Development of a novel and cost-effective redox sensor for voltammetric determination of pantoprazole sodium during pharmacokinetic studies

A pencil graphite electrode modified with poly (bromocresol green (BCG)) was prepared by electro-polymerization process for the determination of pantoprazole sodium. The surface morphology and structure of poly (BCG) film were characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. The determination of pantoprazole sodium in Britton–Robinson buffer (pH 7.0) was carried out by square wave adsorptive stripping voltammetric technique. Under optimum conditions, the linear response of the peak with concentration of the cited drug was in the range of 6.6–360 × 10−8 M with limit of detection of 2.2 × 10−8 M. Moreover, the poly (BCG)-modified electrode has been successfully applied to determine pantoprazole sodium in tablets, vials and during pharmacokinetic studies.


Introduction
Pantoprazole sodium (PAN sodium) is a member of the benzimidazole class of proton pump inhibitors. It is commonly used in the management of gastrointestinal ulcers and reflux esophagitis [1]. Its efficacy as antiulcer and anti-secretory agent has been well established. Several methods have been previously used for its determination in pharmaceutical dosage forms and biological fluids including UV-visible spectrophotometry [2,3] Electrical contact with the lead was achieved by soldering a metallic wire to the metallic part that holds the lead in place inside the pencil. Unless stated otherwise, the pencil was fixed so that about 3 mm of its length is immersed into the solution. Measurements were performed in a 10 ml glass cell containing 6 ml of supporting electrolyte solution. Stirring was achieved with a magnetic stirring bar. The pH values of solutions were measured using Hanna pH meter (Hanna Instruments Brazil, São Paulo Brazil) with a combined electrode. The solutions were sonicated using Bransonic ultrasonic cleaner Branson UL Transonics Corporation Danbury, USA. Surface morphology studies of the modified electrode were carried out using a scanning electron microscope (SEM) JEOL JSM-5400 LV instrument (Oxford, USA). A Nicolet 6700 FTIR advanced Gold Spectrometer supported with OMNIC 8 software (Thermo Electron Scientific Instruments Corp. WI, USA) was used for data processing.

Preparation of standard solutions
An accurately weighed amount of PAN sodium was transferred into a 100 ml calibrated flask and dissolved in about 10 ml methanol. The solution was completed to the mark with distilled water to provide a stock solution containing 1.0 mM of PAN sodium. The working standard solutions were prepared by further dilution of the suitable aliquots of the stock solution with B.R. buffer (pH = 7.0).

Real sample preparation
The contents of 10 tablets were accurately weighed, finely powdered and thoroughly mixed in a mortar. Portions equivalent to about 1.0 mM of each drug was accurately weighed and dissolved in 20 ml methanol. The contents were sonicated for 20 min to assure complete solubility. The excipients were separated by centrifugation at 3000 r.p.m. for 5 min. The residue was washed three times with distilled water.
For vials, accurate weighed amount of the powder equivalent to about 1.0 mM for PAN sodium was dissolved in methanol shaken well for 5 min and sonicated for further 5 min. The filtrate was transferred into a 100 ml calibrated flask and diluted to a final volume with distilled water. Appropriate working solutions were prepared by taking suitable aliquots from these stock solutions and diluting them with the B.R. buffer solutions (pH = 7.0).
Drug-free rabbit plasma samples were obtained from six healthy rabbits stored at −20°C and analysed the next day after collection without any further pretreatment. One millilitre of the plasma was pipetted into a voltammetric cell containing 6 ml B.R. buffer (pH = 7.0).

Procedure for application in vivo (pharmacokinetic study)
The pharmacokinetic of PAN (Pantazol ® injection) was evaluated in plasma using six healthy male rabbits (1800-2200 g) following a single intra-peritoneal injection (I.P.). Blood samples were collected pre-dosing and at 10, 15, 30 min and 1, 2, 4, 8, 12 h (s) after the I.P. administration into heparinized tubes. The blood samples were centrifuged immediately at 3000 r.p.m. for 15 min, and then plasma fractions were rapidly separated and stored in cooled polypropylene tubes at −20°C. The plasma samples were analysed using the described square wave adsorptive stripping voltammetric (SWAdSV).

Study of poly (bromocresol green) film on pencil graphite electrode
Scanning electron microscopy was used to investigate the interface morphology of the electrode surfaces. Figure 2a and b shows the surface morphology of bare PGE and poly (BCG)/PGE, respectively. A smooth surface was clearly observed on the bare PGE. However, after electro-polymerization of BCG the surface of PGE was covered with a uniform flake shaped film indicating that the BCG film was successively modified on the electrode surface. FTIR spectroscopy has been also used to further confirm modification of the electrode (figure 2c). The close inspection of the spectra has revealed that the characteristic υ (C=C), υ (C-H) and υ (OH) bands at 1628, 2923 and 3457 cm −1 , respectively, of the bare electrode were clearly down shifted to 1627, 2890 and 3446 cm −1 in the polymerized electrode. These shifts clearly further confirm the polymerization process.  cited drug (figure 3a). After this concentration, the peak current of PAN sodium decreased, this may be attributed to blocking of PGE surface by excess BGC. The maximum currents were achieved after the cycles increase up to 10 cycles, after that the current was decreased that may be attributed to blocking PGE surface by increasing amount of BCG.

