Electrochemical determination of methocarbamol on a montmorillonite-Ca modified carbon paste electrode in formulation and human blood

Bioelectrochemistry 79 (2010) 241–247

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Bioelectrochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o e l e c h e m

Electrochemical determination of methocarbamol on a montmorillonite-Ca modified carbon paste electrode in formulation and human blood Enass M. Ghoneim ⁎, Hanaa S. El-Desoky Analytical and Electrochemistry Research Unit, Chemistry Department, Faculty of Science, Tanta University, 31527-Tanta, Egypt

a r t i c l e

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Article history: Received 25 January 2010 Received in revised form 19 May 2010 Accepted 15 June 2010 Available online 18 June 2010 Keywords: Methocarbamol Montmorillonite-CPE Voltammetry Tablets Human blood

a b s t r a c t Utilizing the fascinating strong adsorptive ability, high chemical and mechanical stability properties of montmorillonite-calcium (MMT-Ca) clay, a MMT-Ca modified carbon paste electrode was developed for the sensitive determination of methocarbamol. Methocarbamol has been oxidized in buffered solutions at the developed MMT-Ca-modified CPE in a single 2-electron irreversible anodic peak. A simple and sensitive square-wave adsorptive anodic stripping voltammetric method was optimized for determination of methocarbamol in bulk form, pharmaceutical formulation and in spiked human serum using the developed modified CPE. This was carried out without the necessity for samples pretreatment and/or time-consuming liquid–liquid or solid-phase extraction steps prior to the analysis. The developed electrode exhibited an excellent sensitivity and selectivity towards methocarbamol even in the presence of 102–104-fold of its active ingredient “ibuprofen,” common excipients, common metal ions or co-administrated drugs. Limits of detection of 3 × 10−9 and 1.2 × 10−8 M and limits of quantitation of 1 × 10−8, and 4 × 10−8 M methocarbamol were achieved in the bulk form and in spiked human serum, respectively. Moreover, the developed method was successfully applied for determination of methocarbamol in human plasma of subjects following administration of an oral dose of Ibuflex® tablets. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Carbon paste electrode (CPE) is a mixture of an electrically conducting graphite powder and a pasting liquid. It has been widely used in electro- and electro-analytical chemistry as a working electrode. This is because it possesses various advantages e.g. a wide potential range, simple and fast preparation, convenient surface renewal, porous surface and low residual current, besides it is inexpensive. Unfortunately, the sensitivity of bare CPE is relatively poor for determination of inorganic and organic species. In order to improve this, a fascinating and effective way is to modify bare CPE by mixing with some other unique substances. Montmorillonite is natural clay which is one of the very promising modifiers. It belongs to the smectite group of clays with a layer lattice. Because montmorillonite has high chemical and mechanical stability, a welllayered structure and strong adsorptive properties attributed to the expandability of the internal layers, it has been successfully used in electro-analytical chemistry as a modifier in carbon electrodes for assay of different organic [1–4] and inorganic [5–9] species. Methocarbamol (Scheme 1), is a central muscle relaxant used to relax muscles and relieve pain and discomfort caused by strains, sprains, and other muscle injuries [10–12] for human and other mammals. Methocarbamol is usually available in its pharmaceutical ⁎ Corresponding author. Fax: + 20 40 3350804. E-mail address: [email protected] (E.M. Ghoneim). 1567-5394/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2010.06.005

formulations individually or as binary mixtures with other active ingredients. Ibuflex® tablet contains a combination of 2 active ingredients, methocarbamol and ibuprofen (Scheme 1). Ibuprofen [2-(4-(2-methylpropyl)phenyl) propanoic acid] is a non-steroidal anti-inflammatory drug. Methocarbamol is rapidly absorbed and has an onset of action of about 30 min after oral administration. Following the administration of an oral dose (150 mg) of methocarbamol, its peak plasma concentration (23.1 ± 8.2 mg L−1) in human blood was attained at 1.10 h after dosing. The terminal elimination half-life of methocarbamol averaged 1.14 to 1.24 h [10–12]. Drug analysis, an important branch of analytical chemistry, plays important role in drug quality control. Therefore, the development of sensitive, simple, rapid and reliable method for the determination of active ingredient is of great importance and interest. Only few analytical methods were reported in the literature for determination of methocarbamol in combination with other drugs in pharmaceutical formulations or in biological fluids. These include high performance liquid chromatography (HPLC) [12–17], reverse phase liquid chromatography (RPLC) [18,19], supercritical fluid chromatography (SFC) [20], second derivative spectrophotometry [21,22], and 1H NMR spectroscopy [23]. Most of these methods are costly and require expertise in addition to the time-consuming pretreatment steps. To the best of our knowledge, electrochemical determination of methocarbamol is not reported in literature to date. This paper presents the utilization of montmorillonite-calcium clay as modifier of carbon paste electrode for determination of the

