Journal of Electroanalytical Chemistry 698 (2013) 9–16

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Journal of Electroanalytical Chemistry

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A novel voltammetric sensor based on p-aminothiophenolfunctionalized graphene oxide/gold nanoparticles for determiningquercetin in the presence of ascorbic acid

1572-6657/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.jelechem.2013.03.016

⇑ Corresponding author at: Ankara University, Faculty of Science, Department ofChemistry, Ankara, Turkey. Tel.: +90 3122126720; fax: +90 3122232395.

E-mail address: aliosman.solak@gmail.com (A.O. Solak).

Mehmet Lütfi Yola a,b, Necip Atar c, Zafer Üstündag c, Ali Osman Solak d,e,⇑a Sinop University, Faculty of Arts and Sciences, Department of Chemistry, Sinop, Turkeyb Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkeyc Dumlupınar University, Faculty of Arts and Sciences, Department of Chemistry, Kutahya, Turkeyd Ankara University, Faculty of Science, Department of Chemistry, Ankara, Turkeye Kyrgyz-Turk Manas University, Faculty of Engineering, Department of Chemical Engineering, Bishkek, Kyrgyzstan

a r t i c l e i n f o

Article history:Received 15 October 2012Received in revised form 5 March 2013Accepted 18 March 2013Available online 3 April 2013

Keywords:QuercetinGraphene oxideGold nanoparticlesSquare wave voltammetryValidation

a b s t r a c t

In this study, gold nanoparticles (AuNPs) with the diameters of maximum 25 nm were self-assembledonto the surfaces of p-aminothiophenol functionalized graphene oxide (ATPGO) sheets simply by mixingtheir aqueous dispersions. The prepared graphene oxide (GO), ATPGO and AuNPs–ATPGO compositeswere characterized by a transmission electron microscope (TEM), X-ray photoelectron spectroscopy(XPS), reflection–absorption infrared spectroscopy (RAIRS), the X-ray diffraction (XRD) method andRaman spectroscopy. The electrochemical determination of quercetin (QR) has been studied using squarewave voltammetry (SWV) on glassy carbon electrode (GCE) modified with AuNPs–ATPGO composite(AuNPs–ATPGO/GCE). QR gave rise to a single oxidation peak in the potential interval from 200 to600 mV in 0.1 M acetate buffer (pH 5.5). The well-defined peaks were observed at the optimized instru-mental parameters for SWV such as frequency, amplitude and potential increments. The developedmethod was validated according to the ICH guideline and found to be linear, sensitive, specific, preciseand accurate. The linearity range of QR was 1.0 � 10�12–1.0 � 10�11 M with the detection limit (S/N = 3) of 3.0 � 10�13 M under optimum conditions. The validated method was applied successfully forthe determination of QR in pharmaceutical preparations.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

QR belongs to the flavonoid family that is distributed widely infruits and vegetables. Its molecular structure is as shown inScheme 1, which contains several hydroxyl groups located in thedifferent aromatic rings. It is reported that QR has antiviral, anti-cancer, anti-inflammatory, anti-allergic and anti-tumor activityand antioxidant properties [1–4].

Recently, graphene/graphene oxide has been considered a ‘‘ris-ing star’’ carbon material because of its unique properties, includ-ing superior mechanical strength [5–7], low density and high heatconductance [8,9]. Many applications have been developed basedon its mechanical, electrical and chemical properties. Some ofthese applications are graphene–polymer composites [10–12],batteries [13], fuel cells [14–17], drug delivery and biosensors[18–22].

In the past decades, many papers have studied nanostructuredmaterials, describing their interesting chemical, optical, adsorptionand electronic properties [23–25]. Among all the nanomaterials,AuNPs are used frequently for electrode surface modification inthe fabrication of sensors/biosensors [26–29]. In addition, thenano-sized AuNPs have the ability to enhance the electrodeconductivity, facilitate the electron transfer rate and improve theanalytical sensitivity [30–33]. However, the electrocatalytic prop-erties of AuNPs mostly depend on the size, shape and supportingmaterials [34].

