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imultaneous voltammetric determination of paracetamol andomperidone based on a graphene/platinum nanoparticles/nafionomposite modified glassy carbon electrode
ramod K. Kalambatea, Bankim J. Sanghavib, Shashi P. Karnac, Ashwini K. Srivastavaa,∗
Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (East), Mumbai 400 098, IndiaDepartment of Electrical and Computer Engineering, University of Virginia, Charlottesville, VA 22904, USAU.S. Army Research Laboratory, Weapons and Materials Research Directorate, ATTN: RDRL-WM, Aberdeen Proving Ground, MD 21005-5069, USA
r t i c l e i n f o
rticle history:eceived 12 September 2014eceived in revised form 22 January 2015ccepted 16 February 2015vailable online 26 February 2015
Graphene oxide and hexachloroplatinic acid were electrochemically reduced on a glassy carbon elec-trode (GCE) surface so as to form a graphene (Gr)–platinum nanoparticles (PtNP) composite. This nanocomposite was then coated with nafion (NAF) film so as to form NAF/PtNP/Gr/GCE. In this work, anelectrochemical method based on adsorptive stripping square wave voltammetry (AdSSWV) employ-ing NAF/PtNP/Gr/GCE has been proposed for the subnanomolar determination of paracetamol (PCT) anddomperidone (DOM) simultaneously. The electrode material was characterized by scanning electronmicroscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction. The electrochemical perfor-mance of PCT and DOM on modified electrode was investigated by cyclic voltammetry, electrochemicalimpedance spectroscopy, and chronocoulometry. A sixteen fold enhancement in the AdSSWV signal wasobserved at NAF/PtNP/Gr/GCE in pH 6.0, phosphate buffer, as compared to GCE. Under the optimized con-ditions, the method allowed simultaneous determination of PCT and DOM in the linear working range of
−6 −9 −10 −10
afion 8.2 × 10 –1.6 × 10 M with detection limits (3 × SD/s) of 1.06 × 10 and 4.37 × 10 M for PCT andDOM respectively. The practical analytical utilities of the modified electrode were demonstrated by thedetermination of PCT and DOM in pharmaceutical formulations, human urine, and blood serum samples.This proposed method was validated by HPLC and the results are in agreement at the 95% confidencelevel. Simultaneous voltammetric determination of PCT and DOM has been reported for the first time.
Paracetamol (PCT) is widely used all over the world as aharmaceutical analgesic and antipyretic agent [1]. DomperidoneDOM), chemically, known as 5-chloro-1-[1-[3-(2,3-dihydro--oxo-1H-benzimidazol-1-yl) propyl]-4-piperidinyl]-1,3-dihydro-H-benzimidazol-2-one. DOM is a dopamine antagonist used as anntiemetic for the short term treatment of nausea and vomiting ofarious etiologies [2]. A combination of PCT and DOM works in aynergistic manner. PCT relieves symptoms of migraine and DOMncreases the contractions of the stomach and intestines, which
ids in a quick absorption of PCT. However, an overdose of DOMay results in high accumulation of PCT. Symptoms of DOM over-
ose also include drowsiness, dizziness, confusion, twitching, and
irregular heartbeats. Therefore, the development of a sensitive andselective method for their simultaneous determination is highlydesirable for analytical applications and diagnostic research.
Literature survey reveals several analytical methods for simul-taneous determination of PCT and DOM including capillaryelectrophoresis [3], chromatography [4], and spectroscopy [5].However these methods are lengthy, overpriced, complicated,require expert knowledge, and often need the pretreatment stepthat makes them unsuitable for routine analysis. On the other hand,electrochemical methods are used extensively over other methodsdue to simplicity, low cost, and relatively short analysis time. Thereare several voltammetric methods reported for individual deter-mination of PCT [6–11] but only two voltammetric methods areavailable for DOM determination [12,13]. There is no official elec-trochemical method reported for simultaneous determination of
PCT and DOM. Hence, it is essential to develop simple yet sensitivemethod for simultaneous determination of PCT and DOM.
Over the past two decades, chemically modified electrodes(CMEs) have attracted broad interest in sensor development due
o low background current, wide range of potential window, easyurface renewal, lower detection limit, and low sensitivity toissolved oxygen. Because of these advantages CMEs have beensed in various analyses [14–17]. In Recent years, our group haseported chemically modified electrodes for determination of var-ous organic [18–24] as well as inorganic species [25,26].
