Adsorptive stripping differential pulse voltammetry determination of rivastigmine at graphene nanosheet-gold nanoparticle/carbon paste electrode

Adsorptive stripping differential pulse voltammetry determination of
rivastigmine at graphene nanosheet-gold nanoparticle/carbon paste electrode
Pramod K. Kalambate a
, Madan R. Biradar a
, Shashi P. Karna b
, Ashwini K. Srivastava a,
⁎
a
Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (East), Mumbai 400 098, India
b
U.S. Army Research Laboratory, Weapons and Materials Research Directorate, ATTN: RDRL-WM, Aberdeen Proving Ground, MD21005-5069, USA
a b s t r a c ta r t i c l e i n f o
Article history:
Received 1 July 2015
Received in revised form 20 August 2015
Accepted 18 September 2015
Available online 25 September 2015
Keywords:
Rivastigmine
Graphene
Gold nanoparticles
Adsorptive stripping voltammetry
The study of graphene nanosheet (GNS)–gold nanoparticle (AuNP)–carbon paste electrode (GNS–AuNP–CPE) as
an electrochemical sensor for the determination of rivastigmine (RIV) in pharmaceuticals formulations, blood
serum, and urine samples is presented. The GNS–AuNP composite is prepared by in situ simultaneous reduction
of graphene oxide and chloroauric acid using sodium borohydride as a reducing agent. The GNS–AuNP composite
was characterized by X-ray diffraction, UV–Vis spectroscopy, and scanning electron microscopy. Electrochemical
characterization of the GNS–AuNP–CPE electrode surface was carried out by cyclic voltammetry, electrochemical
impedance spectroscopy, chronocoulometry, and adsorptive stripping differential pulse voltammetry. This study
shows that oxidation of rivastigmine is facilitated at the GNS–AuNP–CPE electrode and remarkably increase in
current compared to the bare electrode due to enhanced adsorption of the former on electrode surface. Under
the optimized conditions, the peak current (Ip) is found to be proportional to the RIV concentration in the
range of 2.0 × 10–7–
6.0 × 10−4
M with a detection limit of 5.3 × 10−8
M. The proposed sensor shows a very
high level of sensitivity, selectivity, and a very good reproducibility for RIV determination. A good recovery
level obtained for real samples suggests practical utility of the GNS–AuNP–CPE as an effective and reliable elec-
trochemical sensor for RIV detection.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Rivastigmine (Exelon), chemically known as (S)-N-ethyl-N-methyl-
3-[(1-dimethylamino) ethyl]-phenyl carbamate hydrogen tartrate
(RIV) is an acetylcholine esterase inhibitor of the carbamate type ap-
proved for the treatment of Alzheimer's disease [1], which is a progres-
sive, degenerative brain disorder that affects reason, judgment and
memory. Over a period, people with Alzheimer's disease lose their abil-
ity to think and reason clearly, judge situations, solve problems, concen-
trate, remember useful information, take care of themselves, and even
speak [2,3]. People with mild Alzheimer's disease usually require close
supervision and help with everyday tasks; and those with severe dis-
ease can do little on their own and require complete full-time care.
Alzheimer's disease severely impacts the quality of life of the patient,
their family and caregivers.
Currently, there is no cure for Alzheimer's disease but some drugs
are used to keep symptoms from getting worse for a limited time [4].
Drug treatments include rivastigmine (Exelon), donepezil (Aricept),
Rivamer and galantamine (Reminyl). These drugs affect the level of a
neurotransmitter in the brain called acetylcholine. Rivastigmine (RIV)
is one of the most widely used reversible cholinesterase inhibitor for
treatment of Alzheimer's disease. However, an overdose is toxic and
leads to several side effects viz., chest pain or discomfort, increased
sweating, increased watering of the mouth, slow or shallow breathing,
nausea, dizziness, severe vomiting, pale or blue lips, light headedness,
stomach pain, and trouble sleeping etc. It is thus necessary to develop
a fast, sensitive, and cost-effective method to determine RIV level in var-
ious samples, viz.; pharmaceutical formulations, blood serum, and urine
samples.
Currently used analytical methods for determination of RIV include
liquid chromatography–tandem mass spectrometry (LC–MS/MS) [5,6],
high performance thin layer chromatography (HPTLC) [7], headspace
solid-phase microextraction (HS-SPME), capillary gas chromatography–
mass spectrometry (GC–MS) [8], miniaturized membrane sensor [9],
spectrophotometric and spectrodensitometric methods [10]. However,
most of these methods are lengthy, expensive, require complicated pro-
cedure and expert knowledge and often need the pretreatment step
that make them unsuitable for routine analysis. Electrochemical methods
are used extensively due to their simplicity, low cost, and relatively short
analysis time.