Several methods have been reported for determining QR,including chromatography [35], spectrophotometry [36], capillaryelectrophoresis [37] and spectrofluorimetry [38]. In the literature,several voltammetric methods were found for the determinationof QR [39–43]. For example, Xiao et al. investigated the electro-chemical oxidation of QR at a multi-walled carbon nanotubes-paraffin oil paste electrode [39]. Gutierrez et al. studied thesensitive detection of QR using glassy carbon electrodes modifiedwith multi-walled carbon nanotubes dispersed in polyethyleni-mine and poly(acrylic acid) [40]. The detection limit of QR was

Scheme 1. Chemical structure of quercetin.

10 M.L. Yola et al. / Journal of Electroanalytical Chemistry 698 (2013) 9–16

found to be 0.2 lM. Lin et al. presented reversing differential pulsevoltammetry for the simultaneous determination of QR and rutin,which was based on multi-wall carbon-nanotube paste electrode[41]. The linearity range of QR was found as 0.05–5 lM (in thepresence of 10 lM rutin). Xu and Kim studied electrochemicaldetection of QR, carried out on glassy carbon electrodes modifiedwith carbon nanotubes and Nafion (GC/Nafion-CNT) [43]. Thedetection limit of QR was found to be 0.02 lM. Recent papersdiscussed the electrochemical grafting of QR onto the glassy carbonsurfaces for various purposes [44,45].

The aim of this study is to prepare gold nanoparticle/grapheneoxide composite and develop a new SWV method for determiningQR in real samples. Moreover, the validation parameters of thedeveloped method and reproducibility and stability properties ofproposed electrode are evaluated.

2. Experimental

2.1. Materials

QR was purchased from Fargem Company (Düzce, Turkey) andused as received. The stock solution of QR (1.0 mM) was preparedby dissolving it in 5 mL ethanol and then diluting it with ultra purequality water to 25 mL. The working solutions were prepared bydiluting the stock solution with 0.10 M acetate buffer (pH 5.5).Graphite powder (Sigma–Aldrich), hydrogen tetra-chloroauratehydrate (HAuCl4) (Sigma–Aldrich), p-aminothiophenol (ATP)(Sigma–Aldrich), trisodium citrate dihydrate (Na3C6H5O7�2H2O)(Sigma–Aldrich), 1-ethyl-3-[3-dimethylaminopropyl] carbodiim-ide hydrochloride (EDC) (Sigma–Aldrich), HPLC grade acetonitrile(MeCN) (Sigma–Aldrich), ethanol (Sigma–Aldrich), isopropyl alco-hol (IPA) (Sigma–Aldrich), hydrogen peroxide (H2O2) (Sigma–Al-drich), potassium permanganate (KMnO4) (Sigma–Aldrich),sulfuric acid (H2SO4) (Sigma–Aldrich), activated carbon (Sigma–Aldrich), potassium persulfate (K2S2O8) (Merck), phosphorus pent-oxide (P2O5) (Merck), acetic acid (Merck), sodium acetate (Merck)and other chemicals were reagent grade quality and were usedas received. All the processes were performed in aqueous mediaand the preparation of the aqueous solutions were carried outusing ultra pure quality water with a resistance of 18.3 MX cm(Human Power 1+ Scholar purification system).

2.2. Instrumentation

All electrochemical experiments were performed using a BAS-100B electrochemical analyzer (Bioanalytical System Inc., Lafay-ette, IL, US) equipped with C3 cell stand and Gamry Reference600 work-station (Gamry, US).

The infrared spectra of the composites were recorded withBruker Tensor 27 FT-IR DTGS detector by using a Ge total reflectionaccessory (GATR; 65� incident angle relative to surface normal,Harrick Scientific).

A Rikagu Miniflex X-ray diffractometer using mono-chromaticCu Ka radiation operating at a voltage of 30 kV and current of15 mA was used for X-ray diffraction measurement of the samples.

A scanning speed of 2�2h/min and a step size of 0.02� were used toexamine the samples in the range of 5–75�2h.