Graphene (Gr) is a two dimensional (2D) one atom thickanomaterial consisting of sp2 hybridize carbon, has attractedremendous attention due to its unique properties, such as highurface area, excellent electrical conductivity, and well electrocat-lytic activity [27,28]. Because of these properties graphene haseen used as an ideal electrode material in supercapacitors [29],eld effect transistors [30], and chem/bio sensors [31]. Grapheneas been used for the sensitive determination of various drugsolecules, due to their excellent conductivity because of �–�
tacking and synergetic effects with other materials [32–34]. Plat-num nanoparticles (PtNPs), on the other hand, due to their largespect ratio (surface area to volume), biocompatibility, and highlectrical conductivity have also been widely employed as a mod-fier in voltammetry for analysis of organic molecules [35,36].he introduction of metal nanoparticles into the dispersion ofraphene sheets could inhibit the aggregation of graphene sheets,nd result in mechanically jammed, exfoliated graphene agglom-rate with very high surface area [37]. Nafion, a perfluorinatedulphonated cation exchanger with properties of excellent antifoul-ng capacity, chemical inertness, and high permeability to cations,as been extensively employed as an electrode modifier for organicolecules [38–40]. Thus, a synergistic effect of Gr, PtNP, and NAF
omposite film modified GCE can enable a sensitive determinationf PCT and DOM.
The present study deals with simultaneous determinationf PCT and DOM using nafion/platinum nanoparticles/grapheneodified glassy carbon electrode employing adsorptive stripping
quare wave voltammetry (AdSSWV). The morphological and elec-rochemical characterization of the electrode material has beenarried out by using various techniques, such as scanning electronicroscopy (SEM), energy dispersive X-ray spectroscopy (EDX),-ray diffraction (XRD), cyclic voltammetry (CV), electrochem-
cal impedance spectroscopy (EIS), and chronocoulometry (CC).he developed AdSSWV method based on nanocomposite showsood sensitivity and selectivity for determination of PCT and DOMn pharmaceutical formulations, urine, and blood serum samples.
oreover, the proposed voltammetric method has been validatedy HPLC and the results obtained are in good accordance with thosebtained by the proposed method.
. Experimental
.1. Chemicals and instrumentation
All chemicals were of A. R. grade and were used as received with-ut any further purification. Paracetamol and domperidone (≥98%)ave been procured from Sigma Aldrich. Graphite powder (99%race metals basis) was purchased from S. D. Fine (India). Chloro-latinic acid hydrate (H2PtCl6·×H2O) was obtained from Sigmaldrich. Nafion (NAF, 1100EW, 5 wt% aqueous alcoholic solution,ldrich) was prepared as 0.1% solution by dilution with ethanol.ll solutions were prepared using double distilled water of specificonductivity (0.3–0.8 �S). Phosphate buffer solution (PBS; 0.1 M,H 6.0) was employed as a supporting electrolyte. The developedethod was employed for analysis of the following pharmaceut-
T (5 mg), Acedome (PCT: DOM = 500 mg: 10 mg), Acemol-D (PCT:OM = 500 mg: 10 mg), and Grenil (PCT: DOM = 500 mg: 20 mg).
The voltammetric measurement were performed using anutolab PGSTAT 30 equipped with USB electrochemical interface
tuators B 213 (2015) 285–294
using GPES software, version 4.9.005 and frequency responseanalyzer, software version 2.0 respectively. Conventional three-electrode system employing, a GCE (diameter = 3 mm) was usedas working electrode, platinum wire, and Ag/AgCl (sat. KCl) wereused as counter and reference electrodes, respectively. The pHmeasurements were performed using ELICO LI 120 pH meter.HPLC used for validating the method was an Agilent model 1100.XRD analysis was carried out on an X-ray diffractometer (Shi-madzu 7000S, Shimadzu Analytical, Japan) equipped with CuK�
radiation (� = 0.154 nm). Surface morphology of the materials wasinvestigated by SEM.