Over the past two decades, chemically modiﬁed electrodes (CMEs)
have attracted broad interest in biological and pharmaceutical sensing
development due to low background current, wide range of potential
window, easy surface renewal, lower detection limit, and low cost.
Due to these advantages, the electrochemical sensing using CMEs have
Journal of Electroanalytical Chemistry 757 (2015) 150–158
⁎ Corresponding author.
E-mail addresses: aksrivastava@chem.mu.ac.in, akschbu@yahoo.com (A.K. Srivastava).
http://dx.doi.org/10.1016/j.jelechem.2015.09.027
1572-6657/© 2015 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Journal of Electroanalytical Chemistry
journal homepage: www.elsevier.com/locate/jeac

been successfully used in determination of various organic [11–17] as
well as inorganic [18,19] species.
Graphene, a two dimensional one atom thick nanomaterial
consisting of sp2
hybridized carbon, has attracted tremendous attention
due to its unique properties, such as high surface area, excellent electri-
cal conductivity, and good electrocatalytic activity [20,21]. Because of
these properties graphene has been used as an ideal electrode material
in supercapacitors [22,23], field effect transistors [24], and chem/bio
sensors [25,26]. The introduction of metal nanoparticles into the disper-
sion of graphene sheets also helps inhibit the aggregation of graphene
sheets and result in mechanically jammed exfoliated graphene agglom-
erate with very high surface area. At the same time, AuNP have been
widely used in electrochemical detection because they enhance the
electrode conductivity and facilitate electron transfer by virtue of quan-
tum size effects [27,28].
Rivastigmine is an electroactive compound which can be oxidized
electrochemically. Consequently the development and application of
electrochemical sensors and methods for the determination of
rivastigmine have received considerable interest in the past few years
[29,30]. In this paper we report the application of a graphene-gold
nanoparticle-carbon paste electrode (GNS–AuNP–CPE) for sensitive de-
termination of RIV in pharmaceutical formulations, urine and blood
serum samples with adsorptive stripping differential pulse voltammetry
(AdSDPV). The GNS–AuNP composite was synthesized in two steps. In
the first step graphene oxide was prepared by modified Hummers
method. In the second step, graphene oxide and chloroauric acid were
simultaneously reduced using sodium borohydride to form graphene
and gold nanoparticles. The characterization of composite was carried
out by various techniques viz.; X-ray diffraction (XRD), UV–Visible
spectroscopy, scanning electron microscopy (SEM), and energy disper-
sive X-ray spectroscopy (EDX). The GNS–AuNP modified carbon paste
electrode was used for the determination of RIV employing AdSDPV. In
addition, the electrochemical characterization was performed using cy-
clic voltammetry (CV), electrochemical impedance spectroscopy (EIS),
and chronocoulometry (CC). By employing AdSDPV, determination of
RIV is carried out in pharmaceutical formulations, blood serum, and
urine samples. To the best of our knowledge only two voltammetric
methods have been reported for determination of RIV [29,30].
2. Experimental
2.1. Materials
All chemicals used were of analytical reagent grade and used with-
out any further purification. Rivastigmine hydrogen tartrate (≥98%)
was obtained from Sigma-Aldrich, USA. Graphite powder (99.5%, parti-
cle size b50 μm) and chloroauric acid were purchased from SD Fine-
Chem Ltd. and used as received. Mineral oil (IR spectroscopy grade)
was procured from Sigma-Aldrich, USA. Potassium permaganate
(≥99.0%), sodium nitrate (≥99.0%), and sodium borohydride (99%)
were procured from Sigma-Aldrich, USA. Double distilled water was
used throughout the study. Rivamer 1.5 and Rivamer 3.0 tablets were
obtained from local drug stores. Human blood serum and urine samples
are obtained from Local Pathology Lab, Mumbai, India. The supporting
electrolyte used throughout the analysis was phosphate buffer solution
(PBS; 0.1 M pH 7.0). The pH of the buffer solutions were adjusted with
1 M NaOH and 0.5 M HCl. The stock solution (8 × 10−3
M RIV) was pre-
pared in phosphate pH 7 (0.1 M) buffer and stored under refrigeration.
The working standard solutions were prepared using serial dilutions of
stock standard solution using phosphate pH 7.0. All electrochemical
measurements were carried out at room temperature (24 ± 2 °C).
