Sensors and Actuators B 237 (2016) 196–205

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Sensors and Actuators B: Chemical

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

Voltammetric determination of pyrazinamide at graphene-zinc oxide

nanocomposite modified carbon paste electrode employing
differential pulse voltammetry
Pramod K. Kalambate, Chaitali R. Rawool, Ashwini K. Srivastava ∗
Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (East), Mumbai-400 098, India

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

Article history: Pyrazinamide (PZN) also called as Pyrazinecarboxamide is a widely used drug for the treatment of
Received 14 January 2016 Tuberculosis. In the present work, we proposed a sensitive method for determination of PZN using
Received in revised form 29 May 2016 Graphene–Zinc oxide-Carbon paste electrode (GNS-ZnO-CPE). The surface characterization of elec-
Accepted 2 June 2016
trode materials was done by using X-Ray diffraction, Scanning electron microscopy, Energy dispersive
Available online 3 June 2016
X-ray spectroscopy and transmission electron microscopy. The electrocatalytic response of PZN at
GNS-ZnO-CPE was measured using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and
Keywords:
electrochemical impedance spectroscopy (EIS). In addition, electrochemical impedance studies revealed
Pyrazinamide
Graphene
that the smaller Rct value was observed at GNS-ZnO-CPE as compared to that of CPE, which authenticates
Zinc oxide nanoparticles its good conductivity. Under the optimized conditions, Ip (␮A) was proportional to PZN concentration in
Differential pulse voltammetry the range of 1.5 × 10−7 to 4.0 × 10−4 M with a detection limit of 4.31 × 10−8 M. The proposed electrochem-
Pharmaceutical samples ical sensor showed excellent recovery in pharmaceutical formulations, urine and blood serum samples
which revealed the promising practicality of sensor for PZN detection.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction rarely porphyria and fever. Therefore, development of sensitive

Pyrazinamide (PZN) also called as pyrazinecarboxamide is a
first-line oral drug used to treat tuberculosis (TB). The drug is
mainly used for treatment of tuberculosis caused by mychobac-
terium tuberculosis [1–3]. Generally, PZN is used in combination
with other anti-TB drugs viz; isoniazide, rifampicin, and etham-
butol hydrochloride. It has been used for treatment of TB since
1952 and the exact mechanism of action is not well known. PZN
is converted to pyrazinoic acid, the active form of the drug, by
the pyrazinamidase enzyme (PZase) produced by mychobacterium
tuberculosis. It is assumed that pyrazinoic acid has ability to lower
the pH to an acidic medium that the organism will not toler-
ate [4,5]. The use of PZN results in shortening the duration of
therapy from nine to six months. However, continuous dosage of
PZN leads to various side effects. The most serious side effect is
hepatotoxicity. The other side effects include nausea, vomiting,
exanthema, anorexia, sideroblastic, liver injury, anemia, skin rash,
urticaria, pruritus, dysuria, intestinal nephritis, malaise, dark urine,
∗ Corresponding author.
E-mail addresses: aksrivastava@chem.mu.ac.in, akschbu@yahoo.com
(A.K. Srivastava).
and selective method for determination of PZN in body fluids is
of tremendous importance.
Literature survey reveals several analytical methods for deter-
mination of PZN such as chromatographic methods [6–8], UV–vis
method [9] and capillary electrophoresis [10]. However, most of
these methods are lengthy, overpriced, complicated, require expert
knowledge and often need the pretreatment step that make them
unsuitable for routine analysis. To overcome the drawbacks associ-
ated with the aforementioned methods electrochemical methods
such as cyclic voltammetry (CV), differential pulse voltammetry
(DPV) and stripping methods are used extensively due to their sen-
sitivity, selectivity, simplicity, low cost and relatively short analysis
time. To the best of our knowledge, few electrochemical methods
are reported for determination of PZN [11–16].
Over the past two decades, chemically modified electrodes
(CMEs) have attracted broad interest in sensor development due
to low background current, wide range of potential window, easy
surface renewal, lower detection limit, low sensitivity to dissolved
oxygen and low cost. Due to such advantages CMEs have been used
in various analyses. In recent years, our group has reported chem-
ically modified electrodes for determination of various organic
[17–23] as well as inorganic [24,25] species. Carbon has been widely
used as an electrode material since many years with or without

