Scholarly article on topic 'Adsorptive square wave voltammetric determination of the antiviral drug valacyclovir on a novel sensor of copper microparticles–modified pencil graphite electrode'

Adsorptive square wave voltammetric determination of the antiviral drug valacyclovir on a novel sensor of copper microparticles–modified pencil graphite electrode Academic research paper on "Chemical sciences"

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{Valacyclovir / "Copper microparticles–modified pencil graphite electrode" / "Adsorptive square wave stripping voltammetry" / Tablets}

Abstract of research paper on Chemical sciences, author of scientific article — Gamal A. Saleh, Hassan F. Askal, Ibrahim H. Refaat, Ahmed H. Naggar, Fatma A.M. Abdel-aal

Abstract To overcome the well-known problems encountered with surface fouling of carbon electrodes that occurred with many compounds, we report a simple and rapid method for the preparation of a disposable sensor depending on electrodeposition of porous Cu-microparticles on pencil graphite electrode (Cu-PGE) that can be applied for sensing antiviral compound valacyclovir (VAL). The bare and porous Cu-modified pencil graphite electrodes were characterized by cyclic voltammetry and SEM. The porous Cu-modified pencil graphite electrode displayed distinct electrocatalytic activities in response to the electrochemical redox reaction of Cu2+ ion in the Cu-VAL complex. Adsorptive square wave stripping voltammetry (AdSWV) was used for the direct electrochemical determination of VAL. Under experimental conditions, the modified electrode had a linear response range from 2.0×10−9 M to 1.0×10−8 M VAL with a detection limit of 1.78×10−10 M. The procedure was applied to the assay of VAL in tablets with mean percentage recoveries of 100.28%. The porous Cu-modified pencil graphite electrode (Cu-PGE) is fabricated utilizing a simple single method, and at an extremely low cost with high stability, sensitivity and offering a promising tool for sensing VAL in quality control laboratories and in stability studies.

Academic research paper on topic "Adsorptive square wave voltammetric determination of the antiviral drug valacyclovir on a novel sensor of copper microparticles–modified pencil graphite electrode"

Arabian Journal of Chemistry (2015) xxx, xxx-xxx

King Saud University Arabian Journal of Chemistry

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Adsorptive square wave voltammetric determination of the antiviral drug valacyclovir on a novel sensor of copper microparticles-modified pencil graphite electrode

Gamal A. Saleha, Hassan F. Askala, Ibrahim H. Refaata, Ahmed H. Naggarb, Fatma A.M. Abdel-aala*

a Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut University, 71526 Assiut, Egypt b Chemistry Department, Faculty of Science, Al-Azhar University, Assiut Branch, 71524 Assiut, Egypt

Received 6 April 2015; accepted 14 August 2015

KEYWORDS

Valacyclovir;

Copper microparticles-modified pencil graphite electrode;

Adsorptive square wave stripping voltammetry; Tablets

Abstract To overcome the well-known problems encountered with surface fouling of carbon electrodes that occurred with many compounds, we report a simple and rapid method for the preparation of a disposable sensor depending on electrodeposition of porous Cu-microparticles on pencil graphite electrode (Cu-PGE) that can be applied for sensing antiviral compound valacyclovir (VAL). The bare and porous Cu-modified pencil graphite electrodes were characterized by cyclic voltammetry and SEM. The porous Cu-modified pencil graphite electrode displayed distinct elec-trocatalytic activities in response to the electrochemical redox reaction of Cu2+ ion in the Cu-VAL complex. Adsorptive square wave stripping voltammetry (AdSWV) was used for the direct electrochemical determination of VAL. Under experimental conditions, the modified electrode had a linear response range from 2.0 x to 1.0 10~8M VAL with a detection limit of

1.78 x 10~10 M. The procedure was applied to the assay of VAL in tablets with mean percentage recoveries of 100.28%. The porous Cu-modified pencil graphite electrode (Cu-PGE) is fabricated utilizing a simple single method, and at an extremely low cost with high stability, sensitivity and offering a promising tool for sensing VAL in quality control laboratories and in stability studies. © 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author. Fax: +20 88 2080774. E-mail addresses: 3amora2013@gmail.com, famo207@yahoo.com (F.A.M. Abdel-aal).

Peer review under responsibility of King Saud University.