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Scheme 1. Structure formulae.

muscle relaxant drug methocarbamol in the bulk form, pharmaceutical formulation and human blood. 2. Experimental 2.1. Materials and solutions Bulk methocarbamol and its pharmaceutical formulation “Ibuflex® tablets” that claimed to containing 750 mg methocarbamol and 400 mg ibuprofen (Global Napi Pharmaceuticals, Egypt) were the substances of our interest in the present investigation. Standard stock solutions (1 × 10−3 M) of each of bulk methocarbamol and bulk ibuprofen were prepared in methanol (Merck) and stored at 4 °C. The desired solutions (1 × 10−8 to 1 × 10−4 M) were prepared by appropriate dilution of the standard stock solutions with methanol. Ten Ibuflex® tablets were weighed and the average mass per tablet was determined. A portion of the finely ground tablets was transferred accurately into a 100 mL-volume calibrated flask contains 70 mL methanol (Merck). The content of flask was sonicated for about 15 min and then filled up with methanol. The solution was then filtrated through 0.45 μm milli-pore filter (Gelman, Germany). The desired concentrations of methocarbamol were obtained by accurate dilution with methanol. 2.2. Solutions of human serum A serum sample of a healthy volunteer was stored frozen until assay. Samples of the human serum (each of 1 mL) were fortified with various concentrations (1 × 10−8 to 1 × 10−4 M) of methocarbamol in small centrifugation tubes (3 mL polypropylene micro-centrifuge tubes). Each of these samples was then completed to 2 mL with methanol to denature and precipitate proteins. After vortexing each of the serum samples for 2 min, the precipitated proteins were separated by centrifugation for 3 min at 14,000 rpm. The clear supernatant layer was filtered through 0.45 μm milli-pore filter to obtain protein-free spiked human serum samples. Then the analysis was followed up as indicated in the general analytical procedure. 2.3. Supporting electrolytes Britton–Robinson (B-R) universal buffer (pH 2–11) [24] and acetate buffer (pH 3.8–6.3) were prepared in de-ionized water and were used as supporting electrolytes. All the chemicals used were of analytical grade and were used without further purification. 2.4. Preparation of the modified carbon paste electrode An amount (4.5 g) of graphite powder (1–2 μm, Aldrich, Milwaukee, WI, USA) and 0.5 g of montmorillonite-Ca clay (Fine powderb 5 μm, ECC America Inc., Southern Clay Products Subsidiary, Gonzales, Texas, USA) were mixed uniformly by milling in a small agate mortar. Then 1.8 mL Nujol oil (Sigma, d = 0.84 g mL−1) was added and milled again to give a homogenous 10% (w/w) MMT-Ca-modified carbon paste. Various modified carbon pastes containing different mass percentages of