2.2. Apparatus
All voltammetric measurements were performed using an Autolab
PGSTATE 30 equipped with USB electrochemical interface using GPES
software, version 4.9.005 and frequency response analyzer, software ver-
sion 2.0 respectively. Conventional three-electrode system employing, a
modified carbon paste electrode as working electrode, platinum wire
and Ag/AgCl (sat. KCl) as counter and reference electrodes, respectively
was used for measurements. Scanning electron microscopy was per-
formed on FEI Quanta-200. The pH measurements were done by using
ELICO LI 120 pH meter. X-ray diffraction analysis was carried out on an
X-ray diffractometer (Shimadzu 7000S, Shimadzu Analytical, Japan)
equipped with CuKα radiation (λ = 0.154 nm). The UV–Visible spectro-
scopic study was carried out on a Shimadzu UV-2450 spectrophotometer
with samples in a quartz cuvette operated from 200 to 800 nm. The mag-
netic stirrer used for stripping analysis was REMI 1 MLH. The Mettler
Toledo (AB 204) balance was used for weighting solid materials.
2.3. Synthesis of graphene-gold nanoparticles (GNS–AuNP) composite
Graphite oxide was prepared from natural graphite by modified
Hummers method [31]. 2.0 g of graphite was mixed with 96.0 ml con-
centrated H2SO4 acid and 1.0 g NaNO3 in ice bath for half an hour
using a magnetic stirrer. A 6.0 g of KMnO4 was slowly added (small
amount at each time) into the mixture within 1 h while keeping the
temperature of the mixture not exceeding 5 °C. Then the mixture was
heated up to 60 °C and was maintained at 60 °C for 30 min. It was
followed by addition of 150.0 ml of double distilled water into the mix-
ture and heating was continued for additional 30 min at the same tem-
perature. Finally, the oxidation reaction was terminated by the addition
of 240.0 ml of double distilled water and 10.0 ml 30% H2O2 solution.
Then the mixture was filtered and the product was washed several
times with 10% hydrochloric acid followed by double distilled water
and dried in a vacuum oven for 24 h. 100 mg of dried graphite oxide
was measured using an analytical balance. It was dispersed in
100.0 ml of double distilled and sonicated for 1 h. The formation of
graphene oxide (GO) took place at this step. GO dispersion (1 mg/ml)
prepared in above step was transferred to a 500 ml round bottom
flask and chloroauric acid (1 × 10−3
M, 25 ml) was added to the disper-
sion. Now, the solution containing both GO and chloroauric acid was
treated with 1.5 g of sodium borohydride. The solution was heated to
95 °C for 12 h. The solution was filtered and residue was first washed
with ethanol for several times then with double distilled water. The res-
idue was dried at 60 °C for 12 h. This composite mainly contains gold
nanoparticles and graphene. The graphene was synthesized by same pro-
cedure using sodium borohydride as a reducing agent. The illustration of
the preparation procedure for GNS–AuNP nanocomposites is shown in
Scheme 1. It is shown in Scheme 1 that graphite oxide has intercalated
oxygen's; the exfoliation of graphite oxide gives graphene oxide. The
major difference between graphite oxide and graphene oxide is the num-
ber of layers. Graphite oxide is a multilayer system and in graphene oxide
a few layer flakes and monolayer flakes can be found.
2.4. Preparation of the GNS–AuNP modified carbon paste electrode (GNS–
AuNP–CPE)
The carbon paste electrode (CPE) was prepared with composition of
70:30 (graphite:mineral oil) using mortar and pestle. The paste was
then homogenized for 24 h. The paste was filled in Teflon micropipette
tip and silver wire was dissected for an electrical contact. Fresh elec-
trode surface was obtained by squeezing out paste from the micropi-
pette tip and scrapping off the surface against butter paper until
surface had a shiny appearance. GNS–AuNP–CPE was prepared by incor-
porating GNS–AuNP in to graphite and mineral oil with varying ratio of
GNS–AuNP composite from 1 to 12%. Best results were obtained when
10% of GNS–AuNP composite (Fig. S1) was used along with graphite
and mineral oil. Therefore, optimized electrode with composition of
60:10:30 (graphite powder:GNS–AuNP:mineral oil) was used for deter-
mination of RIV. For comparison, a bare CPE and GNS–CPE were also
prepared by the same procedure.
151P.K. Kalambate et al. / Journal of Electroanalytical Chemistry 757 (2015) 150–158

2.5. Experimental procedure
Adsorptive stripping differential pulse voltammetry (AdSDPV) was
used to record the voltammograms. For AdSDPV, appropriate quantity
of stock standard solution of RIV was taken in to 25 ml volumetric
ﬂask and diluted up to the mark with phosphate buffer, pH 7.0. The so-
lution was then added to the electrochemical cell where the measure-
ments were carried out. No oxygen interference was found in the
anodic window, thus, no deaeration was carried out. A magnetic stirrer
was used to facilitate accumulation of RIV on to the electrode surface. An
Fig. 1. Representative XRD patterns for (A) GNS; (B) GNS–AuNP composite; UV–Vis spectra for (C) GNS and (D) GNS–AuNP composite.
