Electro oxidation and analytical applications of valacyclovir at reduced graphene oxide modified carbon paste electrode

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ScienceDirect Materials Today: Proceedings 18 (2019) 550–557

www.materialstoday.com/proceedings

ICN3I-2017

Electro oxidation and analytical applications of valacyclovir at reduced graphene oxide modified carbon paste electrode Aravind Todakara, Nagaraj P. Shettib,*, Umesh S. Devarushic, Suresh M. Tuwarc a b

Department of Biotechnology, B.V.B college of Engineering and Technology, Hubballi 580031, Karnataka, India

Electrochemistry and Materials Group, Department of Chemistry, K.L.E. Institute of Technology, Gokul, Hubballi-580030, affiliated to Visvesvaraya Technological University, Karnataka, India. c

Department of Chemistry, Karnatak University’s Karnatak Science College, Dharwad-580001, Karnataka, India.

Abstract Electrochemical investigation of valacyclovir (VCH), an antiviral drug was investigated at carbon paste sensor modified with reduced graphene oxide (rGO/CPE). Cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques are inherited to determine the electrochemical behavior of VCH. Modified and bare carbon surfaces were characterized by Atomic force microscope (AFM). The technique dependence on current, potential, supporting electrolyte pH, concentrations, sweep rate, and excipients were evaluated to optimize the experimental conditions. Contrasted with prior reports, this study is very impressive on account of modifiers utilized and expedient because of its affectability, selectivity and LOD value.

© 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017).

Keywords: Graphene oxide; Valacyclovir; Voltammetry; Pharmaceutical samples; Atomic force microscope.

* Corresponding author. Tel.: +91 9611979743; fax: 0836 – 2330688. E-mail address: [email protected] 2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017).

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Nomenclature A Surface area of the electrode (cm2) * C0 Concentration (mol dm-3) D0 Diffusion coefficient (cm2s-1) Ep Peak potential (V) F Faraday constant (C mol-1) Ip Peak Current (μA) k0 Standard Heterogeneous Rate Constant (s-1) R Gas constant (J K-1mol-1) υ Scan rate (mV s-1)  Transfer coefficient RSD Relative Standard Deviation S Standard deviation of the peak currents T Temperature (K) LOD Limit of Detection (mol dm-3) LOQ Limit of Quantification (mol dm-3) n Number of electrons transferred rGO Reduced graphene oxide CPE Carbon paste electrode VCH Valacyclovir 1.

Introduction

Valacyclovir (VCH) (Scheme 1) is an antiviral bio-molecule used in the treatment of infections caused by virus, like varicella-zoster and herpes simplex in human beings [1]. VCH is having highly selective inhibitor activity, because of its thymidine kinase (TK) enzyme affinity which encodes VZV and HSV. VCH is a pro-drug of acyclovir, consists similar characters of acyclovir [2-4]. On the contrary, VCH has higher oral bioavailability than acyclovir, i.e., 3-5 times higher. The electrochemical techniques have more advantageous over the other in sensing any bioactive molecules due to its astonishing qualities [5, 6]. Till now no research was found on the electrosensing of VCH using reduced graphene oxide (rGO) modified carbon paste electrode (CPE). The present work aspires to fabricate a sensor using rGO as modifier. Already the CPEs have magnetized a wide range of researchers, in the area of modified sensors chemically owing to its distinctive properties such as small residual background current, huge applicable potential window (applications to both oxidations and reductions), ease of fabrication, good stability, quick surface renewal, and safe disposability after the use with diverse sorts of modulators [7, 8].

Scheme 1. Chemical Structure of valacyclovir

According to the literature survey, till now few works were reported on the voltammetric determination of VCH. In the present work we used reduced graphene oxide paste electrode (rGO/CPE) to investigate the electrochemical behaviour and its analysis. In prolongation of our work, the experimental conditions for the identification of VCH were optimized and its analysis in tablets as well as in spiked human urine sample was done.