TEM measurements were performed on a JEOL 2100 HRTEMinstrument (JEOL Ltd., Tokyo, Japan) to examine the morphologyof AuNPs and GO.

XPS analysis was performed on a PHI 5000 Versa Probe (U UL-VAC-PHI, Inc., Japan/USA) model X-ray photoelectron spectrometerinstrument with monochromatized Al Ka radiation (1486.6 eV) asan X-ray anode operated at 50 W. The pressure inside the analyzerwas maintained at 10�7 Pa. The sample for XPS measurement wasprepared on a clean glass slide by placing one drop of the nano-structure, and then allowing it to air dry.

A DeltaNu Examiner Raman microscope (Deltanu Inc., Laramie,WY) with a 785-nm laser source, a motorized microscope stagesample holder and a CCD detector were used to detect for charac-terization of GO.

All the pH measurements were made with Metler Toledo MA235.

2.3. Cleaning procedure for the glassy carbon electrode surface

GCE was cleaned and prepared by polishing it to a mirror-likefinish with fine wet emery paper (grain size 4000). The electrodeswere polished successively in 0.1 lm and 0.05 lm alumina slurries(Baikowski Int. Corp., US) on microcloth pads (Buehler, Lake Bluff,IL, US). The electrodes were sonicated first in ultra pure water twotimes and in 50:50 (v/v) IPA + MeCN solution purified over acti-vated carbon. After removal of trace alumina from the surface byrinsing with water and a brief cleaning in an ultrasonic bath (Ban-delin RK 100, Germany) with water and then with IPA + MeCNmixture purified over the activated carbon, they were rinsed withMeCN to remove any physisorbed or unreacted materials from theelectrode surface.

2.4. Preparation of AuNPs

HAuCl4 and Na3C6H5O7�2H2O were used as a gold nanoparticleprecursor and a reducing agent, respectively. Twenty mL of1.0 mM HAuCl4 was added into a 50 mL volumetric flask on astirring hot plate. During stirring 2 mL of Na3C6H5O7�2H2O (1%)was slowly added into the gold precursor solution at 65 �C. Thesolution was well mixed at the same temperature until the colorof solution changed to red, and then it was boiled for 20 min.The mean diameters of the AuNPs are maximum 25 nm and theAuNPs were stabilized with ethanol [46].

2.5. Preparation of GO

GO was synthesized by a modified Hummers method [47–49]. Amixture containing 25 mL of H2SO4 (98%) with 5 g of K2S2O8, 5 g ofP2O5 and 5 g of graphite was placed in a flask and was kept at 80 �Cfor 6 h. The mixture was cooled to 20 �C and diluted with 1 L ofultra pure water and left for 12 h. The pre-oxidized carbon materialwas filtered and washed with ultra pure water. The pre-treatedgraphite was diluted 250 mL H2SO4 (98%) under 0 �C. Thirty gramsKMnO4 was added in the suspension and was cooled to 20 �C. Afterthe KMnO4 feeding was finished, the flask was heated to about35 �C and kept at this temperature for an additional 30 min. Themixture was stirred under this temperature for 4 h and thendiluted 500 mL with ultra pure water in the ice bath. The last mix-ture was stirred for 2 h. The mixture was diluted to 2 L with ultrapure water. The suspension was further treated with 40 mL of H2O2

(30%). The color of the suspension changed to brilliant yellow frombrownish and the mixture was stirred to until the bubblingstopped. The synthesized GO was filtered and washed with 0.1 MHCl and distilled water three times, respectively. The GO was

M.L. Yola et al. / Journal of Electroanalytical Chemistry 698 (2013) 9–16 11

precipitated by an ultracentrifuge and was dried under the atmo-spheric air conditions.