Scheme 1. Illustration of the preparation procedure for GNS–AuNP nanocomposites.
152 P.K. Kalambate et al. / Journal of Electroanalytical Chemistry 757 (2015) 150–158

accumulation potential of 0.6 V with accumulation time of 60 s, was
employed for RIV determination, while the solution was stirred at
250 rpm. The stirring was then stopped, and after 15 s the voltammo-
gram was recorded by scanning potential towards positive direction
from 0.55 V to 1.25 V using differential pulse voltammetry employing
a step potential of 5 mV and modulation amplitude of 50 mV. The cyclic
voltammetric experiments were carried out by sweeping potential from
0.5 to 1.4 V.
2.6. Treatment and determination of samples
Determination of RIV was carried out in pharmaceutical formula-
tions, blood serum, and urine samples. The Rivamer capsules containing
1.5 mg and 3.0 mg of RIV were obtained from local drug store. Five cap-
sules were selected randomly, ground, and mixed. An appropriate quan-
tity was weighed, sonicated for 30 min and filtered through Whatman
filter paper No. 1. All samples were diluted to 100 ml with phosphate
buffer pH 7.0. Quantitative determination was carried out by standard
addition method. Recovery tests were carried out by spiking standard
solutions of RIV in to pharmaceutical formulations. The blood serum
and urine samples were obtained from local pathology laboratory and
stored under refrigeration. Both samples were prepared by adding
50 μl of sample and diluted to 25 ml with phosphate buffer pH 7.0. No
pretreatment step was carried out for both the samples. Samples
cleaning were carried out by filtering through 0.22 μm PVDF syringe fil-
ter (Millex, Millipore Corporation). Electrochemical determination of
RIV was done by using DPV by spiking standard solutions of RIV to
urine and serum samples.
3. Results and discussion
3.1. XRD and UV–Vis spectroscopy
The XRD patterns of GNS and GNS–AuNP are shown in Fig. 1(A) and
(B). Graphene exhibit (Fig. 1(A)) the characteristics diffraction peaks at
25.6° and 43.5°. The two diffraction peaks in this pattern can be indexed
to the (002) and (111) reflection. On the other hand, AuNPs give five
peaks (Fig. 1(B)) at 38.11°, 43.80°, 64.50°, 77.50°, and 81.66° which cor-
respond to (111), (200), (220), (311) and (222) planes [32], respectively.
Hence, XRD pattern of GNS–AuNP confirmed formation of gold nanopar-
ticles and graphene successively. UV–Vis absorption spectra of GNS and
GNS–AuNP are shown in Fig. 1(C) and (D). GNS exhibits absorption
band at 271 nm [33]. This band is due to absorption of an aromatic л sys-
tem in the graphitic structure. Composite material shows two absorption
bands at 271 nm and 540 nm, respectively (Fig. 1(D)). The band at
271 nm is due to GNS and 540 nm is due to AuNPs [32] which further
confirms the formation of GNS and AuNP in the composite material.
3.2. SEM and EDX study
The surface morphology of the as prepared material was studied by
means of SEM (Fig. 2). It can be seen that graphene (Fig. 2(A)) shows
sheet like structure and composite (Fig. 2(B)) shows that gold nanopar-
ticle are uniformly coated on the graphene sheets. An EDX result of GNS
(Fig. 3(A)) shows elemental peak for carbon at 0.24 keV confirming for-
mation of graphene. However, no additional peaks were found in
graphene which confirms complete reduction of graphene oxide to
Fig. 3. EDX spectra for (A) GNS; (B) GNS–AuNP composite.
Fig. 2. SEM images for (A) GNS; (B) GNS–AuNP composite.

graphene. An EDX spectrum (Fig. 3(B)) reveals presence of all the
components of composite viz.; C, O, and Au confirming formation of
GNS–AuNP composite.
3.3. Effect of pH and supporting electrolyte
The effect of pH on peak current and peak potential for 2.0 × 10−4
M
RIV was investigated on carbon paste electrode employing Britton-
Robinson (B.R.) in the pH range 2–11 by differential pulse voltammetry.
As pH of the medium increases, the peak potentials shift towards less
positive values, indicating involvement of proton in the reaction.
There was no peak found in the pH range 2 to 4. It is also found that
the peak current was maximum (Fig. 4(A)) at pH 7.0 and hence was
used for further studies.