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2. Experimental 2.1. Instrumentation and chemicals An electrochemical analyzer (CHI Company, D630, USA) is utilized to carry out voltammetric measurements. The analyzer was incorporated by three electrode system, main functioning sensor as rGO modified carbon paste electrode (rGO-CPE), auxiliary sensor as platinum wire, and reference sensor as an Ag/AgCl (3.0 M KCl) correspondingly. Both the bare and new fabricated CPE sensing surfaces were redeveloped via polishing with the use of filter paper and then water wash was followed, prior to each measurement. By utilizing pH meter (Elico Ltd., India) the pH measurements were performed. Current work utilized phosphate buffer saline (PBS) solution [9]. VCH, graphene and all other chemicals purchased from Sigma Aldrich and were used as without any additional purification. In this whole experiment double distilled water was used. Double distilled water is used to prepare the analyte stock solution of VCH (5 × 10-5 M) and stores at low temperature. 2.2. Preparation of modified electrode Fabrication of CPE was done by homogeneous blending of powder form of graphite with paraffin oil in 7:3 ratios. The resultant paste was firmly packed in a hollow polytetrafluoroethylene tube (PTFE), and the surface was smoothened. After every measurement, the paste was carefully removed prior packing a new paste. Likewise, the rGO-CPE was prepared by adding reduced graphene oxide particles in pertinent amount to the blended mixture during the paste preparation [10-12]. 3. Results and discussion 3.1. Calculation of surface area and characterization of the modified electrode To calculate the active surface vicinity of the new fabricated electrode, Randles-Sevcik equation was utilized as usual. To acquire result, KCl (0.1 M) was taken as supporting electrolyte and the test solution was K3Fe (CN)6 with concentration of 1.0 mM and diffusion coefficient (D0) of 7.6 x 10-6 cm2 s-1 [13]. From the slope of the plot of Ip vs. υ1/2, the area of the electrode surface was calculated to be 0.064 cm2. The surface area of bare and modified electrodes were characterized by AFM analysis as shown in Fig. 1. Ip = (2.69 × 105) n3/2 A D01/2 ν1/2 C0*

(1)

Fig. 1: AFM images of (A) Bare CPE and (B) rGO-CPE

3.2. Electrochemical behaviour of VCH and accumulation time effect The study of preconcentration time impact was carried out in a range of 20-70 s (Fig. 2). At 20 s the maximum oxidation peak was notified. This effect indicates saturated adsorption on the modified electrode was achieved at 20 s, hence the same accumulation time was inherited for further studies.

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Fig. 2 Accumulation time a) 20 b) 30 c) 40 d) 50 e) 60 f) 70 Seconds at pH 4.2, 100 mV/S and 5x10-5 [VCH]

Fig. 3 indicates CVs of CPE and rGO-CPE, in the presence and absence of 5 × 10-5M VCH at 100 mVs−1 in pH 4.2. An accentuated oxidation peak was observed at rGO-CPE while at bare CPE a deprived peak was found. In the active presence of 5 × 10-5M VCH, an irreversible behaviour through moderately weak peak currents occurred at anodic potential for bare CPE. While at rGO-CPE, the same behaviour through an accentuated anodic peak observed correspondingly. The high surface area, large conductivity, elevated electrocatalytic activity and some promising features of rGO accelerates the electron transfer during electrooxidation which is directly responsible for the enhancing effect of rGO-CPE in VCH determination.

Fig. 3. Cyclic voltammogram behavior of VCH (5 × 10-5 M), phosphate buffer (pH = 4.2 , I = 0.2 M), (A) blank CPE, and (B) VCH at CPE (C) VCH at rGO/CPE; scan rate: 100 mVs-1.

3.3. Effect of modified electrode on pH The electrochemical response of VCH (5 × 10-5 M) was studied by CV method, over the pH range of 3.0-11.2 in 0.2 M PBS (Fig. 4). At pH 4.2, highest peak current was noticed for VCH detection and therefore, for further analytical studies pH 4.2 was chosen (Fig. 4 A). From the plot Ep versus pH (Fig. 4B), acquired linear equation is as follows; Ep= -0.04x + 1.188 R² = 0.996. The slope value indicates that in the VCH oxidation, protons and electrons were involved in equal number.

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Fig. 4 Influence of pH on the shape of anodic peak of VCH in pH a) 3.0 b) 4.2 c) 5.0 d) 6.0 e) 7.0 f) 8.0 and g) 9.0 pH (A) variation of peak current with pH (B) Variation of peak current with pH at rGO/CPE

3.4. Effect of scan rate The VCH (5 × 10-5 M) voltammograms were recorded on the rGO-CPE surface at varying scan rates in pH 4.2 by CV (Fig. 5). From the study, it was indication that the peak current (Ip) was linearly augmented with sweep rate (υ) improvement (Fig. 5A) with regression equation; Ip (µA) = 0.47 + 0.3; R2 = 0.990. Further the slope of log Ip of anodic peak current vs. log υ plot, which proves that the present electrochemical reaction is diffusion controlled process [14]. In addition a good linear relation was observed between Ep and log ν [15] (Fig. 5B). Scan rate and Ep relationship for a process involved by sensor can be stated by Laviron’s theory [16].