1. Introduction

Substituted purines represent an important category of compounds actively studied as potential therapeutics against viral infection. Because they constitute the essential components of biologically important compounds such as polynucleic acids, the knowledge of voltammetric behavior of these

http://dx.doi.Org/10.1016/j.arabjc.2015.08.015

1878-5352 © 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

compounds is of biological interest (Brett and Matysik, 1997; Visor et al., 1985).

It is therefore important to examine the electrochemistry of a recently developed guanine analogue, valacyclovir (Scheme 1) that is used for the treatment of diseases caused by herpes simplex and varicella zoster virus infections. This compound is a prodrug and converted rapidly and extensively to acyclovir, the active antiviral component of valacyclovir, and l-valine, an essential amino acid, probably in the liver and the intestine by hydrolysis, after oral administration. Because of the low bioavailability of acyclovir (only 15-30% of the dose is absorbed from the gastrointestinal tract), valacyclovir has been synthesized, which is readily absorbed (about 50% of the dose), after its oral administration so it could not be detected in plasma or urine (Brunton et al., 2011).

There have been only few reports dealing with the determination of valacyclovir based on spectrophotometric analysis (Kumar et al., 2010; Reddy et al., 2011; Srihari et al., 2013, 2011) and high performance liquid chromatography (Jadhav et al., 2007; Lahari, 2013; Sahoo and Sahu, 2014; Sugumaran et al., 2001). Recently, complexation has often been used to influence biological processes that are metal dependent. Valacyclovir was determined also via its complexa-tion with copper by Golcii et al. The complexation process was studied under different conditions using UV-Vis, IR and mass spectra, magnetochemical, thermogravimetric, atomic absorption, conductivity, and elemental analysis data (Golcu and Dolaz, 2006). These reported methods are complicated, not sensitive and time and solvent-consuming. From the electrochemical point of view, only one study has been made on this compound (Uslu et al., 2006). So the electrochemical determination of the present drug is favorable due to its simplicity, portability, fast responses, good sensitivity and high selectivity. However, the electrode should be modified with electrocatalyst or electron mediator to detect the drug as the previously used electrode shows sluggish electrocatalytic properties and poor sensitivity for electrochemical determination of the drug (Uslu et al., 2006). Also because these methods are less sensitive to matrix effects than other analytical techniques, the sensitive determination of drugs in complex biological fluids is possible without time-consuming extraction procedures being necessary before the voltammetric measurement. The use of carbon based electrodes for electroanalysis has gained popularity in recent years because of their applicability to the determination of substances that undergo oxidation reactions (Abdulkadir Levent* and Senturk, 2009; Ashrafi et al., 2013; Bilibio et al., 2014; Levent et al., 2014). When compared with other carbon-based electrodes, PGEs have the following advantages, such as high electrochemical reactivity, commercial availability, good mechanical rigidity, disposability, low cost, low technology and ease of modification (Wang et al., 2000). In addition, it was reported that pencil lead electrodes

^O7^7^ o

Scheme 1 Chemical structure of valacyclovir (VAL).

offer a renewal surface which is simpler and faster than polishing procedures, common with solid electrodes, and result in good reproducibility for individual surfaces (Wang et al., 2000). Disposable pencil graphite electrode when compared with others forms of carbon electrodes such as ''low tech" pencil graphite electrode is extremely inexpensive and provides an attractive alternative to ''high tech" carbon electrodes (Bond et al., 1997).

The experimental procedure for the modification of carbon based electrodes can be simplified if the modifying agent (as microparticles layer) is formed as a layer on the electrode, which is termed in situ modification. The in situ modification has the advantage of shortening or eliminating the preparation steps before the analysis. Metal and metal oxide microparticles are the most widely employed materials owing to excellent physical and catalytic properties. These materials are being employed in electrochemistry to improve the performance of electrochemical techniques due to excellent electrocatalytic properties (Mirkhalaf et al., 2009).

To the best of our knowledge, no study has been reported for the determination of VAL by using PGE. In this study we report the preparation and application of Cu-microparticles modified PGE for the determination of VAL without any additional modification and its application for determination of VAL in its dosage form.

Therefore the aims of this study were, first, to demonstrate the usefulness of a pencil graphite electrode in presence of metals (e.g. Cu) on the electrochemical behavior of VAL, and second, to develop a novel method for the analysis of VAL in its tablets.