The relationship between peak potential (Ep) and pH was linear
(Fig. 4(B)) and is given by the following equation:
RIV : Ep Vð Þ ¼ −0:056 pH þ 1:325 R2
¼ 0:992

Form the equation, the slope of −0.056 V is very close to the expect-
ed slope of −0.059 V. Hence, it was confirmed that equal number of
Fig. 5. (A) Cyclic voltammograms of 6.0 × 10−5
M RIV at three different electrodes:(a) CPE( ), (b) GNS–CPE( ),and (c) GNS–AuNP–CPE ( ).Voltammetric conditions: scanning
electrode potential with a scan rate of 100 mV s−1
between +0.5 and +1.4 V in pH 7.0 phosphate buffer (0.1 M); (B) cyclic voltammograms for RIV 5.0 × 10−5
M obtained in phosphate
buffer (pH 7.0) employing varying scan rates (mV s−1
): (1 to 9) 10, 50, 100, 200, 400, 600, 800, 900, and 1000; (C) Ipvs scan rate plot for the data obtained from Fig. 4 (B); (D) Nyquist plots
for EIS measurements (1 × 10−3
M) K3[Fe (CN)]6/K4[Fe(CN)]6 at CPE ( ), GNS–CPE ( ), GNS–AuNP–CPE ( ) and in the box on the left upper side is the equivalent circuit used for
data fitting.
Fig. 4. (A) A plot of peak current (ip) vs. pH for 2.0 × 10−4
M RIV at CPE employing DPV; (B) a plot of peak potential (Ep) vs. pH for 2.0 × 10−4
M RIV at CPE employing DPV; step potential =
5 mV and modulation amplitude = 50 mV.

protons and electrons are involved in the oxidation of RIV. Additional
supporting electrolytes including phosphate, tris, HEPES, and citrate-
phosphate were studied at pH 7.0 in order to find out best supporting
electrolyte suitable for RIV using differential pulse voltammetry. The
best response, in terms of peak current and peak shape was obtained
when phosphate pH 7.0 (0.1 M) was employed (Fig. S2). Hence, selected
for further studies.
3.4. Effect of modifier
The modifier played a very important role in oxidation peak current
of RIV. The effect of the amount of GNS–AuNP as a modifier was studied
by varying its composition in the range of 1–12% with respect to graph-
ite. The peak current increases until 10% loading of the modifier; after
which it saturates. Therefore, 10% modifier loading was used for the
preparation of the modified GNS–AuNP–CPE. For comparison, CPE and
GNS–CPE were also prepared by the same procedure.
3.5. Cyclic voltammetry
The area of the electrodes was calculated by cyclic voltammetry
using Randles-Sevcik formula (1) in 1 mM K3Fe(CN)6/K4Fe(CN)6.
Ipa¼2:69 x 105
n3=2
A D1=2
υ1=2
C ð1Þ
Where, Ipa (A) is the anodic peak current, n is electron transfer num-
ber, A (cm2
) is surface area of the electrode, D (cm2
s−1
) is diffusion co-
efficient, ʋ (V s−1
) is scan rate, and C (mol cm−3
) is the concentration of
K3Fe(CN)6/K4Fe(CN)6. For 1 mM K3Fe(CN)6/K4Fe(CN)6 in the 1 M KNO3
electrolyte, n = 1 and D = 7.6 × 10−6
cm2
s−1
. The electrochemical re-
sponse signal (Fig. S3) of 1 mM K3[Fe(CN)]6/K4[Fe(CN)]6 is significantly
enhanced at GNS–AuNP–CPE which reflects the improvement of both
the shape of redox peak and the magnitude of the peak current Ip.
Thus, the sensitivity increases on employing GNS–AuNP–CPE. The calcu-
lated surface area is 0.013 cm2
, 0.034 cm2
and 0.070 cm2
, respectively
for CPE, GNS–CPE and GNS–AuNP–CPE. In case of GNS–AuNP–CPE the
area is increased by 5.38 times than that of simple CPE. This increase
in area is responsible for highly sensitive electrochemical sensor for de-
termination of RIV.
The electrocatalytic activity of the electrode was studied using CV by
sweeping the potential from 0.5 to 1.4 V. The cyclic voltammograms for
6.0 × 10−5
M RIV in phosphate pH.
7.0 on different electrodes are shown in Fig. 5(A). It is observed that
GNS–AuNP–CPE gives maximum current and well defined peak shape
compared to GNS–CPE and CPE, suggesting that oxidation of RIV is
more facile on GNS–AuNP–CPE than on the later two electrodes. It is
also observed from the Fig. 5(A) that oxidation of RIV is completely irre-
versible in nature.