MMT-Ca clay (0.0, 5, 15 and 20 % w/w) were similarly prepared. The body of the carbon paste-working electrode was a Teflon rod with end cavity (3 mm diameter and 1 mm deep) bored at one end for paste filling (BASi-MF-2010). Contact was made with a copper wire through the centre of the rod. An amount of the prepared MMT-Ca-modified carbon paste was pressed into the end cavity of the electrode body and leveled off with a spatula. Surface of the constructed MMT-Ca-modified carbon paste electrode (MMT-Ca-modified CPE) was manually smoothed by polishing on clean paper before use. 2.5. Apparatus Computer-controlled Potentiostats Models 263A and 394-PAR (Princeton Applied Research, Oak Ridge, TN, USA) with the software 270/250-PAR were used for the voltammetric measurements. A voltammetric cell consisting of a C-2 stand with a carbon paste-working electrode body (BASi-MF-2010, 3 mm in diameter and 1 mm in depth), an Ag/AgCl/KCls reference electrode (BASi-MF-2079), and a platinum wire counter electrode (BASi-MW-1032) was used in the present investigation. A magnetic stirrer with a Teflon-coated magnet was used to provide the convective transport during the preconcentration step. A pH meter (Crison, Barcelona, Spain) was used for the pH measurements. An Eppendorf centrifuge (Model 5417 C, Hamburg, Germany) was used for separation of the precipitated proteins from human plasma and serum samples prior to the assay. A micropipetter (Eppendorf-Multipette® plus) was used for transferring the analyte solutions throughout the present experimental work. Deionized water was used throughout the present work. 2.6. General analytical procedure 10 mL volume of acetate buffer solution (pH =4) was introduced into the micro-electrolysis cell and a smoothed MMT-Ca-modified CPE was then immersed in the supporting electrolyte. Then several cyclic voltammetric sweeps were applied to obtain a low background current. Then aliquots of the analyte solution were introduced into the electrolysis cell, and then a selected preconcentration potential was applied to the developed MMT-Ca-modified CPE for a selected preconcentration time while the solution was stirred at 400 rpm. At the end of the preconcentration time, the stirring was stopped and a 5 s rest period was allowed for the solution to become quiescent. The voltammograms were then recorded by scanning the potential towards the positive direction using the square-wave potential-waveform. After each measurement, the used modified carbon paste was carefully removed and a new MMT-Ca-modified CPE was constructed as described in Section 2.4. For determination of methocarbamol in human plasma and serum samples, medium exchange treatment for the working electrode was performed after the preconcentration step by immersing it in the blank electrolyte [25]. This is to avoid interferences from low molecular weight proteins which may remain after centrifugation. The mean percentage recovery (%R) for the found concentrations was calculated as a percent of the nominal concentrations in the standard solutions. Accuracy; was expressed as relative error (RE% =

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Cyclic voltammograms of 5 × 10−6 M methocarbamol were recorded at 300 mV s−1 in the B-R universal buffer (pH b 10), and in acetate buffer (pH 3.8–6.3) at both bare CPE and CPE modified with ≤ 15 % (w/w) MMT-Ca clay. The voltammograms recorded in both buffer solutions at the bare CPE exhibited no any voltammetric peak over the entire pH range (e.g. Fig. 1, curve a). Whereas the voltammograms recorded in both buffer solutions at the modified MMT-Ca CP electrodes exhibited a single irreversible anodic peak over the entire pH range. However, this peak was sharp and better developed in acetate buffer of pH 4 when using CPE modified with 10% (w/w) montmorillonite-calcium clay (Fig. 1, curve b). The anodic peak was attributed to oxidation of the –OH group of the aliphatic chain via the transfer of two electrons per analyte molecule.

On the other side, cyclic voltammogram of 5 × 10−6 M methocarbamol in the acetate buffer of pH 4 were recorded at 300 mV s−1 following preconcentration of the analyte by adsorptive accumulation for 400 s at −0.3 V onto both the bare CPE and a developed 10% (w/w) MMT-Ca-modified CPE. The voltammograms recorded at the bare CPE exhibited very ill-defined irreversible anodic peak (Fig. 2, curve a). Whereas, the voltammograms recorded at the developed 10% (w/w) MMT-Ca modified CPE exhibited a well-defined enhanced irreversible anodic peak (Fig. 2, curve b). The enhancement of the anodic peak current (1st cycle, curve b) compared to that at the bare CPE (Fig. 2, curve a) indicated the strong adsorption behavior of methocarbamol onto the MMT-Ca-modified CPE. A substantial decrease of the monitored voltammetric peak was observed in the 2nd cycle (curve c) indicating desorption of methocarbamol out of the MMT-Ca-modified CPE surface. On the other hand, cyclic voltammograms of 5 × 10−6 M ibuprofen (Scheme 1) were recorded under the same previous conditions at either the bare CPE or the developed MMT-Ca modified CPE (without and following preconcentration). The voltammograms exhibited no any voltammetric peak, indicated that ibuprofen is electro-inactive at both electrodes under the experimental conditions. Further, cyclic voltammograms of 5 × 10−6 M methocarbamol at different scan rates (20–500 mV s−1) were recorded at the developed MMT-Ca modified CPE. The peak current (ip) increased upon the increase of scan rate (v). According to Randles–Sevcik's simplified equation (ip = −constant v1/2) [26], a linear plot of log ip versus log v was obtained. Its corresponding regression equation was: log ip / μA = 0.94 ± 0.01 log ν / (mV s−1) − 1.31 ± 0.005 (r= 0.998 and n = 6). The slope value of 0.94± 0.01 (μA / mV s−1) is very close to the expected theoretical value (1.0) for an ideal reaction of surface species [27]. Also, as the scan rate was increased, the peak potential shifted towards more positive potentials as expected for an irreversible oxidation process [28]. A linear Ep / V versus log ν / (mV s−1) plot of slope value of 40± 0.05 mV/ decade was obtained; from which value of αna (product of symmetry transfer coefficient α and number of electrons na transferred in the ratedetermining step) was estimated and found to equal 1.475 ± 0.02. Since, the number of electrons na transferred in the rate-determining step of the electro-oxidation of the OH group of the analyte equals 2 (na = 2) [29] the transfer coefficient α should be 0.737 ± 0.01.