2. Experimental

2.1. Reagent and chemicals

Valacyclovir hydrochloride was gifted from Hekma pharmaceuticals (Cairo, Egypt) and was used without further purification.

Phosphoric acid (85%), glacial acetic acid, boric acid, sodium hydroxide, potassium nitrate and copper sulfate were purchased from (Sigma, Cairo, Egypt). Britton-Robinson (BR) buffer solutions in the pH range 2.1-6.0 were prepared from 0.04 M phosphoric acid, 0.04 M boric acid and 0.04 M acetic acid containing 0.2 M potassium nitrate in deionized water. The pH of the solutions was adjusted by adding 0.2 M NaOH. All solutions were prepared using deionized water.

2.2. Instrumentation

A Princeton VersaSTAT MC (VersaSTAT 3, Model RE-1, Princeton Applied Research, AMETEK, USA) connected to a three-electrode cell was used for the electrochemical measurements. In all measurements, the reference electrode was an Ag/ AgCl (3 M KCl), the auxiliary electrode was a platinum wire and a simple pencil graphite electrode coated with Cu microparticles electrodeposited in situ as the working electrode. The pH values of solutions were measured using Hanna pH meter (Hanna Instruments Brazil, Sao Paulo, SP, Brazil) with a combined electrode. Surface morphological studies of the modified electrode were carried out using scanning electron

microscope (SEM), JEOL JSM-5400 LV instrument (Oxford, USA).

2.3. Electrode preparation

A pencil lead with a diameter of 0.5 mm (Ultra-Polymer, H) and a total length of 60 mm (Tombow, Japan), and a mechanical pencil Model T 0.5 (Rotring, Germany), which was used as the holder for the pencil lead, were purchased from a local bookstore. Electrical contact to the lead was obtained by wrapping a metallic wire to the metallic part of the holder. For each measurement, a total of 10 mm of lead was immersed into the solution. The length of pencil lead was measured with a ruler as 10 mm into the supporting electrolyte and then was dipped into a tip of the electrochemical cell. Electrodeposition of Cu atoms at PGE from 5 x 10—4M CuSO4 solution was carried out in galvanostatic mode by applying direct current at —0.6 V as a deposition potential and 60 sec. as a deposition time and then washed with deionized water.

2.4. Preparation of the standard solutions

A stock solution of VAL (10—2 M) was freshly prepared in deionized water. The required concentration of VAL was then prepared from the stock standard solution. A previous publication (Pham-Huy et al., 1999) reported that valacyclovir was partially hydrolyzed in aqueous solution to acyclovir after storing at +4 0C for several weeks. For this reason, all solutions were kept in the dark in a refrigerator and were used within several hours to avoid hydrolysis. Deionized water was used to prepare all solutions. All experiments were carried out at the ambient temperature of the laboratory (25 ± 5 0C).

2.5. Procedure

In the electrochemical measurements, Cu-PGE was used directly and the electrochemical behavior of VAL at Cu-PGE was investigated in the presence of free Cu2+ ions by recording cyclic voltammograms in BR buffer solution in the pH range of (2.1-6.0) with 0.2 M KNO3. For this, 7 mL of supporting electrolyte was placed in the electrochemical cell and a total of 10 mm of Cu-PGE (previously prepared in situ) was immersed into the supporting electrolyte. The cyclic voltammograms of 1 x 10—4M CuSO4 were recorded at Cu-PGE in the absence and presence of 5 x 10—5 M VAL at a scan rate of 100mVs—1 using an applied potential from —0.6 V to +1.5 V. The square wave voltammetric technique was used for the determination of VAL in the presence of free Cu2+ ions on Cu-PGE. Square wave voltammograms were recorded in a potential range between —0.6 V and +1.5 V.

2.6. Sample preparation

Ten tablets of Valysernex® (each one contains 1000 mg vala-cyclovir) were weighed and powdered in a mortar. A weighed portion of the tablet powder was prepared to obtain a solution of 10—3M of valacyclovir. It was transferred into a 100 mL calibrated flask and completed to the volume with deionized water. The contents were sonicated for 10 min to affect complete dissolution and then filtrated. Appropriate solutions were

prepared by taking suitable aliquots of the clear filtrate and diluting with deionized water, and the voltammetric procedure was followed.