http://dx.doi.org/10.1016/j.snb.2016.06.019
0925-4005/© 2016 Elsevier B.V. All rights reserved.
 P.K. Kalambate et al. / Sensors and Actuators B 237 (2016) 196–205
modification. The wide applications of carbon electrodes started
when carbon paste electrode (CPE) was invented by Adams in 1958
[26]. Graphene (GNS), a two-dimensional honeycomb lattice struc-
ture of graphite is extensively used for development of sensor as
well as supercapacitor due to its large surface area, very high elec-
trical and thermal conductivity, great mechanical strength and low
manufacturing cost [27–31]. These properties make graphene as
a suitable modifier for modification of different electrodes. Metal
oxide nanoparticles have been widely explored in the field of elec-
trochemical sensors as they offer many advantages such as high
abundance, low cost, easy synthetic procedures, high electrical con-
ductivity, etc. Due to such advantages, many metal oxides such
as TiO2 , Fe3 O4 , MnO2 , ZnO, Cu2 O, Co3 O4 , etc. have been widely
used for the electrochemical detection of various species. Among
all these metal oxide nanoparticles, ZnO is widely used because of
its stable chemical and physical characters, easy route of synthesis,
high surface activity and stability. In addition to this, ZnO is bio-
chemically stable material, which makes it ideal for direct use in
biomedical applications without the need of surface passivation.
ZnO has a large excitonic energy, low-cost synthesis, biocompati-
bility, good electrochemical activities, non-toxicity, high-electron
communication features and high mechanical strength. Nanosize
zinc oxide (ZnO) is a thermally stable semiconductor with a wide
band gap (3.37 eV) and it is used widely in the field of chemi-
cal sensors, gas sensors, solar cells, catalyst, electrical and optical
devices etc [32–36]. The ZnO has been widely used in sensor appli-
cations due to its high surface to volume ratio, which results in
greater interaction of an analyte with the sensing part of device.
Graphene sheets suffer from agglomeration and restacking due to
van der Waals interaction which leads to great loss of effective
surface area of electrode. But when graphene is used along with
ZnO nanoparticles, it weakens the interactions between graphene
sheets preventing agglomeration, which leads to increase in surface
area of the electrode. The conductivity of ZnO is enhanced when it
is used along with GNS [37]. Therefore, synergistic effect of GNS and
ZnO leads to a highly sensitive sensor for determination of PZN.
In the present study, we propose a simple method for fabrica-
tion of graphene-Zinc oxide carbon paste electrode (GNS-ZnO-CPE)
for sensitive and selective determination of PZN. The graphene
nanosheets have been synthesized by modified Hummers method
and zinc oxide nanoparticles have been synthesized by precipita-
tion method. To the best of our knowledge there is no report on the
electrochemical determination of PZN using GNS-ZnO-CPE elec-
trode. The GNS-ZnO-CPE electrode has been used for determination
of PZN in pharmaceutical formulations, urine and blood serum
samples. The nanocomposite based electrode displayed excellent
recovery results in synthetic and biological samples indicating its
good practicality. The surface characterization of the synthesized
materials has been carried out by X-ray diffraction (XRD), Scanning
electron microscopy (SEM), Energy dispersive X-ray spectroscopy
(EDX) and Transmission electron microscopy (TEM). Electrochemi-
cal characterization of the electrode material is carried out by cyclic
voltammetry (CV), differential pulse voltammetry (DPV) and elec-
trochemical impedance spectroscopy (EIS).
2. Experimental
2.1. Materials and reagents
Pure pyrazinecarboxamide was acquired from Sigma-Aldrich,
USA. Natural graphite (99.5%, ≤50 ␮m) was provided by SD fine,
India. Zinc chloride (≥98%), hydrazine hydrate (50–60%), potas-
sium permanganate (≥99.0%) and sodium nitrate (≥99%) were
obtained from Sigma-Aldrich, USA. Mineral oil (IR spectroscopic
grade) as a binder was purchased from Sigma-Aldrich, USA. The
197
double distilled water was used throughout analysis. All reagents
used were of analytical reagent grade and used as received with-
out any further purification. A stock solution of 8.0 × 10−3 M PZN
was prepared in double distilled water and kept under refrigera-
tion at 5 ◦ C. The supporting electrolyte used throughout analysis
was Britton Robinson-buffer (B.R.) of pH 7.0 (0.04 M). The work-
ing standard solutions of various concentrations were prepared by
diluting appropriate quantity of stock solution to desired volume by
using 0.04 M Britton-Robinson (B.R.) universal buffer (0.04 M boric
acid, 0.04 M acetic acid and 0.04 M phosphoric acid) (pH 7.0) .The
pharmaceutical formulations were obtained from local pharmacy
store and used as received. The tablets containing PZN (Pyzina:
500 mg/Tab and PZA-CIBA: 1000 mg/Tab) were crushed and diluted
to appropriate quantity with B.R. (pH 7.0) buffer. The blood serum
and urine samples were obtained from local pathology laboratory
Mumbai, India.
2.2. Apparatus
All voltammetric, cyclic voltammetry (CV) and differential pulse
voltammetry (DPV), and electrochemical impedance spectroscopy
(EIS) measurements were performed using an Autolab PGSTATE
30 equipped with USB electrochemical interface using GPES soft-
ware, version 4.9.005 and frequency response analyzer, software
version 2.0 respectively. The voltammetric measurements were
carried out with a conventional three electrode system employing,
GNS-ZnO-CPE as working electrode, Ag/AgCl (sat. KCl) as refer-
ence electrode and platinum wire as counter electrode. Scanning
electron microscopy (SEM) was carried out on FEI Quanta-200
with an operating voltage of 15 kV. The TEM analysis was car-
ried out on PHILIPS-CM 200 electron microscope with operating
voltages of 20–200 kV and resolution of 2.4 A◦ . X-ray diffraction
(XRD) analysis was carried out on X-ray diffractometer (Shimadzu
7000S, Shimadzu Analytical, Japan) equipped with CuK␣ radiation
(␭ = 0.154 nm). A METTLER balance (Toledo AB 204) was used to
weigh solid materials. The pH measurements were carried out by
using ELICO LI 120 pH meter, calibrated with standard buffers of
specific pH. The working standard solutions are stable at room tem-
perature and there was no change in composition with time. All
experiments were carried out at room temperature of 25 ± 1 ◦ C.
2.3. Synthesis of graphene sheets
Graphite oxide was synthesized from natural graphite by mod-
ified Hummers method [38]. Briefly, 2.0 g of graphite powder and
1.0 g NaNO3 were mixed, and then put into 96.0 mL Conc. H2 SO4
in an ice bath. Under vigorous stirring, 6.0 g KMnO4 was added
gradually. The temperature of the mixture was maintained below
20 ◦ C. The ice bath was removed and mixture was stirred in a water
bath for 2 h. To the brownish color pasty liquid, 150 mL of water
was added. To maintain temperature below 50 ◦ C water was added
continuously till total volume of water was 240 mL. To the above
mixture, 5 mL of 30% H2 O2 was added and it was observed that the
solution color transformed into brilliant yellow along with bub-
bling. The mixture was stirred for 2 h; it was filtered and washed
with 10% HCl aqueous solution, water, and ethanol. The product
obtained was dried under vacuum at 60 ◦ C. For the synthesis of
reduce graphene oxide, 100 mg of graphite oxide was dispersed
in 100 mL of water and sonicated for 1 h. In this step conversion of
graphite oxide to graphene oxide (brown dispersion) took place. To
the above dispersion 2.0 g of hydrazine hydrate in 5 mL water was