Fig. 1. Cyclic voltammograms of 5 × 10− 6 M methocarbamol in acetate buffer of pH 4 recorded at a bare CPE (a) and at the developed MMT-Ca-modified CPE (b) without prior preconcentration; scan rate = 300 mV s− 1.

Fig. 2. Cyclic voltammograms of 5 × 10− 6 M methocarbamol in acetate buffer of pH 4 recorded following preconcentration by adsorptive accumulation at Eacc = −0.3 V for 400 s onto a bare CPE (a) and the developed MMT-Ca-modified CPE {1st cycle (b) and 2nd cycle (c)}; scan rate = 300 mV s− 1.

([Cfound / Ctaken] − 1) × 100) while precision was assessed from the relative standard deviation in percentage (RSD %) of the mean recovery. 2.7. Pharmacokinetic study Two healthy male volunteers of 40-45 years old took part in this study. Subjects were caffeine and alcohol free for at least 12 h before the administration of the drug. The two volunteers gave their written informed consent prior to anticipating in the study (at Ramadan Specialized Hospital, Tanta City, Egypt). Each study subject received an oral dose of 1.5 g methocarbamol (two Ibuflex® tablets) in the morning, after an overnight fast. Blood samples of the two subjects (1 mL each) were collected at 0 (pre-dose), 0.5, 1.0, 1.5, 2, 3, 4, 6, 9, 12 and 24 h after the oral administration. The blood samples were centrifuged immediately at 3000 rpm for 10 min and then the plasma fractions were rapidly separated and stored in coded polypropylene tubes at −20 °C until the assay. Following separation of proteins by methanol, the plasma samples were analyzed using the described square-wave adsorptive anodic stripping voltammetry (SW-AdASV) method utilizing the developed MMT-Ca modified CPE. 3. Results and discussion 3.1. Electrochemical behavior

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3.2. Quantification studies Based on the adsorption behavior of methocarbamol onto the developed MMT-Ca-modified CPE surface, a square-wave adsorptive anodic stripping voltammetry (SW-AdASV) method was optimized for its trace quantitation in the bulk form. The various parameters affecting the SW-AdASV peak current magnitude at the developed MMT-Ca modified CPE were optimized. 3.2.1. Composition and stability of the MMT-Ca modified CPE SW-AdAS voltammograms of 1 × 10−6 M methocarbamol in the acetate buffer of pH 4 at CPE modified with various mass percentage (% w/w) of MMT-Ca clay were recorded following preconcentration by adsorptive accumulation at Eacc = −0.4 V for 400 s. As shown in Fig. 3, both the shape and current magnitude of the peak of methocarbamol are strongly influenced by the percentages of MMTCa in the modified CPE. The peak current (ip) magnitude increased upon the increase of the percentage (% w/w) of MMT-Ca clay in the modified CPE up to 10% (w/w), and then decreased. Such enhancement of stripping peak current magnitude was expected due to the strong adsorptive properties of MMT-Ca clay. At higher percentage (w/w) of MMT-Ca clay in the modified CPE the peak current decreased (Fig. 3, inset).This behavior may be attributing to the decrease of conductivity of the modified CPE with the increase of percentage of the non-conductive MMT-Ca clay, which leads to hinder the electron transfer process and increase the background current. Therefore, 10% (w/w) MMT-Ca modified CPE was used in the rest of the present analytical study. On other side, reproducibility of results utilizing repeating fabrication of 10% MMT-Ca modified CPE was examined by comparing the SW-AdAS voltammetric oxidation peak current of 1 × 10−6 M methocarbamol obtained utilizing five fabricated MMT-Ca modified CP electrodes. It was found that the oxidation peak current magnitude remained the same with standard deviation of 0.15 using the same stock of the prepared modified graphite paste. Furthermore, sixteen repetitive analysis of 1 × 10−6 M methocarbamol was carried over a month using five fabricated 10% (w/w) MMT-Ca modified CP electrodes (prepared from the same stock modified graphite paste,