3. Results and discussion

3.1. Surface morphology study

Fig. 1 shows SEM (a) PGE bare (b) PGE coated with copper microparticles. Copper microparticles with near spherical shape with a diameter ranged from 250 to 500 nm can be observed. (c) PGE coated with copper microparticles in the presence of adsorbed layer of Cu-VAL complex, some aggregations of Cu-VAL complex were observed which changed the surface morphology of copper microparticles.

3.2. Cyclic voltammetric studies

The electrochemical behavior of VAL was investigated at Cu-PGE. Cyclic voltammograms of 5.0 x 10—5M VAL were recorded in the BR buffer solution at the optimum pH (2.7) containing 0.2 M KNO3 and at a scan rate of 100 mV s—1 in the absence and presence of 1.0 x 10—4M CuSO4. In the absence of free Cu2+ ions no peaks were observed for VAL on PGE bare or on Cu-PGE. In the presence of 1.0 x 10 4 M CuSO4, the behavior of free Cu2+ ions was observed as a weak peak at —0.05 V for Cu2+ ions reduction and after addition of VAL to Cu2+ salt, there was an increase in the Cu2+ cathodic current quantitatively with the increase of VAL concentration due to the reduction of Cu2+ ions in the electroactive Cu-VAL complex in addition to the appearance of ill defined broad peak at 1.00 V for VAL oxidation (Fig. 2a). The very weak oxidation peak of VAL can't be used for quantitative determination of VAL. So we proposed the quantitative indirect measurement of VAL as mentioned before. The remarkable enhancement in the Cu2+ cathodic current peak current indicates the electrocatalytic activity of the electrodeposited porous Cu-microparticles. This electrocatalytic effect and higher sensitivity can be explained by the porosity of PGE, which is formed by a mixture of graphite and clay and the large surface area introduced by porous-Cu-microparticles.

3.3. Effect of scan rate

The influence of scan rate (o) on the voltammetric peak is investigated in the range from 10 to 1000 mV s—\ Useful information involving electrochemical mechanism usually can be acquired from the relationship between the peak potential and the scan rate. It was found that the increase in Cu peak on addition of VAL is linear to the scan rate (Fig. 3a) according to the following equation:

Ip (iA) = 520.98o + 32.144 (r = 0.999)

The plot of log I versus log of scan rate (log o) was found according to the following linear regression equation:

LogIp — 2.685 + 0.6284 log o (r = 0.995)

The plot of the peak potential Ep versus log of scan rate (log o V s—*) followed the linear regression equation:

Ep(V) — 0.0253 + 0.07log o (r = 0.998)

Figure 1 Scanning electron micrographs (a) PGE bare, (b) PGE coated with copper microparticles and (c) PGE coated with copper microparticles in the presence of adsorbed layer of Cu-VAL complex.

-0.35 0 0.35

E (V) vs. Ag/AgCl electrode

Figure 2 (a) Cyclic voltammograms of BR buffer at the optimum pH (2.7) containing 0.2 M KNO3. (1) 5.0 x 10-5 M VAL on PGE bare [no peaks]. (2) 1.0 x 10~4 M CuSO4 on Cu-PGE. (3) 5.0 x 10~5 M VAL + 1 x 10~4 M CuSO4, (a) oxidation peak of VAL, (b) increase in Cu2+ ions reduction peak after formation of Cu-VAL complex onto Cu-PGE, scan rate: 100 mV s_1; t(acc): 60 sec. (b) Successive Cyclic voltammograms of mixture of 1 x 10~4 M CuSO4 and 5.0 x 10~5 M VAL on Cu-PGE in BR buffer solution (pH: 2.7) containing 0.2 M

KNO3, scan rate: 100 mV s-1; t

60 sec.

For the irreversible electrode process, the relationship between the reduction peak potential and scan rate (Fig. 3b) can be used to calculate number of electrons transferred in the rate determining step by the following equation (Laviron, 1979):

Slope of the graph between Ep and log o = (2.303RT/anF)

where T is the temperature (298 K), a is the transfer coefficient, n the number of electrons transferred in the rate determining step, o is the scan rate and F is the Faraday constant

(a) (b)

Scan Rate (V/sec.) Log Scan Rate /V S-1

Figure 3 (a) Dependence of the reduction peak current (lA) on scan rate (u V s_1). (b) Dependance of the reduction peak potential (V) on log scan rate u/V s_1.