2.303RT RTk0 2.303RT + log Ep = E + nF nF nF

log ʋ

0

(2)

According to Bard and Faulkner,  value can be evaluated by Eq. 3. So, from the calculation we got the value of α to be 0.56. Further, the number of electron (n) transferred in the electro-oxidation of VCH was calculated to be 2 (Scheme 2).

p= Ep - Ep/2=

47.7

(3)

mV

n

O O N

H2N

O

N

N

N

CH3

O O

H

1e-

CH3

H2N

NH2

H N

N

O N

N

O

CH3

O

H

CH3

NH2

H+ OHO H N

N H2N

O O

N H

N

O

O O

CH3 CH3 NH2

1e-1 -H+

H N

N H2N

OH N

N

O

H

Scheme 2. Possible mechanism of VCH

O O

CH3 CH3 NH2

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Fig. 5. Effect of scan rate on electro chemical behaviour of VCH: a) 0.06, b) 0.08, c) 0.15, d) 0.25 e) 0.30 f) 0.40 (A) Plot of Peak current (ip/µA) vs. scan rates (υ) (B) Plot of peak potential Ep vs log scan rate (υ)

3.5. Concentration variation SWV technique give well defined and sharper peaks even at small concentration of VCH (Fig. 6), therefore SWV only utilized for concentration study to get low LOD with high precision. The VCH voltammograms obtained in SWV were lies in the range of 5.0×10-5 to 1.0 × 10-8 M. The linear equation was found calculate LOD and LOQ value of this method [17-18]. The current work recommends highly sensitive method with low detection and quantification limit for VCH compared to former performances by other techniques (Table 1).

Fig. 6. SWV for increasing concentrations of VCH at rGO/CPE in pH = 4.2, (a) 1.0 x 10-8, (c) 3.0 x 10-8, (d) 5.0 x 10-8,(e) 8.0 x 10-7,(f) 1.0 x10-7, (g) 5.0 x 10-6,(h) 7.0 x 10-6, (i) 5.0 x 10-5

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Table 1. Comparison Study Method

LOD

References

Spectroscopy Chromatography RP-HPLC Square wave voltammetry Square wave voltammetry using rGO/CPE

37.4 µM 14.2 µM 25.1 µM 1.40 µM

[19] [20] [21] [22]

1.08 nM

Present work.

3.6. Analytical application: VCH containing commercially obtainable pills were bought from a pharmacy and directly analyzed by SWV technique after the sample preparation process [23, 24]. The clear supernatant liquid of aliquots were taken and diluted with pH 4.2. Standard addition method was adopted for accurate recovery studies. Likewise excipients effect also studied to check the proposed method accuracy. The results shows good agreement with the content marked in the label. In different sample, the recoveries lie around 96.1-97.5 % (Table 2). From healthy candidates, urine samples were collected and then centrifuged (4383 G) at ambient temperature (25 ± 0.1 0C) for 5 minutes. So attained samples go through double dilution using pH 4.2, and the known amount of VCH was spiked with the filtrate to prepare test solution. The results prove good agreement (Table 2). Table 2. Application of SWV for the determination of VCH in pharmaceutical samples and spiked human urine samples. Detected (10-6 M) Recovery Pharmaceutical Samples Spiked (10-6 M) Sample 1 0.1 0.0961 96.1 Sample 2 0.2 0.195 97.5 Sample 3 0.3 0.290 96.6 Urine Samples Spiked (10-4 M) Sample 1 0.05 Sample 2 0.03 Sample 3 0.01 *Average five readings

Detected (10-4 M) 0.0488 0.0275 0.0094

RSD

% RSD

97.6 91.6 94.0

2.79 2.97 2.90

3.7. Effect of excipients Study of excipients scrutinized to check the VCH behaviour in the existence of some general biological metabolites. The result indicates that the potential of the drug changed slightly but not exceed ±5%, which suggests that VCH reactions at the sensing base, does not affects the existence of any metabolites tested. Hence, the fabricated sensor can be capably utilized for VCH detection. 4.

Conclusion In this paper reduced graphene oxide particles were introduced as efficient modifier into the CPE paste and thus fabricated sensing system stood proficient for VCH quantification. The morphology sensing surface was studied by AFM analysis. Compared to bare CPE the modified CPE shows enriched resolution, sensitivity and selectivity towards the analysis, due to the multifarious features of rGO. From the acquired data, diffusion controlled process and equal number of electrons-protons involvement was witnessed. Therefore, to detect VCH in tablets and spiked human urine samples and the proposed method can be profitable. In addition, the existence of general excipients in biological samples was impervious the analytical response

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