Fig. 3. SW-AdAS voltammograms of 1 × 10−6 M methocarbamol in acetate buffer of pH 4 recorded following preconcentration by adsorptive accumulation at Eacc = −0.4 V for 400 s onto CPE modified with various concentrations of MMT-Ca clay: (a) 0.0, (b) 5, (c) 10, (d) 15 and (e) 20 % (w/w). Inset: Peak current (ip) as a function of % (w/w) MMT-Ca clay; f = 80 Hz, ΔEs = 10 mV and Ea = 25 mV.

stored at room temperature). Insignificant differences of peak current magnitudes or its standard deviations (0.14–0.18) were obtained over a month utilizing the developed MMT-Ca modified CP electrodes confirming that the developed MMT-Ca modified CP electrodes were stable and efficient toward the trace determination of methocarbamol. 3.2.2. Pulse parameters Voltammograms of 1 × 10−6 M methocarbamol in acetate buffer of pH 4 following its preconcentration onto the developed 10% (w/w) MMT-Ca-modified CPE for 200 s at −0.4 V were recorded at the various pulse parameters (frequency f = 10–100 Hz, scan increment ΔEs = 2–10 mV, and pulse-amplitude Ea = 5–30 mV). Although the SW-AdAS voltammetric peak current magnitude of methocarbamol was almost directly proportional to each of f, ΔEs, and Ea, however a better developed and symmetrical voltammetric peak were obtained under the following pulse parameters: f = 80 Hz, ΔEs = 10 mV and, Ea = 25 mV. 3.2.3. Preconcentration (accumulation) parameters Effect of varying the preconcentration potential Eacc (−0.5 to 0.2 V versus (Ag/AgCl/KCls) on the peak current magnitude of the SW-AdAS voltammograms of 1 × 10−6 M methocarbamol in acetate buffer of pH 4 was evaluated following preconcentration onto the 10% (w/w) MMT-Ca-modified CPE for 400 s (Fig. 4). The results showed that a better enhanced peak current magnitude was achieved within the potential range of −0.5 to −0.2 V. At more positive preconcentration potentials, significant decrease of peak current magnitude of methocarbamol was observed. Therefore, a preconcentration potential of −0.3 V was applied throughout the present analytical study. On the other side, SW-AdAS voltammograms of 1 × 10−7 and 1 × 10−6 M methocarbamol were recorded at increasing preconcentration time tacc. under the foregoing optimal operational conditions. As shown in Fig. 5, the peak current magnitude was linearly dependent (before saturation of the electrode surface) on both the analyte concentration and the preconcentration time tacc. For 1 × 10−6 M methocarbamol the response was linear up to 400 s; at longer preconcentration time saturation of the electrode surface was achieved at 500 s (curve a). For the lower concentration

Fig. 4. Effect of preconcentration potential (Eacc) on the SW-AdASV peak current (ip) of 1 × 10−6 M methocarbamol in acetate buffer of pH 4, following its preconcentration onto the developed 10% (w/w) MMT-Ca-modified CPE by adsorptive accumulation for 400 s; f = 80 Hz, ΔEs = 10 mV, and Ea = 25 mV.

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mol by the described SW-AdASV method utilizing MMT-Ca modified CPE. This revealed also that detection of very low concentration of methocarbamol is possible even in the presence of 104-fold excess of ibuprofen. Furthermore, the interferences from excipients [31] in determination of methocarbamol were tested by analysis of 5 × 10−7 M bulk methocarbamol in the absence and presence of the common excipients. These excipients were corn starch, FD&C Yellow 6, hydroxypropyl cellulose, hypromellose, magnesium stearate, polysorbate 20, povidone, propylene glycol, saccharin sodium, sodium lauryl sulfate, sodium starch glycolate, stearic acid, and titanium dioxide. The differences in main percentage recoveries and the relative standard deviations obtained by the developed SW-AdASV method were insignificant, since % R ± RSD in the absence of excipients and in their presence were 99.6 ± 1.3 and 98.6 ± 1.9, respectively. These results demonstrated that the described SWAdASV method utilizing the modified CPE is selective and sensitive towards methocarbamol even in the presence of high concentrations of ibuprofen and the other inactive ingredients.