2.6. Preparation of AuNPs–ATPGO composite

The synthesized GO was dissolved in ethanol at a concentrationof 2 mg mL�1 with the aid of ultrasonic agitation for 1 h, resultingin a homogeneous black suspension. To ensure the surface activa-tion of carboxylate groups of GO, the GO suspension was interactedwith 0.2 M EDC solution for 8 h. The activated GO suspension waswell mixed with 1.0 mM ATP at a 1:1 volume ratio for 2 h (ATPGO).In a typical experiment of self-assembly, the aqueous dispersion ofAuNPs (1 mg mL�1) was mixed with the aqueous dispersion ofATPGO sheets (0.1 mg mL�1) at a 1:1 volume ratio and sonicatedfor 15 min to form a homogeneous mixture (AuNPs–ATPGO). Themixture was then kept undisturbed under ambient condition for12 h. The proposed structure of the synthesized AuNPs–ATPGOcomposite is shown in Scheme 2.

2.7. Preparation of AuNPs–ATPGO composite modified glassy carbonelectrodes

GCE (surface area 0.027 cm2) was carefully polished with0.1 lm and 0.05 lm alumina slurries on a cloth pad. After sonica-tion in water and 50:50 (v/v) IPA + MeCN solution purified overactivated carbon to remove the alumina residues, the electrodewas dried under a nitrogen stream. Finally, 10 lL of AuNPs–ATPGOcomposite dispersion was dropped onto the GCE surface and thesolvent was evaporated under an infrared heat lamp.

2.8. Pharmaceuticals and synthetic preparations

Ten capsules (Vitacost Quercetin & Vitamin C) were weighedand powdered. The equivalent amount to one capsule was accu-rately weighed and transferred to a 100 mL volumetric flask. Etha-nol (50 mL) was added and the flask was sonicated for 15 min tocomplete dissolution and diluted to the mark with ultra pure qual-ity water. A 5-mL portion was centrifuged for 10 min. Appropriatesolutions were prepared from the supernatant by dilution with0.10 M acetate buffer (pH 5.5).

Synthetic capsules were prepared by mixing excipients (700 mgof vitamin C known as ascorbic acid (AA), magnesium stearate,

Scheme 2. The structure of A

titanium dioxide) and a labeled amount (250 mg) of QR. Then themixture was transferred to a 50-mL volumetric flask andappropriate solutions were prepared as in the preparation ofpharmaceuticals.

2.9. Electrochemical measurements

Before electrochemical experiments, solutions were purgedwith pure argon gas (99.999%) for 10 min at least and an argonatmosphere was maintained over the solution during the experi-ments. A bare GCE or a GCE modified with GO, ATPGO and AuN-Ps–ATPGO composite was used as the working electrode. Thereference electrode was a Ag/AgCl/KCl(sat) used in aqueous media.The counter electrode was a Pt wire. The square wave voltammo-grams of QR were performed by using the AuNPs–ATPGO/GCE asthe working electrode and 0.1 M acetate buffer as the supportingelectrolyte. A 3.0 mL volume of 0.1 M acetate buffer was purgedwith pure argon gas for 10 min. After the voltammogram of thissolution had been recorded, the prepared working solutions wereadded in the cell by micropipette. Argon was passed through thesolutions for 1 min. The voltammograms were recorded again. Thisprocedure was repeated until the peak height no longer increased.

3. Results and discussion

3.1. Characterizations of GO, ATPGO and AuNPs–ATPGO composite

The morphologies of the GO and the AuNPs–ATPGO compositewere investigated using the JEOL 2100 HRTEM with an acceleratingvoltage of 200 keV. A drop of sample solution was deposited on apolymeric grid and dried at room temperature under an argongas stream. TEM images of the GO and the AuNPs–ATPGO compos-ite are shown in Fig. 1a and b, respectively. As shown in Fig. 1a, theTEM image of the GO shows pellucid and creased GO sheets, exhib-iting its mono- or few-layer planar sheet-like morphology. TheTEM image of the AuNPs–ATPGO shows (Fig. 1b) that the sizes ofthe AuNPs are very similar and the mean diameter is maximum25 nm. In addition, AuNPs appear as dark dots with a mean diam-eter of maximum 25 nm on a lighter-shaded substrate correspond-ing to the planar GO sheet. This creased nature of the GO is highlybeneficial in providing a high surface area on GCE.

uNPs–ATPGO composite.