2.2. Preparation of the nafion/platinumnanoparticles/graphene/GCE (NAF/PtNP/Gr/GCE)
The glassy carbon electrode was polished, at the start of thework, with aqueous slurries of alumina powders (1.0 �m and0.3 �m �-Al2O3) on polishing cloth until mirror-like finish wasobtained. After that, the electrode was ultrasonicated in distilledwater for about 30 s, and finally allowed to dry under infraredlamp. Graphene oxide (GO) was synthesized directly from graphiteby Hummers method [41]. The synthesized graphite oxide pow-der was exfoliated in doubly distilled water by ultrasonicationfor 120 min to form homogeneous GO dispersions with a concen-tration of 1.0 g L−1. Graphene and PtNP were prepared accordingto literature procedure via electroreduction [42,43]. This proce-dure for fabrication of modified electrode is depicted pictoriallyin Scheme 1. Preparation of graphene–platinum nanocompositesis a two-step process. In the first step 10.0 mg L−1 GO solutionwas prepared. The GCE was placed into this solution, and five CVscans between +0.6 V and −1.5 V were carried out so as to con-vert GO to Gr. This modified GCE was then dipped into a solutionof 10 mM H2PtCl6 (in 0.1 M NaCl). The electrochemical reductionto PtNP was performed under magnetic stirring by applying anelectro-deposition potential of −0.7 V for 40 s. After electrochem-ical reduction, the working electrode was washed with doublydistilled water, and dried under I. R. lamp. Finally, NAF modifica-tion was carried out by drop casting (5.0 �L, 0.1%) on to the surfaceof the GCE, and the solvent was allowed to evaporate at roomtemperature. Thus, NAF/PtNP/Gr/GCE was fabricated. For compar-ison, GO/GCE, Gr/GCE and PtNP/Gr/GCE were prepared using samemethod.
2.3. Voltammetric procedures
Stock solutions of PCT and DOM were prepared in double dis-tilled water. The required quantity of the stock solution was placedin to a 25.0 cm3 standard volumetric flask, and the total volume wasmade to 25 cm3 with PBS, pH 6.0 (0.1 M). An AdSSWV was used fordetermination of both the drugs, and optimized adsorptive strip-ping square wave voltammetric parameters were: accumulationpotential: −0.3 V, accumulation time: 90 s, equilibrium time: 15 s,square wave frequency: 100 Hz, step potential: 5 mV, modulationamplitude: 50 mV. The voltammogram was then recorded by scan-ning the potential toward the positive direction from −0.2 to +1.1 V.Cyclic voltammetric experiments were carried by sweeping poten-tial from −0.2 to +1.3 V. Double potential step chronocoulometrywas carried out with a pulse period of 5 s vs. Ag/AgCl. EIS study hasbeen done in the frequency range of 10−2–106 Hz at open circuitpotential with amplitude of 5 mV.
2.4. Treatment and determination of samples
Pharmaceutical formulations, urine, and blood serum sampleswere analyzed for determination of PCT and DOM. Twenty tabletswere weighed, and then finely powdered using mortar and pestle
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n of s
atrppreismss
3
3s
ngFftptt
banlsF
Scheme 1. Schematic illustratio
nd diluted to 100 mL with pH 6.0 phosphate buffer. The portion ofhe resulting solution was taken out, and transferred to voltammet-ic cell. Urine and blood serum samples were obtained from localathology laboratory. For the determination in urine samples, nore-treatment step was carried out. To avoid interferences occur-ing from the serum matrix, a 50 �L serum sample was added to thelectrochemical cell containing 25 mL of buffer solution. The clean-ng of all the samples was done by filtering through a 0.22 �m PVDFyringe filter (Millex, Millipore Corporation) prior to voltammetriceasurements. Recovery tests were carried out by spiking standard
olutions of PCT and DOM in pharmaceutical formulations, blooderum, and urine samples.
. Results and discussion
.1. Characterization of materials by XRD, EDX, SEM, Ramanpectroscopy and FT-IR analysis
The XRD diffraction pattern of the prepared graphene and Ptanoparticles composite is shown in Fig. S1. It can be seen thatraphene shows an XRD peak at a 2� value of 26.8о for (0 0 2) peak.ig. S1 further shows characteristic property of the crystalline Ptace-centered cubic (fcc) phase. The peaks were characterized byhe first (1 1 1) peak, and the next (2 0 0), (2 2 0), (3 1 1), and (2 2 2)eaks to the 2� values of 39.8, 46.1, 67.6, 81.4, and 86.1о, respec-ively. The XRD peaks of Pt nanoparticles are sharp and comparableo those of the corresponding bulk Pt material.
The surface morphology of the electrode material was studiedy means of SEM. Fig. 2(A) shows sheets of graphene oxide. Fig. 2(B)nd (C) are the SEM images of as prepared Gr at two different mag-
ifications and it is seen from the figures that graphene shows sheet
ike structure. Fig. 2(D) is the SEM image for graphene and PtNPshowing the presence of PtNPs as a cluster along with graphene.ig. 2(E) is the image of the NAF/PtNP/Gr composite employed in
tepwise electrode modification.
the present work viz., NAF/PtNP/Gr. The SEM image of NAF/PtNP/Grcomposite shows that the nafion film is uniformly coated on to thegraphene and PtNPs surface. Fig. 2(F) is the energy-dispersive X-rayspectrum for the final composite, which shows the following ele-ments: C from graphene and nafion; Pt from PtNPs and F, O, S fromnafion confirming the presence of all the components of composite.