added and the mixture was refluxed at 100 ◦ C for 24 h under mag-
netic stirring. Finally, the mixture was filtered, washed thoroughly
with water and dried at 60 ◦ C for 12 h.
198 P.K. Kalambate et al. / Sensors and Actuators B 237 (2016) 196–205
2.4. Synthesis of zinc oxide nanoparticles
Zinc oxide nanoparticles were prepared by precipitation
method [39]. In typical synthesis, 0.2 M zinc chloride aqueous solu-
tion was prepared. To this solution ammonium hydroxide was
added drop wise at room temperature with continuous stirring to
obtain precipitate of zinc hydroxide. The precipitate obtained was
filtered from rest of the liquid and it was dried at 100 ◦ C. Finally,
the zinc hydroxide precipitate was calcinated at 450 ◦ C for 3 h. The
formation of Zinc oxide nanoparticles was confirmed by XRD, SEM
and EDX analyses.
2.5. Preparation of graphene-zinc oxide-carbon paste electrode
(GNS-ZnO-CPE)
The carbon paste electrode (CPE) was prepared by mixing
graphite and mineral oil in a very popular ratio 70:30 (w/w). The
graphite powder and mineral oil were grinded in mortal and pestle
for 1 h. The paste was then homogenized for 24 h. The portion of
the resulting paste was filled in a Teflon micropipette tip and elec-
trical contact was made with a silver wire through the centre of the
tip. Fresh electrode surface was obtained by squeezing out paste
from the syringe and scrapping off the surface against zero grade
butter paper until surface had a shiny appearance. For preparation
of GNS-ZnO-CPE, the composition of GNS and ZnO was optimized
by varying amount of both GNS and ZnO in the range of 1–10% with
respect to graphite. It was observed that the peak current increases
until 5% loading of each modifier in the composite after which
it saturates. Therefore, GNS-ZnO-CPE electrode was prepared by
incorporating graphene and zinc oxide in to graphite and mineral
oil with the composition of 60:5:5:30 (graphite: graphene: zinc
oxide: mineral oil). For comparison, GNS-CPE and ZnO-CPE was also
prepared by following the same procedure.
2.6. Experimental procedure
Differential pulse voltammetry (DPV) was used to record the
voltammograms. For DPV, appropriate quantity of stock standard
solution of PZN was taken in to 25 mL volumetric flask and diluted
up to the mark with B.R. buffer (pH 7.0). The solution was then
added to the electrochemical cell where the measurements were
carried out. No oxygen interference was found in the present work
thus, no deaeration was carried out. The voltammograms were
recorded by scanning potential towards negative direction from
−0.6 V to −1.2 V using differential pulse voltammetry employing a
Fig. 1. Representative XRD patterns of (A) GNS; (B) ZnO.
step potential of 5 mV and modulation amplitude of 50 mV. The
cyclic voltammetric experiments were carried out by sweeping
potential from −0.3 to −1.3 V. An electrochemical impedance spec-
troscopy was carried out in 1 mM K3 [Fe (CN)]6 /K4 [Fe (CN)]6 system
in the frequency range 10−1 –106 Hz. The Area of the electrodes was
calculated by Randles-Sevcik formula in 6 mM K3 [Fe (CN)] 6 using
1 M KNO3 as a supporting electrolyte.
2.7. Treatment and determination of samples
Determination of PZN was carried out in pharmaceutical formu-
lations, blood serum, and urine samples. The pyrazinamide tablet
containing 500 mg and 1000 mg of PZN were obtained from local
drug store. Five tablets were selected randomly, ground, and mixed.
An appropriate quantity was weighed, sonicated for 30 min and fil-
tered through Whatman filter paper No.1. All samples were diluted
to 100 mL with B.R. buffer. Quantitative determination was carried
out by standard addition method. Recovery tests were performed
by spiking standard solutions of PZN in to pharmaceutical formula-
tions. The blood serum and urine samples were obtained from local
pathology laboratory and stored under refrigeration. Both samples
were prepared by adding 50 ␮L of sample and diluted to 25 mL
with B.R. buffer (pH 7.0). No pretreatment step was carried out for
both the samples. Sample cleaning was done by filtering through
0.22 ␮m PVDF syringe filter (Millex, Millipore Corporation).
3. Results and discussion
3.1. XRD analysis
XRD diffraction patterns of GNS and ZnO nanoparticles are
shown in Fig. 1. It is observed from XRD pattern that graphene
shows two diffraction peaks at 25.7◦ and 43.5◦ (Fig. 1(A)). The two
diffraction peaks in this pattern can be indexed to the (002) and
(111) reflection. XRD pattern of graphene is agreed well with the
data reported in the literature. Fig. 1(B) shows XRD patterns of zinc
oxide nanoparticles. The peaks at 2␪ = 31.67◦ , 34.31◦ , 36.14◦ , 47.40◦ ,
56.52◦ , 62.73◦ , 66.28◦ , 67.91◦ , 69.03◦ and 72.48◦ were assigned to
(100), (002), (101), (102), (110), (103), (200), (112), (201) and (004)
reflections of ZnO nanoparticles which indicate hexagonal wurtzite
structure. No additional peaks of any impurities were detected,
suggesting that high quality ZnO nanoparticles were synthesized.
 P.K. Kalambate et al. / Sensors and Actuators B 237 (2016) 196–205
Fig. 2. SEM images for (A) CPE; (B) GNS-CPE; (C) GNS-ZnO-CPE; EDX spectra of (D) GNS; (E) ZnO; (F) GNS-ZnO; TEM images for (G) CPE; (H) GNS-CPE; (I) GNS-ZnO-CPE.
3.2. SEM, EDX and TEM study
The morphology of as prepared composites was studied by SEM
and TEM analysis. The SEM of Fig. 2(A) shows multiple sheets like
structure of graphite, (B) shows graphene sheets incorporated in
to graphite and (C) represents ZnO nanoparticles deposited on
graphene sheets. SEM data also revealed some of the zinc oxide
nanoparticles are agglomerated. The elemental analysis of GNS,
ZnO and GNS-ZnO has been carried out by EDX and spectra are
shown in Fig. 2(D), (E) and (F). Graphene shows only strong peak
for C atom (Fig. 2(D)). ZnO shows strong peaks for Zn atoms as
compared to weak peak from O atom (Fig. 2(E)). The GNS-ZnO rep-
resents the prominent peaks for C, Zn and O, indicating presence
of all elements in the composite. The TEM of 2(G) depicts graphite
sheets, (H) shows thin transparent flakes of GNS and (I) represents
deposition of ZnO nanoparticles on GNS surface which attributes
to the increase in surface area of the electrode. TEM data revealed
that the ZnO particles are spherical in shape and size ranges from
14 to 48 nm.
199
3.3. Effect of pH and supporting electrolyte
The effect of pH on voltammetric behavior of PZN was carefully
investigated at CPE in Britton-Robinson (B.R.) buffer by differential
pulse voltammetry. The well defined reduction peaks were found
for 3.0 × 10−4 M PZN in the pH range of 2–10. As pH of the medium
increases, the peak potentials shift towards less positive values,
indicating involvement of proton in the electrochemical reaction.
There were broad peaks found in the pH range 11–12. It is also
found that the maximum sensitivity of the electrode was at pH 7.0
(Fig. 3(A)). Thus, the B.R. solution of pH 7.0 was taken for determi-
nation of PZN. The relationship between peak potential (Ep ) and pH
was linear (Fig. 3(B)) and is given by the following equation:
PZN :E p (V) = −0.074pH + 0.409(R 2 = 0.992) (1)
The Nernstian equation for equal number of proton and elec-
tron transfer process is E/pH = (59.1 mV/n) × N+ H (n = 2) and the
slope value for the Ep vs. pH curve is −74.0 mV pH−1 . Hence, we sug-
gest that the number of protons (N+ H ) transfer could be three. The
above results were coinciding with the electrochemical behavior of
PZN at mercury electrode [40]. The redox reaction of the PZN leads
200 P.K. Kalambate et al. / Sensors and Actuators B 237 (2016) 196–205

