Redox Behavior of Riboflavin and Its Determination in Real Samples at Graphene Modified Glassy Carbo

Physical Chemistry Communications, Volume 3 Issue 2, October 2016 www.bacpl.org/j/pcc

Redox Behavior of Riboflavin and Its Determination in Real Samples at Graphene Modified Glassy Carbon Electrode Atmanand M. Bagoji and Sharanappa T. Nandibewoor* P. G. Department of Studies in Chemistry, Karnatak University, Dharwad‐ 580003, India *Corresponding Author ‐Sharanappa T. Nandibewoor P. G. Department of Studies in Chemistry, Karnatak University, Dharwad‐ 580003, India E‐mail: stnandibewoor@yahoo.com Tel: +91 8362215286; fax: +918362747884

Abstract Improved electrochemical oxidative determination of riboflavin (RF) at a thin graphene film modified glassy carbon electrode (GF‐GCE) has been established using cyclic and differential pulse voltammetric techniques. The graphene was characterized by SEM, TEM and electron diffraction studies. The surface area calculated for modified electrode was higher than the glassy carbon electrode which is responsible for more catalytic activity in the present system. Cyclic voltammetry was employed to unveil the electrocatalytic performance of graphene and two redox peaks were observed for RF with good intensity. The redox voltammetric behavior of RF at the sensor was quasi‐reversible involving two electrons‐two protons. At the surface of modified electrode, the redox reaction was adsorption‐controlled. A probable electro‐redox mechanism was proposed. Under the optimum conditions, a calibration curve of longer linearity for peak current and RF concentration in the range from 1.0 nM to 1.5 × 10‐8 M was obtained with a detection limit of 1.0 × 10‐10 M. The present method was applied to riboflavin determination in pharmaceutical and real samples with good recovery. There was no interference from any excipients, which indicates the specificity of the composite electrode. Furthermore, the fabricated RF chemical sensor exhibited excellent stability, remarkable catalytic activity and reproducibility towards RF determination. The method finds applications in clinical laboratory.

Keywords Riboflavin; Voltammetry; Graphene; Oxidation; Analytical Applications

Introduction Riboflavin (RF) (Scheme 1) is a natural, yellow, essential water‐soluble vitamin. RF is ampiphilic in nature due to the presence of hydrophilic ribityl side chain as well as hydrophobic isoalloxazine ring [1]. RF takes an important role in the formation of two coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).

These coenzymes are responsible for the redox reactions by accepting and donating two electrons in the isoalloxazine ring [2]. These redox reversible reactions are necessary for cellular respiration and energy supplementation [3]. RF also plays vital role in promoting growth, immunity, cancer prevention, cell regeneration and antioxidation [4, 5].

R N

O

N

NH N O R - C5H11O4

SCHEME 1 CHEMICAL STRUCTURE of RIBOFLAVIN

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Graphene is a good material for sensors. Every carbon atom in graphene is in sp2 hybridization responsible to sense changes in its surroundings. Graphene sheets have excellent electron conducting and high electrocatalytic properties [6‐9], they have been employed in optoelectronic devices [10], electrochemical super‐capacitors [11], fabricated field‐effect transistors [12] and batteries [13]. The excellent sensitivity of graphene‐based sensors was tuned to individual and simultaneous voltammetric determination of dopamine, ascorbic acid and uric acid [14], hydroquinone and catechol [15] and cadmium [16].

The origin of the electrochemical interaction depends on the structural aspects of the components under investigation. Amphiphilic character of RF and the π‐like structure of the isoalloxazine ring in RF are the main basic source for the π – π type interaction of RF with other electrochemically active species. A redox and degradation process in the ground‐state of the folic acid/riboflavin system was observed by W.G Santos et al [17] and also electron transfer processes have been observed using riboflavin in the ground‐state or excited‐state [18].

The RF determination in pharmaceuticals as well as in real samples using fluorescence [19] and HPLC/LC‐MS [20] were carried out, but these are of high cost and time consuming methods. Also some electroanalytical techniques using bismuth film electrode [21], silver solid amalgam electrode [22], polythiophene nanotube arrays [23], liquid chromatography‐diode array detection (LC‐DAD )[24], electrically heated graphite cylindrical electrode (EHGCE) [25], poly (3‐ methylthiophene) modified electrode (P3MT) [26], nanocrystalline metallosilicate modified electrode (NME) [4], mesoporous carbon modified electrode (MCE) [27] have been augmented in the literature. However, in order to achieve trace level detection of RF, the development of a reliable, sensitive, selective, low cost and eco‐ friendly method is still necessary.