The effect of scan rate on peak current and peak potential of
5.0 × 10−5
M RIV was studied from 10 mV s−1
to 1000 mV s−1
(Fig. 5(B)). It is observed that increasing scan rate results in regular
increase of anodic peak current along with a shift in peak potential
towards more positive values. The anodic peak current is found to
be linearly dependent on scan rate indicating that the oxidation of
RIV is controlled by adsorption at modified carbon paste electrode
(Fig. 5(C)). A fit to the current is gives the following regression equa-
tion:
Ipa μAð Þ ¼ 0:005ν þ 0:981 R2
¼ 0:994

:
The number of electrons involved in the reaction was calculated
using the equation,
Ep−Ep=2 ¼ 47:7=αnamV at 25
C:
Ep − Ep/2 is found to be 52 mV from CV. If there is no information
available about charge transfer coefficient (α), it can be taken as 0.5
for completely irreversible reaction [34]. Hence, by substituting the
values of α and Ep − Ep/2 in the above equation, the number of electrons
involved in the oxidation of RIV is calculated to be 2. The probable elec-
tron transfer mechanism is shown in Scheme. 2.
Scheme 2. Probable mechanism of RIV oxidation.

It is a two electron and two proton transfer process. In the first step,
the loss of an electron on nitrogen results in the formation of N-radical
cation, which subsequently forms C-radical by losing one proton. The
stability of C-radical increases as the extent of potential delocalization in-
creases. In the next step the C-radical losses one electron leading to the
formation of carbonium ion. The carbonium ion (benzylic carbocation)
is more stable as the positive charge on the central carbon atom gets dis-
persed over other carbon atoms and this renders stability to the carboni-
um ion [35]. The carbonium ion is very reactive intermediate it gets
reacted easily with nucleophilic water molecule to form amino alcohol
after losing one proton. In the final step the formation of ketone and
amine takes place by hydrolysis of the intermediate product.
3.6. Electrochemical impedance spectroscopy (EIS)
Electrochemical impedance spectroscopy technique is a powerful
method for the determination of the surface nature of the solution/elec-
trode [36]. The EIS spectrum has two parts; a semicircular and a linear.
The semicircular part at higher frequency corresponds to the electron
transfer limited process and its diameter is equal to the electron transfer
resistance. The Nyquist plots for K3 [Fe (CN)]6/K4 [Fe (CN)]6
(1 × 10−3
M) at CPE, GNS–CPE and GNS–AuNP–CPE are shown in
Fig. 5(D). The frequency range selected in the experimental studies
was from 10−1
to 106
Hz. As can be seen from the figure, the diameter
of semicircle decreases in going from CPE, to GNS–CPE, to the GNS-
AuNP–CPE electrode. The charge transfer resistance (Rct) value obtained
at CPE, GNS–CPE, and GNS–AuNP–CPE are 0.534 KΏ, 0.401 KΏ, and
0.259 KΏ, respectively. This clearly demonstrates that the charge trans-
fer resistance of the GNS–AuNPs–CPE is considerably small and the
charge transfer rate is higher than at the CPE and GNS–CPE surface.
Accordingly, the double layer capacitance (Cdl) obtained at the GNS–
AuNP–CPE is higher compared to the others two. Specifically, the Cdl is
measured to be 0.010 μF, 0.012 μF and 0.015 μF, respectively for the
CPE, GNS–CPE and GNS–AuNP–CPE.
3.7. Chronocoulometry
Double potential step chronocoulometry was employed to study the
kinetics and mechanism of electrode reactions involved in the electro-
oxidation of 2.0 × 10−5
M RIV at CPE, GNS–CPE, and GNS–AuNP–CPE.
The diffusion coefficient of RIV was calculated from the plot of Q vs. t1/2
using Anson equation [37]. The surface coverage for the three electrodes
is presented in Table 1. The surface coverage is calculated to be maxi-
mum at GNS–AuNP–CPE due to the synergistic effect of GNS and AuNP.
3.8. Adsorptive stripping differential pulse voltammetry (AdSDPV)
The effect of accumulation potential (Eacc) and accumulation time
(tacc) was investigated at GNS–AuNP–CPE for 8.0 × 10−5
M RIV sample.