Scheme 1. Electrochemical reaction of PZN at GNS-ZnO-CPE.

to formation of 1, 4-hidro-pirazinium ion (Scheme 1) which was
clearly explained by the Bergamini et al. [13]. The electrochemical
response of PZN was also studied in different media, namely phos-
phate buffer, tris, HEPES, and citrate-phosphate in pH 7.0. The best
responce in terms of peak current and peak shape was obtained
when 0.04 M B.R. (pH 7.0) was employed (Fig. 3(C)).
3.4. Cyclic voltammetry
The area of the electrodes was calculated by cyclic voltam-
metry (CV) using Randles-Sevcik formula for 6 mM K3 Fe (CN)6
in 1 M KNO3. The calculated surface area is 0.02 cm2 , 0.041 cm2
and 0.076 cm2 , respectively for CPE, GNS-CPE and GNS-ZnO-CPE,
respectively. In case of GNS-ZnO-CPE the area is increased by 3.8
times than that of simple CPE. The increase in area is responsible for
highly sensitive electrochemical sensor for determination of PZN.
The electrochemical response of PZN at the GNS-ZnO-CPE has
been investigated using CV by sweeping the potential from −0.3
to −1.3 V. The cyclic voltammograms for 2.5 × 10−4 M PZN in B. R.
(pH 7.0) at different electrodes are shown in Fig. 4(A). However,
for the GNS-ZnO-CPE the peak current is maximum as compared to
GNS-CPE and CPE. This observation implies that reduction of PZN
is more facile on GNS-ZnO-CPE. As can be seen from Fig. 4(A), the
CV plot exhibits well define anodic and cathodic peaks. The influ-
ence of scan rate on reduction peak current and peak potential of
2.5 × 10−4 M PZN was studied from 10 to 800 mV s−1 (Fig. 4(B)).The
increase in scan rate results in regular increase of peak current along
with a shift in peak potential (Ep ) towards more negative values.