To the best of our knowledge, there are no reports on the electro‐oxidation of RF and its determination at graphene film modified glassy carbon electrode (GF‐GCE). The proposed method aims to establish the appropriate experimental conditions to develop a DPV technique for the determination of RF in pharmaceuticals and real samples.

Experimental Instrumentation Cyclic (CV) and differential pulse (DPV) voltammetric curves at GF‐GCE were recorded with the aid of a CHI630D Electrochemical Analyser (CH Instruments Inc., USA). The measurements were carried out in a 10 ml single compartment three electrode cell with the reference electrode as Ag/AgCl (3M KCl), the auxiliary electrode as a platinum wire and GF‐GCE as the working electrode. The pH values of supporting electrolyte solutions were measured using an Elico LI120 pH meter (Elico Ltd., India).

Chemicals Riboflavin was purchased from Sigma Aldrich India and Lipabol tablets were purchased from local pharmacy store. The phosphate buffers from pH 3.8 – 10.4 were prepared in ultrapure water as described by Christian and Purdy [28]; All reagents were all of analytical grade and prepared using deionized water from a Milli‐Q (Millipore, Bedford, USA) device.

Synthesis of Graphene Graphene oxide (GO) was synthesized from natural graphite powder by the Hummer’s method [29]. The natural graphite powder was treated with concentrated sulfuric acid and nitric acid with potassium chlorate for 96 hours.

After complete oxidation of graphite, the oxidized mixture was added to excess water, washed with 5% HCl and repeatedly treated with water to obtain neutral graphite oxide. Then through extreme heating and continuous splitting of graphite oxide, fabric graphene sheets were obtained.

Preparation of the GF‐GCE The modification was carried out by depositing 5 μL of graphene suspension in ethanol on surface of glassy carbon electrode (GCE) and dried at room temperature. The extra loosely adhered graphene at the electrode was removed by careful rinsing the modified electrode with water and dried in an air stream and stored in room temperature 25ºC.

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Voltammetric Measurements The GF‐GCE in the three‐electrode system was immersed in 0.2 M phosphate buffer (pH 6.0) containing a known amount of RF. Then CV and DPV were performed. The entire CV measurements were of scan rate at 100mVs‐1. The parameters for differential pulse voltammetry (DPV) were initial potential E: ‐0.32 V; final potential E: 0.16V; sample interval: 0.001 V; quiet time: 2 s; sensitivity: 1.0 × 10−6 A/V, with amplitude of 50 mV; voltammograms were recorded with the phosphate buffer in the absence of RF also.

Pharmaceutical Preparation Ten pieces of RF containing tablet, Lipabol were finely powdered in a mortar. An adequate amount of the powder to a stock solution of concentration of 1.0 × 10‐6M was weighed and transferred to a 100ml calibrated flask and dissolved with distilled water. Appropriate solutions were prepared by taking suitable aliquots from this stock solution and diluted them with phosphate buffer solutions. The standard addition method was used for analyzing the pharmaceutical samples.

Analysis of Urine Samples Urine samples were collected from healthy volunteers. 2 ml of aliquot of urine sample was transferred to 100ml calibrated flask and diluted to the mark with phosphate buffer solutions. These urine samples were analyzed immediately or they were stored at −18 °C until analysis. Further, DPV technique has been carried for the analysis of RF in urine samples by using standard addition method.

Results and Discussion Characterization of Graphene Graphene and graphite dispersed in ethanol were characterized by scanning electron microscope (SEM). Fig. 1 (a) a shows the SEM image of the simple graphite powder with unclear surface structure whereas in Fig. 1 (b) i.e. SEM image of graphene powder showed a well cleared surface with spongy type shining structure. This reveals that graphene might have provided larger active surface area for electro catalytic oxidation of RF than graphite powder.

FIG. 1 SEM IMAGES of the SURFACE of (a) GRAPHITE POWDER and (b) GRAPHENE POWDER

FIG. 2 (a) BRIGHT‐FIELD TEM IMAGES of GRAPHENE. (b) ELECTRON DIFFRACTION PATTERNS for THE SEVERAL LAYERS of GRAPHENE

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Further the TEM images of graphene dispersed in ethanol was obtained. Fig. 2 (a) shows the image of the wrinkled graphene sheet with no aggregation, indicating that the functionalized graphene sheets are stable at room temperature for about 3 weeks. Fig. 2 (b) shows an electron diffraction of nanocomposite material yielding a double six‐spot‐ring pattern, which confirms the benzene‐ring pattern of the graphene sheet [30] Active Surface Area of GF‐GCE The Randles‐Sevcik formula (equation 1) was employed to find out the active surface area of the GCE and GF‐GCE by using 1.0mM K4FeCN6 in 0.1M KCl as a standard electrolyte [31].

