Differential Pulse Anodic Voltammetric Determination of Chlorzoxazone in Pharmaceutical Formulation using Carbon Paste Electrode

Differential Pulse Anodic Voltammetric Abstract The electrochemical behavior of chlorzoxazone at the carbon paste electrode was investigated in 0.04 mol/L Brit-ton-Robinson buffer pH 6.50 using cyclic and differential pulse voltammetric techniques. Cyclic voltammetric studies indicated that the oxidation of the drug was irreversible and controlled mainly by diffusion. Experimental and instru-mental parameters were optimized (50 mV/s scan rate, 50 mV pulse amplitude, and 0.04 mol/L Britton-Robinson (BR) buffer pH 6.50 as a supporting electrolyte) and a sensitive differential pulse anodic voltammetric method has been developed for the determination of the drug over the concentration range 0.17–1.68 µg/mL chlorzoxazone, with detection and quantitation limits of 0.05 and 0.16 µg/mL, respectively. The proposed voltammetric method was successfully applied to the determination of the drug in its pharmaceutical formulation (Myoflex tablets), and in spiked human urine samples.


1. Reagents and Materials
All chemicals were of analytical grade. Double distilled water was used throughout all experiments. Pure grade chlorzoxazone and the pharmaceutical preparation Myoflex tablets (250 mg chlorzoxazone and 450 mg paracetamol per tablet) were kindly supplied by the Nile Co., for Pharmaceuticals and Chemical Industries, Cairo, Egypt. Graphite powder (1-2 mm) was from Aldrich, and paraffin oil from B.D.H. As a supporting electrolyte, a series of 0.04 mol/L Britton-Robinson (BR) buffer pH 2.0-11.5 (a mixture of acetic, orthophosphoric, and boric acids), adjusted to the required pH with 0.2 mol/L sodium hydroxide was prepared.

Apparatus
All voltammetric measurements were performed using Metrohm 797 VA Computrace (Herisau, Switzerland) equipped with a Metrohm VA 694 stand. Three electrodes assembly cell (consisted of carbon paste electrode (CPE) as the working electrode, an Ag/AgCl in 3 mol/L KCl as a reference electrode, and platinum wire as an auxiliary electrode) was used. The pH measurements were carried out with Hanna pH 211 microprocessor pH-meter.

3. Preparation of Carbon Paste Electrode
The carbon paste was prepared by thoroughly mixing 5 g of graphite powder with 1.8 mL of paraffin oil in a mortar with a pestle. The carbon paste was packed into the hole of the electrode body and smoothed on a clean paper until it had a shiny appearance. The electrode body was constructed by pressing a small rode of stainless steel (diameter 2 mm) inside a micropipette tip (1 mL volume capacity) leaving a depression at the surface tip approximately 1 mm for housing the carbon paste, and thin wire was inserted through the opposite end to establish electrical contact. 44 The carbon paste electrode was immersed in the supporting electrolyte placed in the cell and several sweeps were applied to obtain a low background current.
The area of the prepared carbon paste electrode was calculated by plotting the relation between the anodic current and square root of the scan rate for 10 -3 M solution of potassium ferricyanide as a probe using cyclic voltammetry. For a reversible process, The Randles-Sevcik equation was used 45 (1) Where I pa is the anodic current, n is the number of transferred electrons, A is the surface area of the electrode, D o is the diffusion coefficient, ν is scan rate, and C o is the concentration of potassium ferricyanide. For 10 -3 mol/L solution of potassium ferricyanide in 0.1 mol/L KCl electrolyte, n = 1 and D o = 7.6 × 10 -6 cm 2 /s, then from the slope of the relation between anodic current and the square root of scan rate, the surface area of the electrode was calculated and found to be 0.036 cm 2 .

4. Procedure
A 10 mL of 0.04 mol/L Britton-Robinson buffer pH 6.5 was introduced into the voltammetric cell and a known amount of the drug solution was pipetted into the cell and differential pulse technique was applied by scanning from 0 to 1.4 V with a scan rate 50 mV/s and pulse amplitude 50 mV.

5. Determination of Chlorzoxazone in
Myoflex Tablets (250 mg Chlorzoxazone and 450 mg Paracetamol per Tablet) Ten tablets were accurately weighed and finely powdered. An adequate amount of the powder corresponding to prepare 1 × 10 -3 mol/L chlorzoxazone was weighed and transferred to a beaker. The tablets powder was dissolved in methanol and filtered to 100 mL calibrated flask using a Whatman 41 filter paper. The residue was washed several times with methanol and the washings were collected in the measuring flask and completed to the mark. Then the analysis was done as described in the general procedure.