Fig. 3. (A) A plot of peak current (Ip ) vs. pH for 3.0 × 10−4 M PZN at CPE employing DPV; step potential = 5 mV and modulation amplitude = 50 mV; (B) A plot of peak potential
(Ep ) vs. pH for 3.0 × 10−4 M PZN at CPE employing DPV; step potential = 5 mV and modulation amplitude = 50 mV; (C) A bar graph for the effect of various supporting electrolytes
employed for 3.0 × 10−4 M PZN at CPE employing DPV; step potential = 5 mV and modulation amplitude = 50 mV.
 P.K. Kalambate et al. / Sensors and Actuators B 237 (2016) 196–205 201

Fig. 4. (A) Cyclic voltammograms of 2.5 × 10−4 M PZN at three different electrodes: (a) CPE(. . .. . ..), (b) GNS-CPE( ) and (c) GNS-ZnO-CPE ( ).Voltammetric
conditions: scanning electrode potential with a scan rate of 100 mVs−1 between −0.3 to −1.3 V in pH 7.0 B.R. buffer (0.04 M); (B) Cyclic voltammograms for PZN 2.5 × 10−4 M
−1
obtained in B.R buffer (pH 7.0) employing varying scan rates (mV s ): (1–7) 10, 50, 100, 200, 400, 600 and 800; (C) Ip vs square root of scan rate plot for the data obtained
from Fig. 4 (B); (D) Nyquist plots for EIS measurements (1.0 × 10−3 M) K3 [Fe (CN)]6 /K4 [Fe(CN)]6 at CPE ( ), GNS-CPE ( ), GNS-ZnO-CPE ( ) and in the
box on the left upper side is the equivalent circuit used for data fitting.