Ipa = (2.69x105)n3/2ADo1/2Coυ1/2 (1) where, Ipa refers to the anodic peak current, n is the number of electrons transferred, A0 is the surface area of the electrode, D0 is diffusion coefficient, ν is the scan rate and C0* is the concentration of K4Fe(CN)6. For 1.0 mM K4Fe (CN)6 in 0.1M KCl electrolyte, n = 1, Do=7.6 × 10−6 cm2s−1. From the slope of the equation 1, the calculated area for GCE and GF‐GCE were found to be 0.0462 and 0.2953 cm2 respectively. This revealed that GF‐GCE provides larger surface area for oxidation‐reduction reaction of RF than bare GCE does.

Electro‐Catalytic Behavior of GF‐GCE for RF RF being readily oxidisable vitamin, its voltammogram was recorded in the potential range from 500 to 200mV in the supporting electrolyte (0.2M) phosphate buffer of pH 6.0 at scan rate 100mV/s. GF‐GCE has no electrochemical activity in the blank supporting electrolyte (Fig. 3 curve (a)). Fig. 3 curve (b) shows a CV profile for 1.0mM RF at GCE with no redox peaks. However, for the GF‐GCE, the voltammogram with two redox pairs was obtained as Epa1 and Epc1 at ‐271 and ‐292mV respectively; Epa2 and Epc2 at ‐134mV and ‐201mV respectively as shown in the Fig. 3 curve (c). The peak to peak separation (ΔEp) for both the pairs were found to be 21 and 67mV respectively, the second redox pair was quasi‐reversible and the ratio of redox peak currents (Ipc2/Ipa2) was 0.98≈1.0 which was also a characteristic of quasi‐reversible electrode process [32]. Since, peak II was more intense than peak I, peak II was considered for further experiments.

FIG.3 CYCLIC VOLTAMMOGRAMS of (a) BLANK 0.2M PHOSPHATE BUFFER at GF‐GCE (b) 1.0mM RF in 0.2M PHOSPHATE BUFFER at GCE and (c) 1.0mM RF in 0.2M PHOSPHATE BUFFER at GF‐GCE

Influence of Ph of the Supporting Electrolyte and Reaction Pathway The selectivity and sensitivity of the voltammetric oxidation of RF at GF‐GCE depends on the pH of the supporting electrolyte. So in order to depict the dependence of peak potential and peak currents on the pH, this study has been carried out. SI Fig. 1 shows CV voltammogram of 1.0 mM of RF in 0.2M phosphate buffer (PB) of different pH from 3.0 to 10.4 with 100mV/s scan rate at GF‐GCE. With the increase in the pH of the medium, the peak potential shifted to more negative values as shown in Table 1 indicating involvement of proton exchange in the electrode reaction of oxidation of RF.

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TABLE 1 VARIATION OF PEAK CURRENT AND PEAK POTENTIAL WITH PH FOR 1MM RF AT SCAN RATE OF 100 MVS‐1

pH

Ipa2 x 10‐5 (A)

Epa2 (V)

Ipc2 x 10‐5 (A)

Epc2 (V)

3.0

4.466

‐0.121

5.251

‐0.1759

4.2

4.487

‐0.1302

4.503

‐0.1841

5.0

7.236

‐0.1309

7.234

‐0.1882

6.0

8.616

‐0.134

8.507

‐0.201

7.0

6.832

‐0.194

6.655

‐0.2379

8.0

3.86

‐0.2795

8.47

‐0.4019

9.2

3.34

‐0.323

8.755

‐0.407

10.4

2.30

‐0.385

11.10

‐0.401

From the SIFig. 2 and Table 1, it was clear that the peak current was maximum for the 1.0 mM RF in the supporting medium of pH 6.0. So, pH 6 was the optimal pH value which is used for further experiments.

The potential (E’ox) of oxidation peak [23] is given by: E’ox = Eox – (2.303 mRT/nF) pH where Eox is the peak potential value at pH 0.0. R, T and F have their usual meanings. The potential – pH diagram was constructed by plotting observed E’ox v/s pH as shown in SIFig.3, which produces two linear segments with slope of ‐5.4 mV/pH and ‐58.3 mV/pH in the pH ranges of 3.0–5.0 and 6.0–10.4, respectively.