Fig. 5. Effect of the preconcentration time (tacc) on the SW-AdASV peak current (ip) of (a) 1 × 10−6 and (b) 1 × 10−7 M methocarbamol in acetate buffer of pH 4 following preconcentration onto the developed 10% (w/w) MMT-Ca-modified CPE by adsorptive accumulation at −0.30 V; f = 80 Hz, ΔEs = 10 mV, and Ea = 25 mV.

(1 × 10−7 M) the response was linear up to 600 s (curve b). Apparently, lower concentration of the analyte required longer preconcentration time. This meant that the choice of preconcentration time was dictated by the sensitivity required. In the present analytical study, preconcentration time of 400 s was applied. This is to avoid the saturation of electrode surface. According to the foregoing results the optimum operational conditions of the developed SW-AdASV method utilizing 10% (w/w) MMT-Ca modified CPE in acetate buffer of pH 4 were: Eacc. = −0.30 V, tacc. 400 s, f = 80 Hz, ΔEs = 10 mV and, Ea = 25 mV. 3.2.4. Method validation 3.2.4.1. Linearity, limit of detection and limit of quantitation. The relationship between oxidation peak current magnitude and concentration of methocarbamol was examined in acetate buffer of pH 4 by the developed SW-AdASV method utilizing the developed 10% (w/w) MMTCa modified CPE. A linear dynamic range of 1 × 10− 8 to 3 × 10− 6 M methocarbamol was obtained following its preconcentration onto the developed 10% (w/w) MMT-Ca-modified CP electrode by adsorption accumulation for 400 s at −0.3 V. Its corresponding regression equation was: ip (μA) = 14.22 ± 0.002 C/(μM) + 0.118 ± 0.014 (n = 12 and r = 0.998). Limits of detection (LOD) and quantitation (LOQ) of bulk methocarbamol were estimated using the expression: k S.D./b [30], where k = 3 for LOD and 10 for LOQ, S.D. is the standard deviation of the blank (or the intercept of the calibration plot) and b is the slope of the calibration plot. LOD and LOQ of 3 × 10−9 M and 1 × 10−8 M methocarbamol, respectively, were achieved by the developed SW-AdASV method utilizing the developed 10% (w/w) MMT-Ca modified CPE. The results indicated the reliability of the developed SW-AdASV method for the trace assay of bulk methocarbamol. −8

3.2.4.3. Accuracy and precision. Accuracy and precision [31] of the described SW-AdASV method were evaluated through intra-day and inter-day assays for three replicate measurements of 5 × 10−7 M methocarbamol following its preconcentration by adsorptive accumulation onto the 10% (w/w) MMT-Ca-modified CPE for 400 s at −0.3 V. Satisfactory results were achieved (Table 1). 3.2.4.4. Robustness and inter-laboratory precision. The robustness [31] of the developed SW-AdASV method for assay of methocarbamol was examined by evaluating the influence of small variations in some of the most important operational conditions (pH 3.8 to 4.5), accumulation potential Eacc (−0.4 to −0.2 V) and preconcentration time (380 to 420 s). As shown in Table 2, the obtained results indicated insignificant effect within the studied range of variation of the optimum operational conditions, and consequently the developed SW-AdASV method was reliable for assay of bulk methocarbamol and it could be considered robust. The inter-laboratory precision [31] of the developed SW-AdASV method for assay of methocarbamol utilizing the MMT-Ca-modified CPE was identified using two PAR Potentiostats, 263A (Lab. 1) and 273A (Lab. 2) at different elapsed time. The results obtained due to Lab.-to-Lab. and even day-to-day were found reproducible (Table 2), since there was no significant difference in the recovery and/or relative standard deviation values. 3.2.5. Application 3.2.5.1. Assay of Ibuflex® tablets. In order to evaluate potential application of the developed SW-AdASV method in analysis of real samples, it was used for determination of methocarbamol in its pharmaceutical formulation (Ibuflex® tablets) using the calibration curve method, without the necessity for samples pretreatment and/or

Table 1 Intra-day and inter-day assay of 1 × 10−7 M bulk methocarbamol by means of the developed SW-AdASV method utilizing the developed 10% (w/w) MMT-Ca modified CPE (n = 3). Day

−6

3.2.4.2. Interferences. Various concentrations (1 × 10 –3 × 10 M) of bulk methocarbamol solution in the presence of 1 × 10−4 M bulk ibuprofen (It is electro-inactive co-formulated drug) were analyzed by the developed SW-AdASV method utilizing the developed MMT-Ca modified CPE. Insignificant differences in percentage recoveries and relative standard deviations (99.7 ± 0.6 to 99.8 ± 1.3) were noticed indicating no interference from ibuprofen in analysis of methocarba-

1 2 3

Intra-day

Inter-day

Recovery

Accuracy

Precision

Recovery

Accuracy

Precision

%R

% RE

% RSD

%R

% RE

% RSD

99.8

−0.2

0.7

99.8 99.4 100.7

−0.2 −0.6 0.7

0.8 1.5 1.5

n: Number of the replicated measurements, RE%: Relative error and RSD%: Relative standard deviation.