The reduction peak current is found to be linearly dependent on considerably small and the charge transfer rate is higher than at the
square root of scan rate indicating that the reduction of PZN is con- CPE and GNS-CPE surface.
trolled by diffusion at GNS-ZnO-CPE (Fig. 4(C)) which is given by
the following regression equation: 3.6. Differential pulse voltammetry (DPV)

I p (A) = 0.6051/2 + 1.124(R 2 = 0.998)
3.5. Electrochemical Impedance Spectroscopy (EIS)
Electrochemical impedance spectroscopy is a powerful method
for determination of surface nature of the solution/electrode [41].
EIS provides information on impedance changes of the electrodes
as a result of the modification process. The Nyquist plots for
1 mM K3 [Fe (CN)]6 /K4 [Fe (CN)]6 at CPE, GNS-CPE and GNS-ZnO-
CPE are shown in Fig. 4(D). The equivalent circuit shown (inset
Fig. 4(D)) is made up of the electrolyte solution resistance (Rs )
in series with the parallel circuit of faradaic impedance (Zf ) and
double layer capacitance (Cdl ). Zf is composed of charge transfer
resistance (Rct ) and Warburg impedance (Zw ).The diameter of the
semicircle for the GNS-ZnO-CPE is smaller than CPE and GNS-CPE
electrodes, which suggests the modification of GNS-ZnO-CPE with
lower charge transfer resistance. The charge transfer resistance
(Rct ) values obtained for CPE, GNS-CPE, and GNS-ZnO-CPE are 0.617
kOhm, 0.553 kOhm, and 0.430 kOhm, respectively. This observation
implies that the charge transfer resistance of the GNS-ZnO-CPE is
(2) The electrochemical reduction of 2.5 × 10−4 M PZN was investi-
gated by the differential pulse voltammetry at CPE, GNS-CPE and
GNS-ZnO-CPE in the B.R. buffer (pH 7.0). As shown in Fig. 5(A), the
best results in terms of peak current are obtained on GNS-ZnO-CPE.
DPV study was also performed on ZnO-CPE, which is compared with
CPE (Fig. 5(B)). However, it is found that the peak current at ZnO-
CPE is lesser than CPE as ZnO alone is electrochemically inactive.
The higher current on the GNS-ZnO-CPE electrode is due to large
effective electroactive surface area of GNS-ZnO and high-electron
communication features of ZnO. This leads to a facile electro reduc-
tion of PZN at the surface of GNS-ZnO-CPE electrode. In order to
confirm applicability of the proposed method the pharmaceutical
tablets were analyzed (Table 1). It is found that amount of PZN
obtained by present method agrees well with the label contents.
3.7. Determination of PZN
In order to develop a voltammetric method for determination
of PZN, DPV was the technique selected due to its high sensitivity
and resolving power. Under optimized parameters established in
the previous sections, the linearity of the method was evaluated
202 P.K. Kalambate et al. / Sensors and Actuators B 237 (2016) 196–205
Fig. 5. (A) DPV of 2.5 × 10−4 M PZN at three different electrodes: (a) CPE (
step potential = 5 mV and modulation amplitude = 50 mV; (B) DPV curves of 2.5 × 10−4 M PZN on CPE and ZnO-CPE; (C) DPV curves obtained at GNS-ZnO-CPE for PZN at
different concentrations: in the range from (a) blank, (b) 1.5 × 10−7 , (c) 4.0 × 10−7 , (d) 6.0 × 10−7 , (e) 2.0 × 10−6 , (f) 4.0 × 10−6 , (g) 8.0 × 10−5 , (h) 2.0 × 10−4 , (i) 3.0 × 10−4 , (j)
4 × 10−4 M; (D) Plot of Ip (␮A) vs C (␮M) for PZN.
Table 1
Determination of PZN by proposed method.
Sample PZN
a b
Pyzina 500 499 ± 2
PZA-CIBA 1000 999 ± 2
a: amount of PZN in a tablet (mg).
b: amount of PZN obtained by the proposed method (mg) ±% RSD (n = 5).
c: amount of PZN obtained by the spectrophotometric method (mg) ±% RSD (n = 5).
by measuring the current response for different PZN concentra-
tions (Fig. 5(C)). The calibration curve was used for evaluating linear
regression equation (LRE) and correlation coefficient (r) by linear
fitting method. Validation of the proposed procedure for assay of
standard PZN was examined via evaluation of limit of detection
(LOD), limit of quantitation (LOQ), reproducibility, precision and
selectivity. Under optimized conditions the peak current was lin-
early proportional to the concentration in the range 1.5 × 10−7 to
4.0 × 10−4 M with a linear regression equation as follows:
I p (A) = 0.022C(M) + 1.184(R 2 = 0.992)
The LOD was calculated using the equation LOD = 3 × SD/s
(where SD is the standard deviation of the blank and‘s’ is the slope
of the linear calibration plot) and it was found to be 4.31 × 10−8 M
(% RSD = 3.19). The results obtained during this study confirmed
), (b) GNS-CPE ( ), and (c) GNS-ZnO-CPE ( ). Voltammetric conditions:
that this technique can be applied for the quantitative analysis of
PZN.
c 3.8. Interference studies, stability, validation and analytical
498 ± 3 applications
998 ± 2
An interference study was conducted using DPV with poten-
tial constituents present in blood serum and urine samples. The
potential interfering species were added one at a time in the pres-
ence of 4.0 × 10−6 M PZN and DPV response was recorded. The
tolerance limit was taken as the maximum concentration of the
potential interfering substances, which caused an approximately
±5.0% relative error in the determination. Some of the commonly
found ions and species in biological samples viz. SO4. 2− , Ca2+ , K+ ,
NH4 + , Na+ NO3 − , Mg2+ and Cl− , ascorbic acid, glucose, uric acid,
dopamine, isoniazide, rifampicine and ethambutol were consid-
ered for interference study. The Na+ , K+ , Cl− ions had no interference
till 220 fold excess. Also, no interference was observed till 110 fold
excess in case of NO3 − , SO4. 2 , Ca2+, Mg2+ . Thus, the commonly found
ions hardly affect the determination of PZN. Ascorbic acid (till 40
(3) fold excess), glucose (25 fold excess), uric acid (45 fold excess),
dopamine (40 fold excess), isoniazide (25 fold excess), rifampicine
(35 fold excess) and ethambutol (40 fold excess) did not interfer-
ence in the determination of PZN. Interference study reveals that,
the determination of PZN is not considerably affected by common
interfering species. This lends confidence in the method presented
 P.K. Kalambate et al. / Sensors and Actuators B 237 (2016) 196–205 203