Keeping tacc = 10 s, the Eacc was determined by changing the potential
from −0.5 to 1.0 V. However, there is no effect of accumulation poten-
tial on peak current up to 0.5 V, after which it increases and observed to
be constant between 0.6 and 0.8 V, thereafter it starts decreasing at
more positive potential which is given in Fig. 6(A). Since, the current ob-
tained was same in the accumulation potential range 0.6 to 0.8 V,
Eacc = 0.6 V is selected. By, keeping Eacc = 0.6 V, tacc was varied be-
tween 10 and 120 s. The oxidation peak current of RIV is found to in-
crease with the deposition time, reaching maximum at 60 s [Fig. 6(B)].
Further, increase in accumulation time shows a decrease in oxidation
peak current due to saturated adsorption of RIV on GNS–AuNP–CPE.
Thus, Eacc of 0.6 V and tacc of 60 s were selected as the optimum accumu-
lation potential and time for determination of RIV. The proposed meth-
od was used for determination of RIV in pharmaceuticals samples
(Table 2). It is found that the amount of RIV obtained by present method
was found to agree well with the label contents.
A comparative study was carried out for 3.0 × 10−5
M RIV employing
AdSDPV on CPE, GNS–CPE and GNS–AuNP–CPE. As shown in Fig. 7(A),
the best results in terms of peak current are obtained on GNS–AuNP–
CPE. The reasons for the notable sensitivity of the GNS–AuNP–CPE elec-
trode are the much favorable properties of the GNS–AuNP composite,
specifically high electrical conductivity, large specific surface area, and
excellent adsorptive and catalytic abilities. This leads to a facile electro
oxidation of RIV at the surface of GNS–AuNP–CPE electrode.
3.9. Determination of RIV
The encouraging findings on the electrochemical and electrocatalyt-
ic performance of the GNS–AuNP composite enabled us to develop
Fig. 6. (A) Influence of accumulation potential on the oxidation peak current of 8.0 × 10−5
M RIV on GNS–AuNP–CPE; (B) Influence of accumulation time on the oxidation peak current of
8.0 × 10−5
M RIV on GNS–AuNP–CPE employing step potential of 5 mV and modulation amplitude of 50 mV in phosphate buffer solution (pH 7.0).
Table 1
Chronocoulometry of 2.0 × 10−5
M RIV at three electrodes.
Molecule Electrode Slope (μC s1/2
) Qads(μC) Surface coverage
(10−12
mol cm−2
)
Diffusion coefficient
(10−6
cm2
s−1
)
RIV CPE 0.22 0.41 2.11 7.16 ± 0.06
GNS–CPE 0.38 1.35 7.0 6.92 ± 0.04
GNS–AuNP–CPE 0.90 2.67 13.8 10.8 ± 0.05

a simple and suitable method for determining RIV employing AdSDPV
at GNS–AuNP–CPE. The optimized parameters established in the
previous sections, were used for calculating the limit of detection
(LOD = 3 × SD/s), where SD is the standard deviation for the intercept
of the regression line and ‘s’ is the slope of the linear calibration plot, lin-
ear working range (LWR), linear regression equation (LRE), and correla-
tion coefficient (r). Validation of the proposed procedure for assay of
standard RIV was examined via evaluation of LOD, limit of quantitation
(LOQ), reproducibility, precision, and selectivity. Under the optimized
conditions, Ip (μA) was proportional to the RIV concentration in the
range (Fig. 7(B)) of 2.0 × 10−7
M − 6.0 × 10−4
M with a LOD of
5.27 × 10−8
M (% RSD = 3.17).
3.10. Interference studies, validation and analytical applications
The effect of possible interferents on the electrochemical determina-
tion of RIV was investigated by adding potential interferents containing
compounds to a solution containing 1.0 × 10−6
M RIV in pH 7.0 phos-
phate buffer. The tolerance limit for interfering species was considered
as the maximum concentration that gave a relative error (%RSD) less
than ±5.0%. Some of the commonly found ions and species in biological
samples involving K+
, Na+
Ca2+
, NO3
−
, NH4
+
, Cl−
and Mg2+
were con-
sidered for interference study. The K+
, Na+
, Cl−
ions did not interfere
in the analysis till 250 fold excess. Moreover, no interference was ob-
served till 130 fold excess in case of Ca2+,
Mg2+
, NO3
−
, SO4
2−
. Glucose,
ascorbic acid did not interfere till 30 fold excess. It is clear that, the de-
termination of RIV is not considerably affected by common interfering
species. This lends confidence in the method presented here to be high-
ly selective and for its use in identifying biological samples. In order to
validate the method, various parameters such as repeatability, repro-
ducibility, precision and accuracy of analysis were obtained by
performing five replicate measurements for standard RIV 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 showed in [Table S1] for the recoveries ob-
tained confirm both high precision of the proposed procedure and the
stability of RIV solutions. For further evaluation of the validity of the
proposed method, recovery tests were carried out in pharmaceutical
formulations, urine and human blood serum samples (Table S2).These
tests gave % R values in the range of 98.55–100.85%. Similarly, recovery
tests were performed on urine sample collected from healthy volunteer.