In order to confirm electroreduction of GO to Gr Raman and IRspectroscopy was carried out. The Raman spectra of GO (Fig. S2(a))shows D and G peaks at 1354 and 1600 cm−1, respectively. Simi-larly, Gr (Fig. S2(b)) show two sharp peaks at 1350 and 1598 cm−1.They are called as D and G bands of Gr. The relative intensity ratio ofboth peaks (ID/IG) is a measure of disorder degree. It is seen from thefigure that ID/IG ratio for Gr is larger than that for GO (1.61 for Gr and1.32 for GO). This suggests that more graphitic domains are formedand the sp2 cluster number is increased after electroreduction ofGO.
FT-IR spectra of GO and Gr are shown in Fig. S3. The presence ofdifferent oxygen functionalities in GO were confirmed at 3417 cm−1
(O H stretching vibrations), at 1720 cm−1 (stretching vibrationsfrom C O), at 1600 cm−1 (skeletal vibrations from unoxidizedgraphitic domains), at 1220 cm−1 (C OH stretching vibrations),and at 1060 cm−1 (C O stretching vibrations). FTIR peak of Grshows that O H stretching vibrations observed at 3417 cm−1 wassignificantly reduced due to deoxygenation. However, stretchingvibrations from C O at 1720 cm−1 were still observed and C Ostretching vibrations at 1060 cm−1 became sharper, which werecaused by remaining carboxyl groups even after electroreduction.
3.2. Effect of pH and supporting electrolyte
The effect of pH on the response of PCT (3.35 × 10−6 M) andDOM (3.70 × 10−6 M) was evaluated using differential pulse vol-tammetry employing glassy carbon electrode. The effect of pH onpeak potential and peak current of PCT and DOM was investigated
288 P.K. Kalambate et al. / Sensors and Ac
oIsicpff
P
D
apToapSifo
3a
tdadsciaptttDpDmoctHtfiNe
Scheme 2. Probable mechanism of paracetamol.
ver the pH range of 2–12 employing Britton-Robinson (B.R.) buffer.t was observed that the peak potentials of both the moleculeshifted toward less positive values as pH of the medium wasncreased. The pH of the supporting electrolytes influences peakurrent and potential of PCT and DOM suggesting involvement ofroton in the oxidation reactions. The plot of Ep vs. pH (Fig. S4) wasound to be linear with the slope of −57.4 mV/pH and −58.2 mV/pHor PCT and DOM respectively regarding following equations:
Hence, it was evident that equal no of protons and electronsre involved in the reaction. It was observed that, the maximumeak current was obtained at pH 6.0 for both the drugs (Fig. S5).hus, this pH was employed for further studies. The effect of vari-us supporting electrolytes on peak current and peak shape of PCTnd DOM was studied by employing various buffers Viz.; phos-hate, tris, citrate-phosphate, and acetate with the same pH (Fig.6). However, pH 6.0 (0.1 M) phosphate buffer gave better responsen terms of peak shape and peak current. Therefore it was chosen forurther study. The probable reaction mechanism for the oxidationf PCT is as given in Scheme 2.
.3. Effect of Gr, PtNP and nafion on the oxidation peak of PCTnd DOM
The effect of modifier is crucial as it changes the properties ofhe electrode surface by lowering the detection limit. The stableispersion of GO was obtained by dispersing 10.0 mg L−1 in water,nd CV scan was run between +0.6 V and −1.5 V. The amount ofeposits was controlled by number of CV cycles, and it is found thatharp increase in oxidation peak current was observed till five CVycles. Therefore, five CV cycles were chosen for further study. Plat-num nanoparticles were electrochemically deposited by applyingccumulation potential of −0.7 V for 40 s. A sharp increase in theeak current for PCT and DOM was observed up to 40 s depositionime. Further increasing the deposition time caused a decrease inhe peak current which suggested that nanoparticles film turnedhicker and hence the electron transfer rate was hindered. PCT andOM have pKa values of 9.5 and 8.0 respectively, thus they exist asositively charged species at pH 6.0. The positively charged PCT andOM exchange with the H+ from NAF, thus facilitating their accu-ulation onto the electrode surface. Thus, optimizing the amount
f nafion as a modifier is necessary. The relationship between peakurrents and the amount of 0.1% NAF is shown in Fig. S7. Initially,
he peak current increases with increasing amount of 0.1% nafion.owever, when the amount exceeds 5 �L, the peak current starts
o decrease. Thus, 5 �L of nafion was used to prepare the modi-ed electrode. NAF/PtNP/Gr/GCE was prepared by dropping 5 �L ofAF onto the PtNP/Gr/GCE surface, and the solvent was allowed tovaporate under IR lamp.