Fig. 1. TEM images of (a) GO and (b) AuNPs–ATPGO composite.

Fig. 2. Raman spectrum of the GO.

12 M.L. Yola et al. / Journal of Electroanalytical Chemistry 698 (2013) 9–16

Fig. 2 shows the Raman spectrum of the GO, which reveals the Dband at 1320 cm�1 that arose from sp3-hybridized carbon as wellas the G band at 1599 cm�1 that indicates in-phase vibration ofthe graphite lattice [50].

The RAIRS measurements were carried out to get informationabout examining the graphite, the GO and the ATPGO composite.In the spectrum of the GO (Fig. 3b), the bands around 3310,1735, 1622, 1168 and 1030 cm�1 are attributed to the oxygen-con-taining functional groups on the GO [51], confirming the successfuloxidation of the graphite. The RAIRS spectrum reveals the verycharacteristic stretching vibrations of C@O (ACOOH) (1735 cm�1)on the edges of the GO planes [52]. Fig. 3c shows the RAIRS

Fig. 3. RAIRS spectra of (a) the graphite (b) the GO and (c) ATPGO composite.

spectrum of the ATPGO and confirms the presence of covalentlyattached ATP on the GO. The C@O (ACOOH) band in the GO at1735 cm�1 shifted to 1746 cm�1 in the ATPGO. This shift is attrib-uted to covalent functionalization of the carboxyl group of the GOwith ATP. The peaks at 1630 cm�1 and 1372 cm�1 are attributed toaromatic C@C double bond stretching and CAH bending vibra-tional frequencies in the ATPGO, respectively [53]. In addition,the existence of the band at 3337 cm�1 for the NAH stretchingvibration bond confirms the covalent functionalization of the car-boxyl group of the GO with ATP [54].

XPS experiments were performed to examine the attachment ofAuNPs to ATPGO. In the XPS spectrum (Fig. 4) of the AuNPs–ATPGOcomposite, C, N, S and Au peaks are prominent, showing that AuN-Ps have been functionalized on the ATPGO. After the chemicalattachment of these organic moieties, the single peak at 285 eVdue to the sp2 hybridized C atoms [55] is transformed into a broadenvelope, and deconvolution reveals that it consists of two compo-nents for the AuNPs–ATPGO composite. The peaks at 285.5 and283.6 eV are assigned to CAN and CAH, respectively [56,57]. TheN1s narrow region XPS spectrum of the AuNPs–ATPGO compositewas curve-fitted with two components at 398.4 and 400.7 eV.The peak located at 398.4 eV is attributable to CAN groups in thecovalent functionalization of the carboxyl group of the GO withthe amino group of the ATP [56]. The peak observed at 400.7 eVcan be assigned to the NAH group in unreacted ATP molecules[58].

The S2p region is characterized by a doublet (2p1/2 and 2p3/2),owing to the spin–orbit coupling. It is thought that the sulfur ofthe AuNPs–ATPGO composite was easily bonded to the AuNPs bythe appearance of sulfur peaks at 167.6 eV. The peak at 162.9 eVindicated a free mercapto group [59]. The Au 4f7/2 peak signal thatappeared at 83.0 eV verified the presence of bonded Au [60].

The successful synthesis of the AuNPs–ATPGO composite is alsoconfirmed by XRD measurements. The XRD patterns of the graph-ite and as-synthesized AuNPs–ATPGO composite are examined andshown in Fig. 5. As shown in the inset of Fig. 5, the XRD patternsindicate a very intense and narrow peak at 2h = 26.5� assigningto the (002) planes of GO layers occurring in graphite. Three weakpeaks at 2h = 42.5�, 44.6�, and 54� correspond to (100), (101),(004) and (002) planes of GO layers occurring in graphite, respec-tively [61]. As shown in Fig. 5, a broad peak at 2h = 20–28� occurs,which is the structure expansion as oxygen-containing groupsincorporate between the carbon sheets during the course of strongoxidation. Two peaks occurring at 2h = 22.0� and 42.5� is typicalXRD patterns of GO. The peaks at 2h = 38.8� and 44.2� indicatethe (111) and (200) crystalline planes of Au [62].