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Table 2 Validation studies for determination of 5 × 10−7 M bulk methocarbamol by means of the developed SW-AdASV method utilizing the developed MMT-Ca modified CPE. Variables

Operational conditions

pH of the medium 3.8 4.0 4.5

Eacc = −0.3 V, tacc = 400 s

Preconcentration potential (Eacc) −0.4 pH = 4, −0.3 tacc = 400 s −0.2 Preconcentration time (tacc) 380 pH = 4, 400 Eacc = −0.3 V 420 Inter-laboratory precision Lab. (1) pH = 4, Lab. (2) Eacc = −0.3 V, tacc = 400 s

% R ± RSD (n = 3) 98.9 ± 0.2 99.8 ± 0.5 98.7 ± 0.5

98.8 ± 0.8 99.8 ± 0.5 98.2 ± 0.7

99.0 ± 0.8 99.8 ± 0.5 100.7 ± 1.2

99.8 ± 0.5 97.8 ± 1.8

time-consuming extraction steps prior to analysis (Table 3). The validity of the described SW-AdASV method was further assessed by applying the standard addition method [32] for three different standard methocarbamol solutions added to pre-analyzed tablet solution (Table 3). The results were statistically compared with that obtained by a reported RPLC method [18]. Since the calculated F-value did not exceed the theoretical one (Table 3), there was no significant difference between the described and reported methods with respect to reproducibility [33]. Also, no significant difference was noticed between the two methods regarding accuracy and precision as revealed by t-test value [33]; Table 3. These results demonstrated that the described SW-AdASV method utilizing the developed 10% (w/ w) MMT-Ca-modified CPE was quite reliable and sensitive enough for methocarbamol determination in real formulation samples in the presence of the co-formulated drug “ibuprofen.” 3.2.5.2. Assay of methocarbamol in spiked human serum. The described SW-AdASV method utilizing the developed MMT-Ca-modified CPE was also successfully applied for assay of methocarbamol spiked in human serum without prior extraction, taking into consideration the medium exchange method described in the Experimental section. SW-AdAS voltammograms of various concentrations of methocarbamol spiked in human serum were recorded (Fig. 6). As shown in Fig. 6, no interfering peaks from endogenous human serum constituents were appeared in analysis of methocarbamol. Variation of the peak current (ip/μA) versus concentration ([C Meth]/μM) of methocarbamol was linear within the range 4×10−8 to 1.2×10−6 M (Fig. 6, inset); its corresponding regression equation was: ip/μA=12.95± 0.011 C/μM+0.066±0.052 (r=0.996 and n=11). The achieved LOD

Fig. 6. SW-AdAS voltammograms for various concentrations of methocarbamol spiked in human serum: 8 × 10− 8, 1 × 10− 7, 2 × 10− 7, 3 × 10− 7, 4 × 10− 7, 6 × 10− 7, 8 × 10− 7 M (from down to up), recorded in the acetate buffer of pH 4 following preconcentration onto the developed 10% (w/w) MMT-Ca modified CPE by adsorptive accumulation at −0.30 V for 400 s. Dotted line is the background; inset: Plot of peak current ip versus concentration of methocarbamol [C Meth] spiked in human serum.

and LOQ [30] were 1.2 × 10−8 and 4× 10−8 M, respectively. Mean percentage recovery of methocarbamol in human serum was 101.3± 1.1% (n=5). On the other side, voltammograms of human serum spiked with a binary mixture (methocarbamol and ibuprofen) showed no interference from ibuprofen. Moreover, in analysis of various samples of methocarbamol by the described SW-AdAS voltammetric method utilizing the developed MMT-Ca modified CPE, insignificant interferences in recoveries and relative standard deviations were noticed from various organic and inorganic species (such as urea, phenylalanine, valine, glutamine, histidine, aspirin, ketoprofen, Ketorolac, pregabalin, gabapentin, carisoprodol, meprobamate, caffeine, Na +, K +, Ca+ 2 , Zn+ 2, Fe+ 3 and Mg+ 2 ) up to concentration of 5 × 10−5 M of each. The obtained mean % R ± RSD (98. 5 ± 1.3–99.4 ± 1.1) confirmed again the reliability of the described SW-AdASV method for assay of methocarbamol in biological fluids.