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3.4. Cyclic voltammetry (CV)
The electrocatalytic activity of NAF/PtNP/Gr/GCE toward the PCTand DOM was studied by cyclic voltammetry in the potential rangeof −0.2 V to +1.3 V. CV plots for 3.6 × 10−7 M PCT and DOM at GCE,GO/GCE, Gr/GCE, PtNP/Gr/GCE, and NAF/PtNP/Gr/GCE are shownin Fig. 1(A). In the CV voltammograms the good peaks with max-imum current was observed at NAF/PtNP/Gr/GCE, which impliesthat the reaction is more facile at the modified electrode. It can beseen from Fig. 1(A) that PCT shows quasireversible wave with oxi-dation peak on forward scan and small reduction peak on reversescan, while DOM is irreversible in nature. The effect on scan rateon peak current and peak potential is shown in Fig. 1(B). It isobserved that oxidation peak current varied linearly with increas-ing scan rate ranging from 10 mV s−1 to 1000 mV s−1 (Fig. 1(C)),which implies that oxidation of PCT and DOM is kinetically con-trolled at NAF/PtNP/Gr/GCE by following equations:
PCT : Ip(�A) = 0.013� + 3.0390 (R2 = 0.9988) (3)
DOM : Ip(mA) = 0.0079� + 2.1894 (R2 = 0.9976) (4)
PCT undergoes a two electron and two proton redox reactionto form N-acetyl-p-quinone imine [6] This is a quasi-reversibleprocess (Scheme 2).
The no. of electrons involved in oxidation of DOM was calculatedby following equation:
Ep − Ep/2 = 47.7/˛na mV at 25 ◦C (5)
Ep − Ep/2 was found to be 98.2 mV from cyclic voltammetry andelectron transfer coefficient (˛) is taken as 0.5 for irreversible reac-tion. Hence, by submitting the values of Ep − Ep/2 and ̨ in aboveequation, n was found to be 1. It is reported in the literature thatthe oxidation of nitrogen atom of amide group is most probablyinvolved in the oxidation, with one electron and one proton pro-ducing a free radical in the rate-determining step [12,13].
3.5. Chronocoulometry (CC) and electrochemical impedancespectroscopy (EIS)
Double potential step chronocoulometry was employed forthe determination of the kinetics, and mechanism of electrodereactions involved in the electro-oxidation of 1.81 × 10−5 M PCTand 5.75 × 10−5 M DOM at GCE, GO/GCE, Gr/GCE, PtNP/Gr/GCE, andNAF/PtNP/Gr/GCE. The diffusion coefficient of PCT and DOM werecalculated from the plot of Q vs. t1/2 using Anson equation [44].The surface coverage for all the electrodes is presented in Table S1.The maximum surface coverage was obtained at NAF/PtNP/Gr/GCEdue to the synergistic effect of Gr, PtNP, and NAF. Electrochemicalimpedance spectroscopy technique is a powerful method for thedetermination of the surface nature of the solution/electrode[45]. The EIS spectrum has two parts semicircular parts andlinear parts. The semicircular part at higher frequency corre-sponds to the electron transfer limited process, and its diameteris equal to the electron transfer resistance. The Nyquist plotsfor K3[Fe(CN)]6/K4[Fe(CN)]6 (0.75 × 10−3 M) at the GCE, GO/GCE,Gr/GCE, PtNP/Gr/GCE, and NAF/PtNP/Gr/GCE are shown in Fig. 1(D).It is clearly visible that bare GCE exhibits large diameter with morecharge transfer resistance, upon addition of modifiers on GCE thediameter of Nyquist plot’s changes, resulting low value of Rct.But, in case of NAF/PtNP/Gr/GCE the charge transfer resistanceincreases due to electrostatic repulsion of negatively charged ferri-cyanide ions. The charge transfer resistance (Rct) for GCE, GO/GCE,