3.2. Cyclic voltammograms of quercetin at different electrode surfaces

Fig. 6 shows the cyclic voltammetry of 1.0 � 10�6 M QR in 0.1 Macetate buffer (pH 5.5) at different electrode surfaces. The anodic

Fig. 4. The narrow region XPS spectra of AuNPs–ATPGO composite for the deconvolution spectra of the C1s, N1s, S2p and Au4f.

Fig. 5. XRD patterns of the AuNPs–ATPGO composite (inset is the XRD patterns ofgraphite).

Fig. 6. Cyclic voltammograms of 1.0 � 10�6 M QR in 0.1 M acetate buffer (pH 5.5)(a) at bare GCE, (b) GO modified GCE and (c) AuNPs-ATPGO/GCE (scan rate is200 mV s�1).

M.L. Yola et al. / Journal of Electroanalytical Chemistry 698 (2013) 9–16 13

and cathodic peaks at the AuNPs–ATPGO/GCE were much greaterthan those at other electrodes while the peak potential separationat the AuNPs–ATPGO/GCE is almost the same as those at otherelectrodes. As Fig. 6 shows, the AuNPs–ATPGO/GCE can greatlyaccelerate the electron transfer rate between QR molecules insolution and the surface. The following reasons might explain theelectrocatalytic response of QR at the AuNPs–ATPGO/GCE. First,the high density of edge-plane-like defective sites on GO may pro-vide many active sites, and it would be beneficial for acceleratingelectron transfer between the electrode and species in solution.Second, these enhanced performances were attributed to the large

surface area and good electrical conductivity of GO, and the syner-gistic effect of GO and metal nanoparticles [63,64]. Therefore theAuNPs–ATPGO/GCE is more suitable for the determination of QRat a low concentration level.

3.3. Influence of pH and scan rate in CV technique

Anodic peak current increases gradually, achieves a maximumat about pH 5.5 and then decreases. Therefore, pH 5.5 was decidedas optimum pH for electrochemical works (Fig. 7). When pHexceeds 9, the anodic peak disappears. As shown below (Scheme 3),the concentration of the anion form of QR increases by increasingpH.

The dependence of the CV peak potential on pH was also inves-tigated. The slope of the Ep–pH plot was �60.3 mV/pH for the AuN-Ps–ATPGO/GCE. This result shows that the numbers of electronsand protons transferred are equal in the electrochemical reaction

Fig. 7. Effect of pH on the anodic peak current of 1.0 � 10�6 M QR in 0.1 M acetatebuffer (pH 5.5) at the AuNPs–ATPGO/GCE (scan rate is 200 mV s�1).

Fig. 8. (A) Effect of concentration on the peak current of QR at the AuNPs–ATPGO/GCE (a) 0.1 M acetate buffer (pH 5.5), (b) 1.0 � 10�12, (c) 2.0 � 10�12, (d)4.0 � 10�12, (e) 6.0 � 10�12, (f) 8.0 � 10�12 and (g) 10.0 � 10�12 M QR. (B) SWV of10.0 � 10�12 M AA + 10.0 � 10�12 M QR in 0.1 M acetate buffer (pH 5.5), which wasregistered by the AuNPs–ATPGO/GCE (frequency of 50 Hz, pulse amplitude of20 mV, scan increment of 3 mV).

14 M.L. Yola et al. / Journal of Electroanalytical Chemistry 698 (2013) 9–16

[65]. Among the hydroxyl groups of QR, the catechol dihydroxylelectron-donating group (ring A) with the highest level of electro-activity may be oxidized at low potentials and show high peakcurrents [66].

The influence of scan rate (v) on the electrochemical response ofQR was investigated. With the increase of scan rate, the redox peakcurrent increased with the positive shift of the anodic peak poten-tial and the negative shift of the cathodic peak potential. Theincrease of DEp indicated that the electrode process is more qua-si-reversible. The plots of log Ipa vs. log v and log Ipc vs. log v inthe scan rate range of 25–500 mV s�1 yielded a straight line witha slope of 0.513 and 0.482. These values are close to the theoreticalvalue of 0.50, which is expected for ideal diffusion-controlled elec-trode processes.