The % R obtained for this sample were in the range of 97.26–100.65%
(Table S2). The recovery tests performed on human blood serum sample
gave the % R values the range of 97.71–101.12% (Table S2). These results
show that the recovery of RIV is not affected significantly, and conse-
quently, the described method is accurate for its assay in complex ma-
trices. For analytical applications, the determination of the amount of
RIV in all samples was carried out by the standard addition method.
The amount of RIV obtained in the pharmaceutical formulations by
the proposed method was found to agree well with the label contents.
The results also showed that interferences from the matrix were
negligible. The long term stability of the GNS–AuNP–CPE electrode
was evaluated by intermittently measuring voltammetric response for
3.5 × 10−5
M RIV for 60 days. The electrode was stored in dry condition
at room temperature when not in use. It is found that after 60 days only
minimal decrease of current sensitivity (Fig. S4) with a relative standard
deviation of 2.3% was observed, which shows long term stability of the
proposed sensor.
3.11. Comparison of proposed method with literature method
A comparison of the analytical performance of the proposed method
with previously reported voltammetric methods for determination RIV
[29,30] is shown in Table 3. The data reveal that the GNS–AuNP–CPE
shows a superior analytical performance in terms of the detection
limit, linear dynamic range, sensitivity, reproducibility, and repeatabili-
ty over the methods reported in literature. In addition, the present
method is simple and does not involve any pretreatment step.
4. Conclusion
The electrochemical behavior of RIV at GNS–AuNP–CPE has been
studied and discussed. The gold nanoparticles (AuNP) were uniformly
deposited onto the surfaces of graphene sheets by reducing simulta-
neously with graphene oxide using sodium borohydride. The proposed
method enables loading a large amount of AuNP on individual graphene
sheets with uniform morphology. We demonstrate that the proposed
adsorptive stripping differential pulse voltammetry using GNS–AuNP–
CPE can be successively used for the determination of RIV in pharma-
ceutical formulations as well as in biological samples. The method is
simple, accurate, and does not require any pretreatment step such as,
extraction of an analyte from pharmaceutical as well as biological sam-
ples. The percentage recovery obtained for pharmaceuticals as well as
biological samples is in the range of 97.71–101.12% showing high
Fig. 7. (A) AdSDPV of 3.0 × 10−5
M RIV at three different electrodes: (a) CPE ( ), (b) GNS–CPE ( ), and (c) GNS–AuNP–CPE ( ). Voltammetric conditions: Eacc = 0.6 V, tacc =
60 s, in phosphate buffer (pH 7.0), step potential = 5 mV and modulation amplitude = 50 mV; (B) AdSDPV curves obtained at GNS–AuNP–CPE for RIV at different concentrations: in the
range from (1) blank, (2) 2.0 × 10−7
, (3) 2.0 × 10−6
, (4) 6.0 × 10−6
, (5) 2.0 × 10−5
, (6) 4.0 × 10−5
, (7) 6.0 × 10−5
, (8) 2.0 × 10−4
, and (9) 6.0 × 10−4
M.
Table 2
Determination of RIV by proposed method.
Sample RIV
a b
Rivamer 1.5 1.5 1.47 ± 3.0
Rivamer 3.0 3.0 2.94 ± 2.3
a: amount of RIV in a tablet (mg).
b: amount of RIV obtained by the proposed method (mg) ± % RSD (n = 5).

selectivity of the proposed method. The proposed method is efficient
and more sensitive compared to the methods reported in the literature
for RIV determination and can be readily adapted to clinical as well as
pharmaceutical analyses.
Acknowledgments
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 and partly by the US Army Internation-
al Technology Center, Tokyo, Japan through grant number FA5209-09-
P-02.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jelechem.2015.09.027.
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Table 3
Comparison between various electroanalytical methods for the determination of RIV with the proposed method.
Electrode Linear working range (M) Limit of detection
(M)
Samples analyzed References
Glassy carbon disk electrode 7.0 × 10−5
to 8.0 × 10−4
1.69 × 10−5
Pharmaceutical formulations [29]
MIP1
-CPE 2.0 × 10−6
to 1.0 × 10−3
4.4 × 10−7
Pharmaceutical formulations, Urine, blood serum [30]
GNS–AuNP–CPE 2.0 × 10−7
to 6.0 × 10−4
5.3 × 10−8
Pharmaceutical formulations, Urine, blood serum This work
1
Molecularly imprinted polymer.

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