6. Determination of Chlorzoxazone in Spiked Human Urine Samples
0.0170 g of chlorzoxazone was dissolved in methanol and introduced into 100 mL volumetric flask, 5 mL of urine from a healthy person was added, and the mixture was completed to the mark by methanol to prepare 10 -3 mol/L chlorzoxazone in the spiked human urine sample. 10 mL of 0.04 mol/L BR buffer pH 6.5 was introduced into the voltammetric cell, different amounts of the above spiked human urine sample were added and the procedure was repeated as described before. The amount of chlorzoxazone was determined using the standard addition method.

1. Cyclic Voltammetric Studies
The repetitive cyclic voltammograms for 3.85 × 10 -5 mol/L solution of chlorzoxazone in 0.04 mol/L BR buffer pH 6.5 and the scan rate of 50 mV/s using carbon paste electrode were illustrated (Figure 1). A well defined anodic peak at 1.01 V was observed which may be due to the oxidation of the hydroxyl group in the chlorzoxazone molecule. No peak was observed in the reverse cathodic scan, indicating that the process is irreversible. A decrease in the oxidation peak current during the successive cyclic vol-tammograms was observed. This decrease in current may be attributed to the fouling of the electrode surface due to the adsorption of the oxidation products on the surface of the electrode. The effect of the scan rate on the peak current and peak potential was tested from 10 to 100 mV/s ( Figure 2). The relation between oxidation current and scan rate and oxidation current and the square root of the scan rate was studied. Linear relationship was found between oxidation current and the square root of the scan rate, which could be represented by the equation, , r 2 = 0.9949. which indicates that the ox-idation process is controlled by diffusion. 46,47 This is confirmed by plotting the logarithm of the peak of oxidation current vs. the logarithm of the scan rate, which gave a straight line relation with a slope of 0.38, which is close to the theoretically expected 0.5 value for a diffusion-controlled process. The relation is represented by the equation , r 2 = 0.9921. Also the peak potential shifts to more positive values on increasing the scan rate confirm the irreversibility of the oxidation process.
In addition, the oxidation peak potential and logarithm of the scan rate showed a straight line relation with slope equal to 0.0647 as represented by the following equation: For an irreversible process according to Laviron 48 Where α is the transfer coefficient, k 0 is the standard heterogeneous rate constant of the reaction, n is the number of transferred electrons, v is the scan rate and E 0 is the formal redox potential. So the value αn can be calculated from the slope of the straight line relation between E p and log ν by substituting the values of R, T, and F. The calculated value of αn was found to be 0.914.
The transfer coefficient α for an irreversible process can be calculated from the relation 38 (4) Where E P/2 is the potential at which the current equals half of the peak current. The value of α was found to be 0.723 and the calculated value of n = 1.26.

2. Differential Pulse Voltammetric Studies
The electrochemical behavior of chlorzoxazone (3.98 × 10 -6 mol/L) in different media such as 0.1 mol/L phosphate, 0.1 mol/L BR, and 0.1 mol/L citrate buffers was tested by differential pulse voltammetry. The oxidation peak is higher in the case of BR buffer than in the case of phosphate buffer, but in the case of citrate buffer, the oxidation peak disappeared ( Figure 3A). And hence, BR was selected as the best medium. The effect of pH on the anodic peak current and oxidation potential was tested in the pH range 2.0-11.0 ( Figure 3B). The oxidation peak current increased with an increase in the pH until it attained its maximum at pH 6.50. The study of the effect of the supporting electrolyte (BR buffer) concentrations (0.02, 0.04, and 0.1 mol/L) indicated that the highest peak current was obtained at 0.04 and 0.1 mol/L BR buffer ( Figure 3A). So 0.04 mol/L BR buffer pH 6.50 was selected as the medium for the de-  termination of chlorzoxazone. The oxidation potential shifted negatively with increasing the pH, suggesting that the protons are involved in the electrode reaction process.
The effect of oxidation current of 3.98 × 10 -6 mol/L chlorzoxazone with the change in pulse amplitude was tested in the range 10-100 mV pulse amplitude (Figure 4). The oxidation current increases from 10 to 50 mV, then remains nearly constant, so 50 mV pulse amplitude was used for this work.