Gr/GCE, PtNP/Gr/GCE, and NAF/PtNP/Gr/GCE were 0.746 ± 1.66,0.303 ± 2.07, 0.261 ± 2.48, 0.209 ± 1.98, and 0.242 ± 2.12 K Ohm,respectively. The double layer capacitance obtained at higherfrequency for GCE, GO/GCE, Gr/GCE, PtNP/Gr/GCE, and NAF/PtNP/
P.K. Kalambate et al. / Sensors and Actuators B 213 (2015) 285–294 289
Fig. 1. (A) Cyclic voltammograms of 3.6 × 10−7 M PCT and DOM at four different electrodes: (a) GCE ( ), (b) GO/GCE ( ), (c) Gr/GCE ( ), (d) PtNP/Gr/GCE( ) and (e) NAF/PtNP/Gr/GCE ( ). Voltammetric conditions: scanning electrode potential with a scan rate of 50 mV s−1 between −0.2 and +1.3 V in pH 6.0p ) obta
( asurem
G box o
Ga
3
mpmctGtrift
3P
3
m
hosphate buffer (0.1 M); (B) Cyclic voltammograms of PCT and DOM (3.6 × 10−7 M
C) Ip vs. scan rate plot for the data obtained from (B); (D) Nyquist plots for EIS me
r/GCE ( ), PtNP/Gr/GCE ( ) and NAF/PtNP/Gr/GCE ( ). In the
AdSSWV was employed for examining the influence of accu-ulation potential and accumulation time on the oxidation
eak current of PCT and DOM at GEC (Fig. S8). After opti-ization of experimental parameters it is found that, peak
urrent was maximum at Eacc = −0.3 V and tacc = 90 s. Fig. 3(A) ishe AdSSWV for the 1.54 × 10−7 M PCT and DOM on the GCE,O/GCE, Gr/GCE, PtNP/Gr/GCE, and NAF/PtNP/Gr/GCE. It shows
hat NAF/PtNP/Gr/GCE gives higher response in terms of peak cur-ent compared to the other electrodes. The enhanced peak currentmplies that electrochemical oxidation of PCT and DOM is moreacile on NAF/PtNP/Gr/GCE, which results in higher sensitivity ofhe present method.
.7. Individual and simultaneous voltammetric determination ofCT and DOM
.7.1. Determination of PCT and DOM individuallyThe above findings enabled us to develop a simple and suitable
ethod for PCT and DOM employing AdSSWV at NAF/PtNP/Gr/GCE.
ents (0.75 × 10−3 M K3[Fe(CN)]6/K4[Fe(CN)]6) at GCE ( ), GO/GCE ( ),
n the right upper side is the equivalent circuit used for data fitting.
The optimized parameters were used for calculating the limit ofdetection (LOD; 3 × SD/s; where SD is the standard deviation forthe intercept of the regression line, and ‘s’ is the slope of the lin-ear calibration plot), linear working range (LWR), linear regressionequation (LRE), and correlation coefficient (r) (Table 1(A)).
3.7.2. Determination of PCT and DOM simultaneouslyUnder optimized conditions, AdSSWV was used for simulta-
neous determination of PCT and DOM in synthetic samples atNAF/PtNP/Gr/GCE. In the first case, the voltammetric response ofdifferent concentrations of PCT and DOM was evaluated keepingone constant and the other changing as shown in Fig. 3(B) and (C).Results revealed that the change in concentration of one drug inthe mixture did not have any effect on the peak current and peakpotential of the other one. In the second case, the concentrationof PCT and DOM were increased simultaneously, and response isshown in Fig. 3(D). The statistical results on these measurementsare summarized in Table 1(B)–(D).
It is noteworthy that the comparison of these results with the
ones obtained for determination of PCT and DOM individually, theobtained LOD and LWR are of the same magnitude, indicating thatthe simultaneous determination of PCT and DOM is as efficient astheir individual determinations.
290 P.K. Kalambate et al. / Sensors and Actuators B 213 (2015) 285–294
Fig. 2. SEM images of (A) Graphene oxide, (B, C) Graphene, (D) PtNP/Gr, (E) NAF/PtNP/Gr composite film and (F) Energy-dispersive X-ray spectrum for NAF/PtNP/Gr.
Table 1Analytical parameters for electrochemical determination of PCT and DOM in pH 6.0 phosphate buffer.