The relationship of the redox peak potentials with the logarithmof scan rate was also obtained with the linear regression equationsas Epa = 0.168 + 0.0151lnv (Epa: V, v: V s�1, R2: 0.9974) andEpc = 0.118–0.0135lnv (Epc: V, v: V s�1, R2: 0.9961), respectively.Based on the following equations [67]:

Epa ¼ E00 þm½0:78þ lnðD1=2k�1s Þ � 0:51 ln m� þ ðm=2Þ ln m;

m ¼ RT=ð1� aÞnF ð1Þ

Epc ¼ E00 �m0½0:78þ lnðD1=2k�1s � 0:51 ln m0Þ� � ðm0=2Þ ln m;

m0 ¼ RT=anF ð2Þ

the electron-transfer coefficient (a) is calculated as 0.525 for threemeasurements.

3.4. Analytical application

The SWV method was used to optimize a rapid and sensitiveelectroanalytical method for determining of QR. This method wasapplied to the drugs in pharmaceutical dosage forms.

The square wave voltammetric responses are directly relatedto the instrumental parameters. To obtain a much more sensitivepeak current, the optimum instrumental parameters such aspulse amplitude, frequency and scan increment, were studiedfor 1.0 � 10�6 M QR in 0.1 M acetate buffer (pH 5.5) at the

Scheme 3. Equilibrium r

AuNPs–ATPGO/GCE. The frequency varied from 10 to 100 Hz.Although the signal response increased with frequency, the peakshape was deformed above 50 Hz. When the pulse amplitude wasvaried in the range of 10–50 mV, the peak current increased withincreasing pulse amplitude. However, broadening was observedabove the 20 mV peak. The scan increment varied from 2 to6 mV. When peak height and peak shape were taken into consid-eration, 3 mV of scan increment was chosen. Hence, the best peakdefinition was recorded when using frequency of 50 Hz, pulseamplitude of 20 mV and scan increment of 3 mV.

3.5. Validation of the proposed method

3.5.1. Linearity rangeThe square wave voltammograms recorded with increasing

amount of QR (Fig. 8A) show that the peak currents increased lin-early with increasing concentration. Each point of the calibration

eaction of quercetin.

Table 1Data of the calibration curves for the proposed method (n = 6).

Regression equation y = 0.0417x + 0.0101Standard error of the slope 0.29Standard error of the intercept 0.51Correlation coefficient (r) 0.9993Linearity range (M) 1.0 � 10�12–10 � 10�12

Number of data points 7LOD (M) 3.0 � 10�13

LOQ (M) 1.0 � 10�12

y = ax + b; y: Peak current/lA, x: QR concentration/pM, a: Slope, b: Intercept, LOQ:Limit of quantification, LOD: Limit of detection.

Table 3The results obtained by SWV for (Vitacost Quercetin &Vitamin C) capsules containing 250 mg QR (n = 7).

QR found/mg per capsule

Capsule number SWV

1 250.962 250.253 248.284 250.965 251.136 249.747 250.31

X 250.38 ± 0.05SD 0.99RSD % 0.40

X: Mean ± standard error, SD: standard deviation,RSD%: relative standard deviation.

M.L. Yola et al. / Journal of Electroanalytical Chemistry 698 (2013) 9–16 15

graph corresponded to the mean value obtained from six indepen-dent measurements. Table 1 gives the data of the calibration curvesfor the proposed method.

3.5.2. PrecisionThree different concentrations of QR (2.0, 6.0, 8.0 � 10�12 M) in

the linear range were analyzed in six independent series on thesame day (intra-day precision) and six consecutive days (inter-day precision) from six measurements of each series (Table 2).The RSD values varied from 0.92 to 1.38 for intra-day and from1.14 to 1.19 for inter-day precision. The low RSD values of intra-day and inter-day indicated that the developed method has a highprecision [68].