Table 2
Precision and Bias of assay for standard PZN solution by the proposed voltammetric procedure (n = 5).

Molecule Concentration (taken) (10−6 M) Concentration (found) (10−6 M) Recovery (%) (n = 5) Bias (%) Precision% R.S.D (n = 5)

PZN Intra − day

3.0 2.97 99.06 0.94 2.31
Inter − day
6.0 5.88 98.0 2.04 3.11

Table 3
Recovery test for PZN in pharmaceutical, urine and blood serum samples.

Sample PZN
Std drug added (10−6 M) Drug found (10−6 M) Recovery%R Average recovery%R ± RSD

Pyzina – 2.30 – 98 ± 0.4

1.21 3.45 98.29
3.21 5.43 98.55
4.63 6.83 98.55

PZA-CIBA – 4.98 – 99 ± 1
2.10 7.00 98.87
3.43 8.31 98.81
4.56 9.41 98.64

Urine sample – NDa – 99 ± 2

1.58 1.55 98.10
3.75 3.69 98.40
4.32 4.33 100.23

Blood Serum sample – ND – 99 ± 2

3.42 3.43 100.29
5.41 5.35 98.89
6.86 6.81 99.27
a
ND: Not detected.

Table 4
Comparison between various electroanalytical methods for the determination of PZN with the proposed method.