(96.480 C moP1), R is the universal gas constant (8.314 J mol-1 K-1). Herein, the slope is 0.07 and an was calculated to be 0.84. Generally, a is assumed to be 0.5 in totally irreversible electrode process. Therefore, the value of n = 1.68 (w2) was obtained for the reduction peak of Cu2+ ions.

Furthermore, it was found that the peak current of Cu-VAL complex showed a remarkable decrease during the successive cyclic voltammetric sweeps as shown in (Fig. 2b). It resulted from the fact that the electrode surface is blocked by the adsorption of the Cu-VAL complex which reduces the effective reaction sites at the modified electrode surface.

4. Optimization of experimental conditions

The resulting reduction peak current of Cu2+ in Cu-VAL complex, using adsorptive square wave stripping voltammetry (AdSWV) technique at the working Cu-PGE, was characterized with respect to the pH, supporting electrolyte composition, accumulation potential, frequency, pulse amplitude, step height and accumulation time.

4.1. Effect of type of metal

3.4. Possible mechanism

Electrodeposition of a Cu-microparticles layer onto PGE surface is a viable tool for improving its sensitivity due to its excellent electrocatalytic properties. In Britton Robinson buffer at the optimum pH (2.7) VAL acts as a legend to complex Cu2 + ions present in the solution forming Cu-VAL complex that adsorbed onto Cu-modified PGE surface. The complexation processes between Cu2 + ions and VAL were studied by Golcii and Dolaz (2006)) that proved formation of mononu-clear Cu-VAL complex. The proposed Cu-VAL complex structure was illustrated in (Scheme 2). The electrodeposited Cu-microparticles act as mediator to shuttle electrons between Cu-VAL complex and PGE surface. This mediator catalyzes the electroreduction of Cu2 + ions present in Cu-VAL complex. This is the principle of the indirect quantitation of VAL concentrations. Totally 2e~ were transferred in the reduction of Cu2 + ions in the complex to Cu metal. The Cu-complexation depends on the guanine moiety present in VAL structure, so the proposed method can be applied for determination of structurally related compounds that contain guanine moiety as acyclovir, ganciclovir and valganciclovir.

Scheme 2 The proposed structure of Cu-VAL complex (Gölcu and Dolaz, 2006).

Adsorptive square wave stripping voltammetry was used to test the effect of different metal salts (Cu2 + , Zn2 + , Co2 + , Ni2 + and Pb2+) in complexation with VAL to give quantitative relation between VAL concentration and current. No metal was found to complex with VAL or enhance its determination via ADSWV. In addition to that copper metal is the only one that is reported to complex with VAL in previous work (Golcii and Dolaz, 2006). It was found that the most suitable one is the copper. Different copper salts (Cu-sulfate, Cu-nitrate, and Cu-acetate) were tested to determine the most appropriate salt that gives higher sensitivity. Copper sulfate was found to be the best one.

4.2. Effect of pH and background electrolyte

The pH of the electrolyte, which influences the distribution of species of not only Cu2 + but also VAL, had a significant effect on the formation of Cu-VAL complex and so had a significant effect on the indirect determination of VAL. The BrittonRobinson buffer solution was used in the pH range from 2.1 to 6.0. The peak current, (Ip), versus pH plot shows that Cu2 + reduction peak current in Cu-VAL complex is maximized at pH 2.7 as shown in (Fig. 4a). These results suggest the importance of an acid medium for Cu-VAL complex formation. The pH also affects Cu2+ reduction peak potential, (Ep). When the pH values were increased from 2.1 to 6.0, a cathodic shift of the potential peak was observed due to the decrease in the tendency for Cu-VAL complex formation (Fig. 4b).

The effects of the other different buffer solutions, such as acetate buffer, Clark-Lubs buffer, Teorell Stenhagen buffer on ACV peak, also were tested. The results showed that the peak of Cu-VAL complex was sharper and sensitive in

(a) (b)

Figure 4 Dépendance of Peak current (a) and peak potential (b) on the pH for the reduction of Cu2+ ions present in Cu-VAL complex in the presence of 1.0 x 10~5 M Cu2+ and 5.0 x 10~6 M VAL in BR buffer solutions (2.1-6.0) containing 0.2 M KNO3. (Pulse amplitude: 25 mV; deposition time: 180 sec; frequency 150 Hz).