3.5.3. AccuracyThe accuracy of an analytical method expresses the closeness

between the reference value and the found value. Accuracy wasevaluated as a percentage of relative error between the measuredmean concentrations and added concentrations for QR (Bias %).Both the results obtained for intra-day and inter-day accuracywere 60.50% (Table 2) [68].

3.5.4. RecoveryThe determination of OR in a synthetic preparation [the mixture

of excipients and labeled amount (250 mg QR)] were made. Therecovery percentage values ranged between 98.10% and 102.30%with RSD < 2.00. The closeness of the results to 100.00% showedthat the recovery of the developed method was very good andthe validated method is recommendable for QR determination inthe presence of AA [68].

3.5.5. SelectivityThe selectivity of this method validated for the determination of

QR was evaluated in the presence of 10.0 � 10�12 M of AA in 0.1 Macetate buffer (pH 5.5), containing 10.0 � 10�12 M of QR. As can beseen in Fig. 8B, the oxidation peak at about �0.1 V was attributedto the oxidation of AA and at about +0.2 V to QR. For the methoddeveloped, the appearance of oxidation peaks at different poten-tials is very practical because AA did not affect the oxidation peakattributed to QR. The standard addition technique was applied tothe same preparations that were analyzed by calibration curves.

Table 2Intra-day and inter-day precision and accuracy results of QR (n = 6).

Added/pM Intra-day

Founda/pM Precisionb (%) Accuracyc

2.0 2.01 ± 0.03 0.92 0.506.0 5.98 ± 0.05 1.06 0.338.0 8.03 ± 0.06 1.38 0.36

a Mean ± standard error.b Precision %: Relative standard deviation (RSD).c Bias %: [(found-added)/added] � 100%.

The regression equation of the standard addition curve wasy = 0.0409x + 2.171 for QR. There was no significant differencebetween slopes of calibration curves and standard addition curves.These results show that there was no interference from matrixcomponents. Therefore the developed method is highly selectivefor QR in the presence of AA.

3.6. Determination of QR in pharmaceutical formulations

To check the applicability of the proposed voltammetric sensor,commercial tablet formulations containing QR (250 mg per tablet)were analyzed. Table 3 gives the results obtained by the developedmethod for determining QR in pharmaceutical preparations. It canbe concluded that the determination of QR in the presence of AA isperformed without using any reagents, which cause interferencesand contaminations.

3.7. Fabrication reproducibility and stability of the AuNPs–ATPGO/GCE

The fabrication reproducibility was also estimated with sixdifferent electrodes that were fabricated independently by thesame procedure. The RSD is 1.12% for peak current measuring in1.0 � 10�6 M QR which demonstrates the reliability of the fabrica-tion procedure. Additionally, the stability of one GCE modified withAuNPs–ATPGO was also investigated. After 1 month, the currentresponse is approximately 96.37% of the original value. The excel-lent long-term stability and reproducibility of the prepared elec-trodes make them attractive in the fabrication of electrochemicalsensors.

4. Conclusions

Gold nanoparticle tagged surfaces of p-aminothiophenol func-tionalized GO sheets, i.e., AuNPs–ATPGO composites, show a cata-lyzing effect for QR oxidation; therefore it is found suitable for itsdetermination. The AuNPs–ATPGO modified GC electrode shows

Inter-day

(%) Founda/pM Precisionb (%) Accuracyc (%)

2.00 ± 0.04 1.17 06.02 ± 0.07 1.14 0.337.96 ± 0.03 1.19 0.50

16 M.L. Yola et al. / Journal of Electroanalytical Chemistry 698 (2013) 9–16

much better performance when compared with unmodifiedelectrodes. Under optimized conditions, the anodic peak currentis linear to QR concentration in the reasonable concentrationranges of 1.0 � 10�12–1.0 � 10�11 M. The proposed method isprecise, accurate, sensitive, rapid, cheap, easy to use and mightbe preferred to the published chromatographic, spectrophotomet-ric and electroanalytical methods for determining QR.

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