Proposed Mechanism
The proposed mechanism for the oxidation of chlorzoxazone (Scheme 2) is one electron, one proton process as reported by Abbar and Nandibewoor 38,39 Figure 3A. Differential pulse voltammograms for 3.98 × 10 -6 mol/L chlorzoxazone in different media: a, 0.04 mol/L; b, 0.1 mol/L, c, 0.02 mol/L BR buffer pH 6.5, and d, 0.1 mol/L phosphate buffer pH 6.5. Figure 3B. Effect of pH on the differential pulse anodic peak current (a), and peak potential (b) of 3.98 × 10 -6 mol/L chlorzoxazone in 0.04 mol/L BR buffer, pulse amplitude 50 mV, and scan rate 50 mV/s.

Analytical Performance of the Proposed Method
On the basis of electrochemical oxidation of chlorzoxazone at the carbon paste electrode under the optimum conditions, differential pulse anodic voltammetric method was proposed for the determination of the drug over the working linear range 0.17-1.68 µg/mL. Figure 5 represents the differential pulse anodic voltammograms recorded using the standard addition method. The linear regression parameters are listed in Table 1. The limit of detection (LOD = 3(SD a )/b) and limit of quantitation (LOQ = 10(SD a )/b) were calculated, 49 where SD a is the standard deviation of the intercept and b is the slope of the calibra-tion graph. The linear range and limit of detection of the proposed method were also compared with the previously reported methods (Table 2). It is observed that the detection limit was better than in the reported HPLC method, 3 and the lower linear range was lower than in the published HPLC methods, 3,4,7,10 spectrophotometric methods, 24,32,33 capillary zone electrophoresis, 37 and the voltammetric method using the gold electrode. 39

Validation of the proposed procedure
The validation of the proposed method was tested via linear range, the limit of detection (LOD), the limit of quantitation (LOQ), repeatability, precision, accuracy, selectivity, and robustness. The linear dependence of the oxidation current versus drug concentration was represented by the following straight line relation I(nA) = 59.72 × C(µg/mL) + 6.41, (r = 0.9991, n = 10), in the linear range 0.17-1.68 µg/mL.
The sensitivity of the proposed method was tested in terms of limit of detection (LOD) and limit of quantitation (LOQ) values. The values of LOD and LOQ were found to be 0.05 and 0.16 µg/mL, and these values indicate that the proposed method could be considered sensitive.
The repeatability of the proposed method was tested on the same day (intra-day) and in three different days (day-to-day) precision from seven repeated measurements of 0.17 µg/mL chlorzoxazone. The intra-day and inter-day precision expressed as standard deviations were found to be 0.29 and 0.73, respectively. The accuracy of the proposed method was determined by calculating the recoveries of 9.99 × 10 -7 and 2 × 10 -6 mol/L chlorzoxazone using the standard addition method. The estimated mean recoveries based on four replicate measurements were 97.18% ± 2.03% and 99.77% ± 2.28%, respectively, which indicates the high accuracy of the proposed procedure.
The selectivity of the method under the optimum conditions for the assay of 9.99 × 10 -7 mol/L of the drug  No interference was found in the presence of 2.5-fold excess of dopamine hydrochloride, 7-fold of uric acid, and 9-fold of ascorbic acid. The robustness 49 of the proposed method was examined by evaluating the effect of small changes in some of the most important procedure parameters, including the pH of the Britton-Robinson (BR) buffer (6.3-6.7) and the pulse amplitude (47-53 mV). None of the changes significantly affected the drug recovery (Table  3); consequently, the optimized procedure was reliable for the assay of chlorzoxazone and it could be considered robust.

4. Determination of Chlorzoxazone in Myoflex Tablets
The proposed differential pulse anodic voltammetric method was successfully applied for the assay of chlorzoxazone in Myoflex tablets (250 mg chlorzoxazone + 450 mg paracetamol per tablet). The percentage mean recovery for four replicate determinations and the relative standard deviation values are listed in Table 4. The anodic differential pulse voltammograms recorded using the standard addition technique for determination of chlorzoxazone in its tablets are depicted in Figure 6 (in the Supplementary Material). The data indicate that there is no interference from the other drug paracetamol present in the tablet or from the excipients which are used in tablets formulation, and the results were in good agreement with the values obtained using the HPLC reference method. 8 Statistical comparison of the accuracy and precision of the proposed method with the reference method (Table 4) was performed using Student's t-and the F-ratio tests at a 95% confidence level. 50 The t-and F-values did not exceed the theoretical values; there is no significant difference in accuracy or precision between the proposed and the reference HPLC method.

Conclusions
The present work describes an effective procedure for the determination of chlorzoxazone. The proposed method has advantages, such as being simple, sensitive, rapid, inexpensive, low detection limit, and ease of preparation and renewable for carbon paste electrode. The developed procedure can be considered as an alternative for HPLC techniques in quality control laboratories.