Britton-Robinson buffer than in other media. Several supporting electrolytes were also tested including KCl, NaClO4 and KNO3 and the results showed that the best one is KNO3. The supporting electrolyte concentration is also effective and so it was optimized. By varying the KNO3 concentration it was found that 0.2 M KNO3 in the solution gave the best results so it was chosen as the optimum one.

4.3. Effect of accumulation potential and time

It is important to fix the accumulation potential (Eacc) and time (tacc) when adsorption studies were undertaken. Bearing this in mind, the effect of Eacc and tacc has been studied by AdSWV method. The influences of the Eacc on the reduction peak of Cu2+ in Cu-VAL complex were studied over the potential range of —0.8 to —0.3 V. The plot of stripping peak current as a function of accumulation potential indicated that the maximum peak current took place at —0.6 V. Thus, accumulation potential of —0.6 V was chosen for subsequent uses as shown in (Fig. 5a).

The influence of accumulation time (tacc) on the response of the electrode in a solution containing 1.0 x 10—5M Cu2

+ 5.0 x 10—6 M VAL was also investigated. Variation of the accumulation time showed that the peak current of Cu-VAL increased with increasing the accumulation time, gradually levelling off at period longer than 180 sec. (the response at 180 sec. is about 90% of 240 sec. deposition time), presumably due to the saturation of the electrode surface with the adsorbed layer of Cu-VAL complex. Thus deposition time of 180 sec. was used throughout, as it combines good sensitivity and relatively short analysis time as shown in (Fig. 5b).

4.4. Effect of square-wave voltammetric parameters

Final optimization of the analytical signal centered upon varying the square-wave parameters such as the frequency, step potential and pulse amplitude.

4.4.1. Effect of frequency

The effect of frequency was studied in the range 10-250 Hz. A linear relationship was obtained between the peak current and frequency of the signal up to 150 Hz due to the increase in the effective scan rate but at higher values of frequency, the peak heights decreased. Hence the frequency of 150 Hz was chosen for entire analysis.

4.4.2. Effect of step potential

The influence of step potential was investigated between 1 and 35 mV. The peak height increased up to 15 mV because the effective scan rate was increased, but at higher values of step potential peak shape was distorted. So 15 mV was chosen as the optimum step potential in the entire analysis.

4.4.3. Effect of pulse amplitude

The analytical signal was dependent on the pulse amplitude even if this parameter seems to be less important than the frequency. Pulse amplitude was examined in the range from 5 to 50 mV. Peak heights increased upon increase of the pulse amplitude up to 25 mv and at higher values the peak shape was distorted. Thus 25 mV was chosen as the optimum pulse amplitude for all subsequent works.

120 -,

ä 80 -

nt 60 -

u 3 40 -

at 60 sec. 1200 -,

at 120 sec. ) 1000 -

at 180 sec. a 800 -

nt e 600 -

u Э <J 400 -200 -0-

-0.8 -0.6 -0.4 -0.2 Initial potential (V)

100 150 200 250 Deposition time (Sec.)

Figure 5 (a) Dependance of the reduction Peak current of Cu2 + present in Cu-VAL complex on the initial potential at different

deposition times. (b) Dependance of the Cu2 + surface.

reduction peak current on the deposition time onto the Cu-microparticles coated PGE

300 350

5. Validation of the proposed method

The method was validated according to International Conference on Harmonization (ICH) guidelines (ICH, Nov 2005) and (Harris, 2003) for sensitivity, precision, accuracy and recovery for the proposed method.

The increase in the cathodic current of Cu2 + is denoted as follows:

where ICu2+ is the current response of Cu + alone, IVALadded is the current response of Cu2 + in the presence of VAL, and AI is the difference between them.

The parameters for analytical performance, such as linearity range, limit of detection, limit of quantification, repeatability, and specificity, were investigated by recording square wave voltammograms.

5.1. Linearity

The applicability of the proposed square-wave voltammetric (SWV) procedures for the determination of VAL was examined by measuring the stripping peak current as function of concentration of the bulk drug for at least five times under the optimized conditions. The calibration plot of the peak current vs. the concentration was found to be linear over the range 2.0 x 10"9 M to 1.0 x 10"8 M. This is the saturation limit because above this concentration there was a consistency in the peak current. The linear regression equation is expressed as follows:

Ip (lA) = (1.5 x 1010)Cval (M)+28.57

The regression plots showed that there is a linear dependence of the current intensity on the concentration over the range as given in Table 1 and Fig. 6.

