPV
C–Ni/GCE DPV
CdPCNF/GC Amperometry
MCPE/PR DPV
3.6. Analytical characterization
Differential pulse voltammetry (DPV) is an effective method for
the trace level detection of AAP because of its certain advantages
like elimination of background current, good resolving power and
low detection limits [43]. Hence, DPV was employed to investigate
sensitivity of MCPE/PR toward AAP. DPVs of AAP were recorded
in 0.1 M acetate buffer solution of pH 5.0 at MCPE/PR after opti-
mizing the operational parameters. Fig. 4a shows the DPVs of
AAP at different concentrations. The peak current an increase lin-
early with concentration and the calibration curve is shown in
Fig. 4b. The linear dynamic range is from 0.7 ␮M to 100 ␮M with
linear regression equation Ipa (␮A) = −0.9052–1.011 CAAP (␮M)
and having R2 = 0.9941. The detection limit of AAP at MCPE/PR is
5.3 × 10−7 M.
3.7. Interference studies
The major interfering molecule that is present in AAP tablets is
4-AP and is a well known fact that this molecule is present either
as degradation product or as synthetic intermediate of AAP. The
presence of this molecule leads to inaccurate determination of AAP
content in its tablets. Since, the main objective of present study is to
estimate AAP content in different pharmaceutical products and bio-
logical fluid; we have examined the specificity of MCPE/PR toward
the AAP in presence of 4-AP. In order to rule out the possibility of
mutual interference of AAP and 4-AP, an experiment was carried
out in 0.1 M acetate buffer solution of pH 5.0 under the previously
optimized parameters. Two separate well defined differential
pulse voltammetric oxidation peaks were observed at 171 mV and
418 mV for 4-AP and AAP respectively with a peak separation of
247 mV. The separation of peak potentials observed at MCPE/PR
was large enough to enable the sensor for their simultaneous deter-
mination in a mixture containing AAP and 4-AP. In the experiments
followed, the concentration of AAP was changed while keeping
the concentration of 4-AP constant. The resulted DPVs are shown
in Fig. 5a. It is evident from Fig. 5a; that only peak current of AAP
changed while the peak current of 4-AP remain the same. The peak
potentials of these two molecules were observed to remain the
same throughout the experiments. Also, the peak potential of each
of the analyte in the mixture remains the same as when studied
independently indicating that the reactions of analytes at the elec-
trode surface are not affected by the presence of each other. Thus,
the oxidations of these molecules are completely independent to
each other at MCPE/PR. Fig. 5b shows the calibration curve of AAP in
binary mixture. Ipa holds a linear relationship with concentration
of AAP in the range of 100–10 ␮M and 8–0.7 ␮M with coefficient of
determinations 0.9936 and 0.9892. The linear regression equations
for the above plots are Ipa (␮A) = −9.003–0.2227 CAAP (␮M) and Ipa
(␮A) = 0.1608–0.9428 CAAP (␮M) respectively. The detection limit
(S/N = 3) of AAP in binary mixture was found to be 5.6 × 10−7 M.
The wide linear dynamic range and low detection limit make this
modified electrode a potential electrochemical sensor for AAP
95
Linear dynamic range (␮M) Detection limit (␮M) Reference
3–300 0.6 [44]
10–100 0.81 [45]
5–250 1.2 [46]
4–400 1.39 [47]
200–1500 120 [48]
50–1500 50 [49]
7.8–110 0.6 [50]
1.64–52.90 2.04 [51]
0.7–100 0.53 Present work
in pharmaceutical formulations and biological fluids. MCPE/PR
was compared with other modified electrodes which are already
reported in literature as shown in Table 1.
Even though a detection limit of 0.6 ␮M was achieved at GCE/f-
MWCNT [44], it requires pre-treatment and activation of the
electrode before use. Unlike BDDE [45], MCPE/PR is inexpensive
apart from giving better results. A nafion/ruthenium oxidepy-
rochloro chemically modified electrode, Nafion/ROPCME/GCE [46]
suffers from complex procedure of modification and thickness of
the nafion film found to control the performance of the electrode.
SPE had to be subjected to electrochemical pretreatment before
film deposition in the case of SPE/PEDOT [47] apart from a higher
over potential for oxidation and the electrode is not validated for its
analytical application for real sample analysis. Cu(II)–DPM/DDT/Au
[48] has a wide linear dynamic range though, MCPE/PR has a better
lower detection limit. At concentrations >4.0 mM of 4-AP, merging
of the peaks due to AAP and 4-AP has been reported at C60 /GCE [49]
on account of interference of 4-AP at this electrode. The reported
interference seems to be a serious setback at C60 /GCE whereas the
merging of peaks were not observed at MCPE/PR even in presence of
1000-folds excess of 4-AP. C–Ni/GCE [50] apart from pretreatment
of the electrode, it involves a longer time for its modification and a
higher over potential for oxidation. In the amperometric detection
of AAP at CdPCNF/GC [51] interference of 4-AP has been reported
at higher concentrations of 4-AP. Looking at it from all angles a less
expensive electrode with easier method of preparation is desired.
Apart from other advantages, the DPV study was more impressive
at MCPE/PR in terms of method of preparation and detection limit.
3.8. Analytical applications
In order to assess the analytical utility of the modified elec-
trode, it is employed in the determination of AAP in real samples by
taking human blood serum in addition to pharmaceutical samples
containing AAP. Different commercial pharmaceutical tablets con-
taining AAP were analyzed to estimate its content in them using
MCPE/PR. The tablets were finely powdered and then dissolved in
water. This solution was diluted with 0.1 M ABS of pH 5.0 so that
AAP concentration lies in the working range of calibration plot and
then DPV was recorded using MCPE/PR under the same optimized
operational parameters as before. From the peak current in cal-
ibration plot and keeping dilution factor into consideration, AAP
present in the tablet was determined. As shown in Table 2 the
obtained values are in good agreement with specified content on
tablet. The RSD shows high reproducibility of the result at MCPE/PR,
which was used for the quantitative analysis of AAP.
Human blood serum was used as biological sample. The serum
samples were 3 times diluted with 0.1 M acetate buffer of pH 5.0
without any pre-treatment. The standard addition method was
used for this purpose. The samples were spiked with known quan-
tities of AAP and its differential pulse voltammetric responses were
recorded at MCPE/PR. Good quantitative recoveries were obtained
as shown in Table 3. The unique advantage of this electrode is its
96 T. Thomas et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 91–96

Table 2 References
Determination of AAP in different pharmaceutical tablets using MCPE/PR.

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