Therefore, the slope value 58.3mV which is in agreement with the theoretical slope (2.303mRT/nF) of 59mV [33] for two electrons and two protons and the corresponding linear equation was Eox (V) = 0.0583 pH + 0.2101 (r = 0.9851) (1) Influence of Scan Rate It is necessary to analyze and evaluate the behavior of electrode reaction for the oxidative determination of the RF. Fig. 4 demonstrates the cyclic voltammograms of GF‐GCE for 1.0 mM RF in 0.2M PB supporting electrolyte at different scan rates in the range from 50 to 600mV. It was observed from Fig. 4 that all the anodic and cathodic peak potentials were shifted to less negative and more negative values respectively with the increase in the scan rate. This study had been carried out to assess whether the electrode reaction was the adsorption or diffusion‐ controlled process.

FIG. 4 CYCLIC VOLTAMMOGRAMS of GF‐GCE FOR 1.0mM RF in 0.2M PHOSPHATE BUFFER of PH 6.0 at SCAN RATE of (a) 50, (b) 100, (c) 150, (d) 200, (e) 250, (f) 300, (g) 350, (h) 400, (i) 450, (j) 500, (k) 550 and (l) 600 mV/s.

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The slope values of the plot of log Ip vs. log v for the oxidation and reduction reactions (Fig.5) were 1.07 and 0.911, indicating that both the reactions were totally adsorption‐controlled processes [34]. The respective linear equations were as follows log Ip = 1.076log v ‐ 1.192(r = 9946) for oxidation (2) log Ip = 0.9118log v ‐ 0.806(r = 9999) for reduction (3)

2.0

2.0 A

B 1.6 log Ipc

log Ipa

1.6 1.2 0.8 0.4

1.2 0.8 0.4

0.0

0.0 0.0

0.5

1.0 1.5 log ˅

2.0

2.5

3.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

log ˅

FIG. 5 LINEAR LOGARITHMIC PLOTS of the (A) ANODIC PEAK CURRENT VERSUS SCAN RATE and (B) CATHODIC PEAK CURRENT VERSUS SCAN RATE

Probable Mechanism The electro‐oxidation of RF involves two electrons and two protons transfer process. The voltammetric oxidation takes place at flavin group of RF as it is capable of undergoing redox reactions [35] and can accept either one electron in two steps or two electrons at once. The reduction is made with the addition of hydrogen atoms to specific nitrogen atoms in the isoalloxazine ring system. The oxidized and reduced forms of RF are in fast equilibrium with semiquinone form. The probable reaction pathway was shown in Scheme 2.

R

R N

O

N

+

2H , 2e

-

N

NH

N

N H

O

O NH

O

H

+

-

,e

-

+,

N + N H

e

H

R

R - C5H11O4

H N

N

O NH

O

SCHEME 2. MECHANISM of REDOX REACTION of RF

Calibration for Detection of RF The determination of RF was carried out by using differential pulse voltammetry (DPV) as it produced current– voltage curves with better intensity and sharp peaks at lower concentration of RF. Under optimum conditions, DP voltammograms were obtained by successive addition of RF in the range from 1.0 nM to 1.9 ×10‐8 M and the peak current increased with the increase in concentration of RF as shown in Fig. 6.

There was a good linear relationship between peak current and concentration of RF in the region 1.0 nM – 1.5 ×10‐

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8

M (Fig. 6 inset). The corresponding linear equation was Ipa(10‐6A) = 0.1102[RF] + 2.408 (r = 0.9827). (4)

The sensitivity of the modified electrode was evaluated by the limit of detection, repeatability, reproducibility and precision values and the parameters of the calibration graph were shown in Table 2.