-0.6 -0.1 0.5 1.0

E (V) vs. Ag/AgCl electrode

Figure 6 Square-wave voltammetric responses for successive additions of VAL (a) 1.0 x 10"8 M CuSO4, (b) a + 2.0 x 10"9 M VAL, (c) a + 4.0 x 10"9 M VAL, (d) a + 6.0 x 10"9 M VAL, (e) a +8.0 x 10"9M VAL and (f) a +1.0 x 10"8 M VAL in 0.04 M BR buffer solution (pH 2.7) at the Cu-microparticles modified PGE. (Inset) the calibration curve corresponding to different values of VAL.

5.2. Detection and quantitation limits

The limit of detection was calculated by the equation: (LOD = 3SD/b) while the quantitation limits were estimated by equation: (LOQ = 10SD/b) where (SD) is the standard deviation of intercept and (b) is the slope of the regression line. The calculated detection and quantitation limits were 1.78 x 10"10M and 5.38 x 10"10 M respectively.

5.3. Repeatability/intermediate precision

Table 1 Analytical parameters for voltammetric determination of valacyclovir.

Parameter SWCAdsV

Linearity range (M) 2 x 10~9 to 1 x 10~s

Slope (iA M"1) 1.5 x 10

Intercept (lA) 28.57

Correlation coefficient (r) 0.998

% RSDa of slope 2.05

% RSDa of intercept 2.82

LOD (M) 1.78 > c 10-10

LOQ (M) 5.38 > c 10-10

Intra-day precision (% RSD)b 2.24

Variance of the slope for intra-day precision 2.66 > c 1017

Inter-day precision (% RSD)b 3.33

Variance of the slope for inter-day precision 3.13 x c 1017

a n = 5 where n is no. of experiments. b % Relative standard deviations of intra- and inter-day precision are not more than these values for three different concentration levels within the linearity range.

For the precision of the method, the intra- and inter-day variation for the determination of valacyclovir was carried out at three different concentration levels covering the low, medium and higher ranges of the calibration curve.

The precision and repeatability of the developed method for valacyclovir was determined in five replicate analyses for each concentration level. The results confirmed both the good precision of the proposed procedure and stability of the drug's solution. The intra-day % RSD of the peak current for different concentration levels was not more than 2.2% while the inter-day % RSD was not more than 3.3%. These results indicate that the method is precise and confident.

5.4. Durability of Cu/PGE

To access the electrocatalytic activity and durability of the catalytic Cu/PGE for routine VAL analysis, we examined its hydrodynamic stability by performing continuous measurements of a sample solution over an extended period of time. At a Cu/PGE, no appreciable signal degradation was observed even after 3 hrs. In fact, a single Cu/PGE is sufficient for a day's analysis. As mentioned before in the results of repeatability, % RSD of the peak current for different concentrations

Table 2 Comparative and recovery studies for VAL from

pharmaceutical dosage forms by the proposed method and

previously reported method (Uslu et al., 2006).

Parameters SWV

Labeled claim 1000 mg VAL per tablet

Amount found 1002.8 mg VAL per tablet

% Recovery® 100.28

% RSDa 2.27

% Biasa 0.15

Student ttestb Calculated °.25 theoretical 2.78

F b F test Fcalculated 1.59 Ftheoretical 6.39

Standard addition method

Added (M) 2.0 x 10-9 4.0 x 10-9 6.0 x 10-9

Found(m) 2.04 x 10-9 3.87 x 10-9 5.88 x 10-9

% Recovery® 102.0 96.80 98.0

% RSDa 2.75 1.63 3.53

% Biasa 1.80 -3.22 -1.96

a n = 5 where n is no. of experiments.

b At confidence level 95%.

repeated at the same electrode surface several times was not more than 2.2%.