No. Molecule LWR (M) LRE R2 LOD (M) % RSD
(A) Statistical data for individual molecules1 PCT 1.55 × 10−9–8.30 × 10−6 Ip (�A) = 0.0371 C (nM) + 1.507 0.9990 1.01 × 10−10 1.502 DOM 1.56 × 10−8–8.25 × 10−6 Ip (�A) = 0.0265 C (nM) + 1.455 0.9963 4.31 × 10−10 1.87
(B) Statistical data for PCT when the concentration of DOM is kept constant (1.95 × 10−7 M)3 PCT 1.57 × 10−9–8.26 × 10−6 Ip (�A) = 0.0370 C (nM) + 1.509 0.9985 1.05 × 10−10 1.58
(C) Statistical data for DOM when the concentration of PCT is kept constant (1.06 × 10−7 M)4 DOM 1.58 × 10−8–8.22 × 10−6 Ip (�A) = 0.0263 C (nM) + 1.455 0.9954 4.35 × 10−10 1.81
(D) Statistical data for PCT and DOM simultaneously5 PCT 1.60 × 10−9 Ip (�A) = 0.0368 C (nM) + 1.511 0.9982 1.06 × 10−10 1.57
to) = 0.0
L n; LR
3
a
DOM 8.20 × 10−6 Ip (�A
WR, Linear working range; RSD, relative standard deviation; LOD, Limit of detectio
.8. Interference and validation studies
The effect of some possible interferents was investigated byddition of the compounds to a solution containing 1.06 × 10−8 M
262 C (nM) + 1.455 0.9949 4.37 × 10−10 1.83
E, Linear regression equation; r, correlation co-efficient.
PCT and 5.78 × 10−8 M DOM in pH 6.0 phosphate buffer. Sincenafion is cation exchange resin most of the interferents like ascor-bic acid, uric acid, and citric acid did not interfere in the analysisbecause all of them are present in anionic form when phosphate pH
P.K. Kalambate et al. / Sensors and Actuators B 213 (2015) 285–294 291
Fig. 3. (A) AdSSWV of 1.54 × 10−7 M PCT and DOM at four different electrodes: (a) GCE ( ), (b) GO/GCE ( ), (c) Gr/GCE ( ), (d) PtNP/Gr/GCE( ) and (e) NAF/PtNP/Gr/GCE ( ). Voltammetric conditions: Eacc = −0.3 V, tacc = 90 s, f = 100 Hz in 0.1 M phosphate buffer (pH 6.0), step potential = 5 mV andm E for P −7 −9
t at d8 ual co
6ednit
rs3aosTtapdapaar
odulation amplitude = 50 mV. (B) AdSSWV curves obtained at the NAF/PtNP/Gr/GCo (9) 8.26 × 10−6 M. (C) AdSSWV curves obtained at the NAF/PtNP/Gr/GCE for DOM.22 × 10−6 M. (D) AdSSWV curves obtained for the oxidation of PCT and DOM at eq
buffer was used. It is found that species like glucose (till 500 foldxcess), K+, Ca2+, NO3
−, NH4+, Cl− and Mg2+ (till 700 fold excess)
id not interfere in the analysis of PCT and DOM. Hence determi-ation of PCT and DOM was not considerably affected by common
nterfering species, which shows that the method is more selectiveoward both drugs.
In order to validate the method, various parameters, such asepeatability, reproducibility, precision, and accuracy of analy-is were obtained by performing five replicate measurements for.50 × 10−8 M standard PCT and 4.75 × 10−8 M standard DOM over
single day (intra-day assay) (n = 5) and for five days over a period ofne week (inter-day assay). The recovery tests were carried out bytandard addition method, and results are shown in (Table S2(A)).he robustness of the proposed procedure (Table S2(B)) was inves-igated by varying pH, accumulation potential, accumulation time,nd frequency, on the recovery of PCT and DOM. Comparison ofroposed method with HPLC method shows that no significanceifference was found between values obtained by proposed methodnd HPLC method. The results are shown in Table 2. Applying a
aired t-test and f-test in the results obtained by this procedurend those claimed on the labels, it was found that all results are ingreement at the 95% confidence level, and within an acceptableange of error.
CT at different concentrations in the presence of 1.95 × 10 M DOM: (1) 1.57 × 10ifferent concentrations in the presence of 1.06 × 10−7 M PCT: (1) 1.58 × 10−8 to (9)ncentrations of each: (1) 1.60 × 10−9 to (9) 8.20 × 10−6 M.
3.9. Determination of PCT and DOM in pharmaceutical, urine andserum samples
The recovery tests for pharmaceutical formulations, urine, andblood serum samples were carried out by spiking specific quan-tity of standard drug in the samples having complex matrices. Asshown in Table S3% recovery obtained for all samples were in therange of 98–102% indicating method is suitable for determination ofPCT and DOM in pharmaceutical formulations as well as biologicalsamples. The amount of PCT and DOM obtained in pharmaceuti-cal formulations agree well with the label contents. Simultaneousdetermination of both the molecules was carried out in pharma-ceutical formulations in the same manner as for the individualsamples.