FIG.6 DIFFERENTIAL PULSE VOLTAMMOGRAMS OF RF of CONCENTRATION (a) 0.0, (b) 1.0, (c) 3.0, (d) 5.0, (e) 7.0, (f) 9, (g) 11, (h) 13, (i) 15, (j) 17 and (k) 19nM in 0.2M PHOSPHATE BUFFER SUPPORTING ELECTROLYTE at GF‐GCE. the INSET SHOWS the CALIBRATION PLOT of the DEPENDENCE of NET PEAK CURRENTS VS. the RF CONCENTRATION. TABLE 2 PARAMETERS OF CALIBRATION CURVE

Linearity range (nM)

1.0 – 16

Intercept (A)

2.408× 10‐6

Correlation coefficient

0.9827

LOD (M)

1.0 × 10‐10

LOQ( M)

3.34× 10‐10

RSD of slope (%)

2.27

RSD of intercept (%)

0.678

Number of data points

10

Repeatability of the peak current (RSD %)

1.26

Repeatability of the peak potential (RSD %)

0.83

Reproducibility of the peak current (RSD %)

1.13

Reproducibility of the peak current (RSD %)

0.91

LOD ‐ Limit of detection LOQ – Limit of quantification RSD – Relative standard deviation

The LOD and LOQ were calculated by using the equations LOD = 3s/m and LOQ = 10s/m [36] respectively, where s is the standard deviation of the peak current of (five runs) of blank 0.2M PB electrolyte and m is the slope of the calibration equation(4). The obtained LOD and LOQ were 1.0 × 10‐10M and 3.43 × 10‐10M. The limits of detection and concentration ranges of previous reports in the literature were listed in the Table 3 [4,

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19‐27]. The proposed method presents lower detection limit and lower concentration range compared with earlier reports which indicates the superiority of the proposed method.

TABLE 3 COMPARISONS OF LINEAR RANGE AND DETECTION LIMITS FOR RF TO PREVIOUS REPORTS

Method

Linearity range (μM)

LOD (μM)

Ref

SFS/PLS

0.018 ‐ 0.143

0.0053

[19]

UHPCL/LC‐MS

‐

0.019

[20]

SWAdSV

0.3 ‐ 0.8

0.1

[21]

m‐AgSAE

0.05 ‐ 0.16

0.00082

[22]

PNA/DPV

0.01 ‐ 65

0.003

[23]

LC‐DAD

0.531 ‐ 5.31

0.1328

[24]

EHGCE

0.4 – 3

0.1

[25]

P3MT

0.1 ‐ 2

0.05

[26]

NME

0.03 ‐ 500

0.005

[4]

MCE

4 ‐ 10

0.02

[27]

GF‐GCE

0.001‐ 0.016

0.0001

Present work

SFS/PLS‐ Synchronous fluorescence technique coupled with partial least squares UHPCL/LC‐MS‐ Ultra High Performance Liquid Chromatography with LC‐MS SWAdSV‐ Square‐wave adsorptive stripping voltammetry m‐AgSAE‐ Mercury Meniscus modified Silver Solid Amalgam Electrode PNA/DPV‐ Polythiophene Nanotube Arrays (DPV) LC‐DAD‐ Liquid Chromatography‐Diode Array Detection EHGCE ‐ Electrically Heated Graphite Cylindrical Electrode P3MT‐ Poly (3‐ methylthiophene) NME ‐ Nanocrystalline Metallosilicate Modified Electrodes MCE‐ Mesoporous Carbon modified Electrode GF‐GCE‐ Graphene Film‐Glassy Carbon Electrode

Reproducibility and Repeatability The repeatability and reproducibility of the modified sensor was examined by repeating five experiments on the same day and in the same standard condition and over 2 days from the different standard solutions. For these studies 1.0 ×10‐6 M RF standard solution were used. The results are given as shown in Table 2. The RSD values of peak potentials and peak currents between day reproducibility are almost identical to that of within a day if the temperature was kept almost constant which could be ascribed to the excellent stability and reproducibility of GF‐ GCE. Effect of Excipients To check the analytical application of determination of RF at the modified electrode, the effect of some common excipients used in pharmaceutical formulation was examined. The acceptable limit for change in peak signal was defined as the maximum concentration of the interfering substance that should not cause an error of 5% for determination of RF. The interference of these excipients on the voltammetric response of RF was obtained by analyzing sample solutions containing a fixed amount of RF (1.0 ×10‐9 M) spiked with various excess amounts of each excipient under the optimized experimental conditions. In our experiment, results showed that a hundred‐ fold excess of citric acid, lactose, dextrose, starch, gum acacia, glucose, sucrose and tartaric acid did not interfere with the voltammetric signal of RF as shown in Table 4. The procedure was able to assay RF in the presence of excipients, and hence it can be considered specific.