Table 3 Assay of Ibuflex® tablets (750 mg methacarbamol + 400 mg ibuprofen/tablet) by means of the developed SW-AdASV method (tacc = 400 s,) and a reported reverse phase liquid chromatography (RPLC) method [18]. Method

SW-AdASV

Reported LC [18]

% R ± RSD (A)

(B)

99.9 ± 1.0 F-value = 2.9 t-test = 0.2 99.7 ± 1.7

98.8 ± 1.1 F-value = 2.4 t-test = 1.0

(A): Using calibration curve method and (B): using standard addition method. Theoretical F-value = 6.4 and t-value = 2.3 at 95% confidence limit for n1 = 5 and n2 = 5.

Fig. 7. Mean plasma concentration–time profiles for two subjects following the administration of an oral dose of 1.5 g methocarbamol (two Ibuflex® tablets).

E.M. Ghoneim, H.S. El-Desoky / Bioelectrochemistry 79 (2010) 241–247 Table 4 Pharmacokinetic parameters estimated for two male volunteers following administration of an oral dose of 1.5 g methocarbamol (two Ibuflex® tablets). Parameter/unit

−1

Cmax (μg mL ) tmax (h) AUC0–12 (μg h mL−1) AUC0–∞ (μg h mL−1) Kel (h−1) t1/2 (h) a

Estimated valuesa Subject (1)

Subject (2)

22.9 1.0 58.5 59.1 0.50 1.4

26.2 1.3 72.9 73.8 0.55 1.3

Average of three measurements.

3.2.5.3. Pharmacokinetic study. Pharmacokinetic studies were performed on the plasma samples of two healthy volunteers following the administration of an oral dose of 1.5 g methocarbamol (two Ibuflex® tablets). SW-AdASV peaks of methocarbamol in plasma samples of the two subjects (collected at 0.5, 1.0, 1.5, 2, 3, 4, 6, 9, 12 and 24 h after the oral administration) were of good shape and no any additional peak was interfered with that of methocarbamol. Plasma concentration–time profiles of the two subjects are shown in Fig. 7. The following parameters were assessed for the period of 0.0– 12 h: Area under the plasma concentration–time curves from time zero to the last measurable sample time (AUC0–12) and to infinity (AUC0–∞); maximum plasma concentration (Cmax); time of the maximum concentration (tmax); elimination constant (Kel) and elimination half-life time (t1/2), Table 4. The pharmacokinetic parameters obtained for the two subjects were in good agreement with those reported in literature [10–12]. The satisfactory results confirmed the reliability of the described SW-AdASV method for assay of methocarbamol also in human plasma utilizing the developed MMT-Ca modified CPE without interferences from the co-formulated ibuprofen. 4. Conclusions Electro-oxidation of methocarbamol was studied in buffered solutions at a developed 10% (w/w) MMT-Ca modified CPE. A simple and precise SW-AdASV method was also developed for determination of methocarbamol in the bulk form, formulation and human blood without prior extraction. The modified electrode exhibited an excellent sensitivity and selectivity towards methocarbamol even in the presence of 102–104 folds of the co-formulated drug “ibuprofen,” various organic species or metal ions. The described SW-AdASV method with the developed MMT-Ca modified CPE could be recommended for use in quality control and clinical laboratories. Acknowledgement The authors express their gratitude to Ramadan Specialized Hospital's staff, (Tanta City, Egypt), for the kind care of the two volunteers and for providing the great facilities for collecting and treatments of the plasma samples required for the present pharmacokinetic studies. References [1] B. Muralidharan, G. Gopu, C. Vedhi, P. Manisankar, Voltammetric determination of analgesics using a montmorillonite modified electrode, Appl. Clay Sci. 42 (2008) 206–213. [2] P. Manisankara, G. Selvanathanb, C. Vedhib, Utilization of sodium montmorillonite clay-modified electrode for the determination of isoproturon and carbendazim in soil and water samples, Appl. Clay Sci. 29 (2005) 249–257. [3] P. Manisankar, G. Selvanathan, C. Vedhi, Determination of pesticides using heteropolyacid montmorillonite clay modified electrode, Talanta 68 (2006) 686–692.

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