Electrode Linear working range (M) Limit of detection (M) Samples analyzed References
−7 −3 −7
Poly-l-methionine/reduced graphene 4.0 × 10 −1.129 × 10 1.6 × 10 Urine, blood plasma [12]
oxide
−7 −4 −7
Poly-histidine 9.0 × 10 to 1.0 × 10 5.7 × 10 Urine [13]
GO/PAG/GCE 2.5 × 10−5 to 1.6 × 10−3 3.28 × 10−6 Pharmaceutical formulations, blood serum [14]
MWCNT/GO/GCE 3.75 × 10−5 to 1.8 × 10−3 5.54 × 10−6 Pharmaceutical formulations, blood serum [15]
PARS /GCEa 5.0 × 10−6 to 1.0 × 10−4 1.2 × 10−6 Pharmaceutical formulations [16]
GNS-ZnO CPE 1.5 × 10−7 to 4.0 × 10−4 4.31 × 10−8 Pharmaceutical formulations, blood serum, Urine sample This work
a
Polyalizarin red S (ARS) modified glassy carbon electrode.

here to be highly selective and for its use in identifying biologi-
cal samples. The proposed method was validated by employing UV
spectrophotometric method [9] (Table 1). The results show that the
amounts of PZN obtained by the proposed and spectrophotomet-
ric method are comparable. The stability of the electrodes was also
tested. The peak current only decreased less than 6% after the elec-
trodes were stored at room temperature for two months. In order
to validate the method, various parameters such as repeatability,
reproducibility, precision and accuracy of analysis were obtained
by performing five replicate measurements for standard PZN over
a single day (intra-day assay) (n = 5) and for five days over a period
of one week (inter-day assay). The recovery tests were carried out
by standard addition method. The results are shown in (Table 2)
which confirm both high precision of the proposed procedure and
the stability of PZN solutions. For further evaluation of the valid-
ity, recovery tests were carried out in pharmaceutical formulations,
urine and human blood serum samples (Table 3). These tests gave%
R values in the range of 98.10–100.29%. These results confirm that
the recovery of PZN is not affected significantly, and consequently,
the described method is accurate for its assay in complex matri-
ces. The results also confirmed that interferences from the matrix
were negligible. The real sample studies demonstrated that the pro-
posed method is suitable and efficient for determination of PZN in
pharmaceutical formulations, urine and blood serum samples with
good accuracy and precision. The electrochemical parameters for
the determination of PZN were compared with the reports in the
literature [12–16] and the results are listed in Table 4. It can be seen
that the present method can offer several advantages like simplic-
ity, high sensitivity, low detection limit, wide linear working range,
high selectivity, along with no need of any pretreatment step.
4. Conclusion
A modified carbon paste electrode has been successfully fab-
ricated by using graphene and zinc oxide nanoparticles. The
GNS-ZnO-CPE exhibits good catalytic activity for reduction of PZN.
It could be found that the electrochemical reduction of PZN had
been greatly improved due to the synergistic effect between GNS
and ZnO nanoparticles. The GNS-ZnO-CPE exhibited superior per-
formance in terms of peak current, linear working range, limit of
detection etc. The sensor was applied for determination of PZN
in real samples such as pharmaceutical tablets, human urine and
blood serum samples. Selective determination of PZN is possi-
ble in the presence of potential interfering species as they did
not change the current response of PZN. The proposed method
is simple, accurate, and does not require any pretreatment step
such as extraction of an analyte from pharmaceutical as well as
biological samples. Additionally, the proposed sensor presented
very good anti-interference ability, reproducibility and stability.
The proposed method is efficient and more sensitive compared to
204 P.K. Kalambate et al. / Sensors and Actuators B 237 (2016) 196–205
the electrochemical methods reported for PZN and can be readily
adapted to clinical as well as pharmaceutical analyses. The above
mentioned advantages testify the proposed sensor can be used as
an electrochemical platform with a bright future for determination
of PZN.
Acknowledgements
The funding for this work is by the University Grant Commission,
New Delhi, India under the University with potential for excellence
scheme to University of Mumbai. The authors wish to acknowledge
National Centre for Nanoscience and Nanotechnology, University of
Mumbai for providing SEM facility.
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Biographies
Pramod K. Kalambate received his M.Sc (2011) degree in
analytical chemistry from University of Mumbai, India. He
is currently a Ph.D student working under the guidance of
Dr. A. K. Srivastava at department of chemistry, university
of Mumbai, India.H is research interests consist of synthe-
sis of nanomaterials for development of electrochemical
sensors and supercapacitors.
 P.K. Kalambate et al. / Sensors and Actuators B 237 (2016) 196–205 205

Chaitali R. Rawool received her M.Sc (2015) degree in
analytical chemistry from University of Mumbai, India.
She is currently a Ph.D student working under the guid-
ance of Dr. A. K. Srivastava at department of chemistry,
university of Mumbai, India.H er research interests con-
sist of nanomaterials for development of electrochemical
sensors and supercapacitors.
Ashwini K. Srivastava is a professor of analytical chem-
istry at the department of chemistry, university of
Mumbai, India. He received Ph.D (1981) degree in chem-
istry from Banaras Hindu University, India. His research
interests are in the field of development of electrochem-
ical (bio) sensors using different functional materials,
supercapacitors and liquid chromatography.