3.10. Stability of the NAF/PtNP/Gr/GCE
In order to study the stability of the NAF/PtNP/Gr/GCE, the elec-trode was kept in pH 6.0 phosphate buffer for 15 days, and then the
CVs were recorded, and compared with the CVs obtained before 15days for the same electrode. It is found that peak current decreasedby 2.71% for electrode, which shows that the electrode has goodstability. The electrode was found to retain 99.6% of its initial peak
292 P.K. Kalambate et al. / Sensors and Actuators B 213 (2015) 285–294
Table 2Comparison between the proposed method and the HPLC method for sample analysis.
a: amount of drug in the sample (mg).b: amount of drug obtained by the proposed method (mg) ± % RSD (n = 5).c: amount of drug obtained by the standard method (mg) ± % RSD (n = 5).
Table 3Comparison between various electroanalytical methods for the determination of PCT and DOM with the proposed method.
Molecule Modified electrodes Linear working range (M) Limit of detection (M) Samples analyzed References
PCT In situ surfactantmodified MWCNTpaste electrode
urrent response for a PCT concentration of 4.3 × 10−8 M by the endf 45 days, which shows the long-term storage stability of thin-filmodifier on the surface of GCE during the determinations in aque-
us solutions (Fig. S9). The results indicate a good stability of theensor and capacity for repeated measurement to be performed onhe same electrode.
.11. Comparison of proposed method with literature methods
A comparison between the analytical performance of the pro-osed method with previously reported methods for determinationCT [6–11] and DOM [12,13] is shown in Table 3. The data revealshat the NAF/PtNP/Gr/GCE shows superior analytical performancen terms of very low detection limit, wide linear dynamic range,igh sensitivity, good reproducibility, and repeatability over meth-ds reported in literature. In addition present method is simple,nd does not involve any pretreatment step.
. Conclusion
In this work, a NAF/PtNP/Gr/GCE based electrochemical sensors prepared by combining unique properties of electrochemicallyenerated PtNP/Gr composite, such as high specific surface area,lectrocatalytic properties, adsorptive properties, and the cationelectivity of the nafion film. The simultaneous determination of
CT and DOM has been reported for the first time employingdSSWV. Various techniques, such as XRD, EDX, SEM, CV, CC,dSSWV, and EIS were employed in the present work to charac-
erize the electrode and electrode processes. The sensitivity was
Pharmaceutical formulations, wastewater [13]
0−10 Pharmaceutical formulations, urine, blood serum This work
enhanced considerably by preconcentration of PCT and DOM onmodified electrode due to the presence NAF, PtNP, and Gr respec-tively. Moreover, the proposed method is highly sensitive havingvery low detection limit, wide linear dynamic range, very goodreproducibility, and repeatability over other methods reported inliterature. The method has been further employed for simultaneousdetermination of PCT and DOM in pharmaceutical formulations,human urine, and serum samples. Since PCT is used for treatmentof migraine headaches and DOM is used to prevent nausea andincrease absorption of PCT in the body, it is expected that theproposed method will be of great help to both clinical as well aspharmaceutical industries.
Acknowledgements
The funding for this work is partly by the University GrantsCommission, New Delhi, India, Reference number UGC/X-PGR23and partly by the US Army International Technology Center, Tokyo,Japan through contract number FA2386-12-1-4086.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.snb.2015.02.090.
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Biographies
Pramod K. Kalambate received his M.Sc (2011) degreein analytical chemistry from University of Mumbai, India.
Mumbai, India. He received Ph.D. (1981) degree in chem-istry from Banaras Hindu University, India. His researchinterests are in the field of development of electrochem-ical (bio) sensors using different functional materials,supercapacitors and liquid chromatography.
94 P.K. Kalambate et al. / Sensors a
Bankim J. Sanghavi received his Ph.D. degree in analyticalchemistry at University of Mumbai, India (2012). He is cur-rently a research associate at university of Virginia, USA.His research interests cover synthesis of nanomaterialsand their applications in sensors and biosensors.
Shashi P. Karna is a senior research scientist – ST at theUS Army Research Laboratory, Maryland, USA. He received
Ph.D. (1983) degree in Chemistry from Banaras HinduUniversity, India. The main focus of his research is a funda-mental understanding of the structure and quantum-sizeproperties of nano-materials and their applications inArmy technologies.
tuators B 213 (2015) 285–294
Ashwini K. Srivastava is a professor of analytical chem-istry at the department of chemistry, university of
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