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TABLE 4‐ EFFECT OF EXCIPIENTS ON THE VOLTAMMETRIC RESPONSE OF 1.0 × 10‐9M RF

Excipient

Observed potential(V)

Signal change

Riboflavin

‐0.1071

0.0

Citric acid + Riboflavin

‐0.1041

‐0.3

Lactose+ Riboflavin

‐0.1027

‐0.44

Dextrose + Riboflavin

‐0.1084

0.13

Starch+ Riboflavin

‐0.113

0.59

Gum acacia + Riboflavin

‐0.1051

‐0.20

D‐Glucose + Riboflavin

‐0.1036

‐0.35

Sucrose+ Riboflavin

‐0.1046

‐0.25

Tartaric acid+ Riboflavin

‐0.1018

‐0.53

Tablet Analysis and Recovery Test Pharmaceutical tablets containing riboflavin with trade name Lipabol were used for this study to investigate the analytical performance of the modified sensor GF‐GCE. A portion of stock solution prepared as described in the experimental section was spiked into the electrochemical cell containing supporting electrolyte. The differential‐ pulse voltammograms were then recorded under exactly similar conditions that were employed while recording differential pulse voltammograms for plotting calibration plot. The precision of the present method was evaluated for the analysis of pharmaceutical assay of RF sample using 5 different aliquots of stock solution. The present method showed good recoveries, low RSD and bias values as shown in Table 5.

TABLE 5 ANALYSIS OF LIPABOL TABLET BY DPV AND RECOVERY STUDIES

Lipabol

DPV

Labeled claim (mg)

100

Amount found (mg)

98.8

Recovered (%)

98.8

Added (mg)a

10.0

Found (mg)

9.87

Recovered (%)

98.7

R.S.D. (%)

2.82

Bias (%)

1.3

a

Average of five determinations

a

Detection of RF in Urine Samples In order to test the sensitivity of the modified electrode GF‐GCE, the differential pulse voltammetric technique was employed for the detection of RF in different aliquots of prepared stock urine sample as described in experimental section. The recoveries from urine were measured by spiking drug free urine with known amounts of RF. A quantitative analysis can be carried out by adding the standard solution of RF into the detect system of urine samples. The calibration graph was used for the determination of spiked RF in urine samples. The detection results of five urine samples were obtained with good recovery range from 96.0 to 103% as shown in Table 6.

TABLE 6 DETERMINATION OF RF IN URINE SAMPLES

Urine

Spiked(nM)

Found(nM)a

Recovery (%)

SD±RSD

Sample 1

1.0

0.96

96.0

0.011±1.17

Sample 2

3.0

3.1

103

0.046± 1.49

Sample 3

5.0

4.87

97.4

0.050±1.03

Sample 4

7.0

7.08

101

0.037±0.522

Sample 5

9.0

8.83

98.1

0.074±0.839

(a) Average of five measurements

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Conclusions The proposed method demonstrates the successful fabrication, characterization and application of the GF‐GCE in electro‐analytical determination of RF. Graphene film upon casting onto the surface of GCE developed a good electrochemical interaction with riboflavin. The π‐like structures of the isoalloxazine ring in RF and sp2 carbon atoms in graphene were the major sources to shuttle the electron between RF and graphene. The voltammetric oxidation of RF was pH dependant, quasi‐reversible and adsorption controlled. A probable redox mechanism is proposed. There was a longer linearity range between the peak current and concentration of RF in the region of 1.0 nM‐1.5×10‐8 M with a low detection limit of 1.0 × 10‐10 M which was better compared with earlier reports in the literature. There was no effect of any excipients on the electrochemical behavior of RF at GF‐GCE. The modified electrode was successfully employed in the determination of RF in the pharmaceuticals as well as in urine samples and obtained good recoveries. Furthermore, the proposed method could possibly be applicable in the pharmacokinetic studies as well as clinical and quality control laboratories.

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Supplementary Figures

SIFIG.1 CYCLIC VOLTAMMOGRAMS OF GF‐GCE FOR 1.0mM RF IN 0.2M PHOSPHATE BUFFER OF PH (a) 3.0, (b) 4.2, (c) 5.0, (d) 6.0, (e) 7.0, (f) 8.0, (g) 9.2, and (h) 10.4 AT SCAN RATE OF 100mV/s

10.0 9.0 8.0

Ip (10‐5A)

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0.0

2.0

4.0

6.0

8.0

10.0

12.0

pH SIFIG.2 VARIATION OF PEAK CURRENT WITH PH

-0.5 -0.4 EʹOX (V)

-0.3 -0.2 -0.1 0.0 0.0

2.0

4.0

6.0

8.0

10.0

12.0

pH SIFIG. 3 THE TWO LINEAR SEGMENTS FOR THE PLOT OF E'OX VERSUS PH.

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