5.5. Robustness

The robustness of the developed stripping voltammetric methods was examined by studying the effect of variation of some of the operational conditions such as pH (2.7 ± 0.1), precon-centration potential (—0.6 ± 0.05 V) and preconcentration time (180 ± 10 sec.) on mean percentage recovery (% Recovery) and relative standard deviation (% RSD) of 4.0 x 10—9 VAL. The obtained mean % R and RSD based on five replicate measurements under the varied conditions were between the following values (98.83 ± 1.14 to 101.55 ± 2.34). Since the mean percentage recoveries and relative standard deviations obtained within the studied range of variation of the operational conditions were insignificantly affected, the developed adsorptive stripping voltammetric method was reliable for quantitation of VAL and could be considered robust. The ruggedness is the degree of reproducibility of the results obtained by analysis of the same sample under a variety of normal test conditions, such as different days and equipments. The results obtained day to day or using different equipments were found to be reproducible and the % R and % RSD were varied between (97.45 ± 1.34 and 100.78 ± 1.87).

5.6. Interferences

The tolerance limit was defined as the maximum concentration of the interfering substances that caused an error less than

±5% for the determination of VAL. Under the optimum experimental conditions, the effects of potential interferents on the voltammetric response of 5.0 x 10—5M VAL in Cu-VAL complex were evaluated. The experimental results show that thousand-fold excess concentration of glucose, starch, sucrose and talc, did not interfere.

6. Application of the proposed method to VAL tablets

The adequacy of the developed method was evaluated by quantifying VAL in its dosage form. The nominal content of the tablet amount was calculated from the corresponding regression equations of previously plotted calibration plots. The concentration of VAL determined in tablets using the proposed method is reported in Table 2. The mean results for the determination of VAL using the proposed method were found very close to the declared value of 1000 mg. The mean recoveries in tablets were found to be 100.28%. The obtained results showed that the proposed method could be applied with great success to VAL assay in tablets without any interference (Table 2). The standard addition method was also carried out for the accuracy studies (Harris, 2003). A solution of 4.0 x 10—9 M of VAL tablets was subjected to three successive additions of 2.0 10—9 M of standard solution, SWV curves were recorded before and after each addition and standard addition dependence was plotted, and corresponding calculations were performed.

For showing the accuracy of the proposed method, the results of VAL tablets were also compared with the previously reported voltammetric method (Uslu et al., 2006). To the best of our knowledge, no official method is described in any pharmacopoeia related to the VAL analysis in tablets. For this reason, SWV method was compared with already published voltammetric method (Uslu et al., 2006) using both Student's t- and F-tests. At 95% of the confidence level, the values calculated from experiments were less than those of theoretical t and F ones showing that there were no statistically significant differences between the performances of both methods.

7. Comparison of the sensitivity of the method with previously reported methods

Table 3 compares the detection limit of the proposed method with the other reported methods (Jadhav et al., 2007; Kumar et al., 2010; Uslu et al., 2006). The proposed method was highly sensitive and rapid compared with other instrumental methods. It did not require derivatization for chromatography or the use of organic reagents for spectrophotometry. The data in Table 3 reveal that the detection limit of the method is lower than all previously reported methods.

Table 3 Comparison of the sensitivity of the proposed method with the other reported method.

Method Linearity range Detection limit Reference no.

Spectrophotometry 20-120 ig/ml High performance liquid chromatography 900-6000 ng/ml Square-wave voltammetry 4 x 10 6-2 x 10 4 M Differential pulse voltammetry 4 x 10 6-2 x 10 4 M Square-wave voltammetry 2 x 10—9-1 x 10—8 M 0.445 ig/ml « 1.37 x 10~6 M 300 ng/ml « 9.25 x 10~7 M 4.6 x 10~8M 1.04 x 10~7M 1.78 x 10~10M Jadhav et al. (2007) Kumar et al. (2010) Uslu et al. (2006) Uslu et al. (2006) This work

8. Conclusion

We successfully fabricated a novel, extremely low-cost, disposable, and easily fabricated VAL sensor based on the electrode-posited Cu-microparticles onto PGE surface. The highly reproducible fabricated sensor exhibits a remarkable electro-catalytic activity toward Cu2 + ions reduction present in the Cu-VAL complex at low detection limit, greater analytical sensitivity and stability. The performance of the novel Cu-PGE proved to be excellent, and found to be suitable for the analytical determination of different VAL methods is applicable in quality control laboratories and in stability studies.

Acknowledgment

The authors gratefully acknowledge Prof. Mahmoud A. Ghan-dour (Chemistry Department, Faculty of Science, Assiut University) for his contribution in the revision of this research article.

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