The effect of initial deposition potential and time on bromocresol green deposition
The effect of adsorption potential and time on the BCG through measuring the anodic peak current of PAN sodium was studied at various adsorption potentials between -1.2 V and +2.0 V. The maximum peak current was obtained at an adsorption potential of −1.0 V and after 5 s.

The effect of scan rate on deposition bromocresol green at electrode surface
The effect of scan rate on the redox behaviour of the deposited poly (BCG) film was investigated in the scan rate range of 25-100 mV s −1 . The cyclic voltammograms of the poly (BCG) film recorded at different scan rates are shown in figure 3b.

Electrochemical characterization of modified electrode using standard potassium ferricyanide
Prior to voltammetric analysis the PGE was evaluated. The cyclic voltammetry (CV) was recorded on PGE wetted with 0.5 M KCl where no voltammetric peaks were recorded. Thus no electro-active interfering species were appreciably released by all graphite sticks. Furthermore, the CV was recorded again after wetting PGE with 1 mM potassium ferricyanide in 0. 5 where D and C°are the diffusion coefficient and bulk concentration of the redox probe, respectively. The electro-active surface area of the bare PGE was 16.5 mm 2 (figure 4a) while that of the poly (BCG)/PGE was 30.3 mm 2 (figure 4b). Compared with the bare PGE the electro-active surface area of the modified PGE increased approximately 83.6%.

Electrochemical characterization of pantoprazole sodium at bare electrode versus modified electrode
The CV and SWAdSV were presented in figure 5a and b, respectively. The CV of PAN sodium displayed only a single irreversible anodic peak at +0.83 V and no cathodic peak in the reverse scan was recorded which means that the oxidation of PAN sodium is irreversible.

The effects of initial deposition potential and time
The effect of adsorption potential and time on the anodic peak current of PAN sodium was studied using poly (BCG) electrode at various adsorption potentials between −0.2 V and +0.6 V for SWAdSV in B.R. buffer (pH 7.0). The maximum peak current was obtained at an adsorption potential of 0.2 V and after 20 s (figure 7). Figure 6b shows the effect of scan rate in the range of 30-150 mV s −1 on the CV response of PAN sodium in B.R. buffer (pH 7.0). With increasing scan rates, the anodic peak slightly shifted to the positive potential side. The peak current is increasing remarkably with increasing scan rates. From the value of the slope, it can be deduced that the electrochemical oxidation process of PAN sodium is a diffusion-controlled process with an adsorption contribution. For an adsorption-controlled electrode reaction, the following equation could apply [39]:

Effect of scan rate
where Q is the peak area that could be obtained under given scan rate; v is the scan rate; F, R and T are constants. From the slope of Ip versus ν the electron-transfer number (n) that was involved in the electrode reaction of PAN sodium was calculated to be 2.

Method validation
The optimum conditions for the determination of PAN sodium using SWAdSV were: adsorption potential = 0.2 V, frequency = 180 Hz, step potential = 20 mV and potential amplitude = 55 mV. Under these optimum conditions, a linear calibration plot was recorded.         proposed method. The low values of limit of detection (LOD) and limit of quantitation (LOQ) reflect that SWAdSV is sensitive for PAN sodium determination. The LOD and LOQ were calculated by using the following equations [40]: LOD = 3.3 s/S and LOQ = 10 s/S where 's' is the mean of standard deviation of intercept and 'S' is the mean of the slope of the calibration curve. The LOD and LOQ are presented in table 1. Obviously, the low values of LOD and LOQ would indicate that the SWAdSV is highly sensitive for the determination of PAN sodium.

Accuracy
The accuracy of the method was determined by adding known amounts of PAN sodium to sample solution (tablets and vials) and calculating the recovery percentages. The results have indicated good accuracy of the proposed method (tables 2 and 3). These results have also proved the absence of the interference owing to common excipients.

Precision
The results of the inter-day and intra-day precision of the proposed method were ranged from 98.0 to 100.8% ± (1.2-1.7). The inter-day and intra-day precisions were evaluated through replicate analysis of the studied drug. The precision of the proposed methods was fairly high as indicated by the low values of S.D. and %RSD.

Selectivity of the method
The effects of common excipients co-administered drugs biologically active compounds and divalent metals were evaluated (table 4). Clearly, the % signal change of 120 × 10 −8 M PAN sodium upon addition of these potential interfering substances has not changed appreciably. This could indicate the selectivity of the method and hence its suitability for the determination of pharmaceuticals in complex matrices.

Application to pharmaceutical dosage forms
The proposed method was applied to the determination of PAN sodium in tablets and vials (table 2). The results were compared with the reported method [10]. The results of the proposed method were found to be comparable with those of the reported method as indicated by t-and F-tests. In addition, recovery studies were performed by using a standard addition method.

Precision
The precision was evaluated by measuring intra-day and inter-day precision. The intra-day values were calculated at three concentration levels in the same day with good coefficient of variation values. The inter-day precision was evaluated at three different days with good values. The results of 97.8-99.3% (± 0.9-1.6) show good repeatability and reproducibility of the proposed method in rabbit plasma.

Pharmacokinetic study
Owing to high sensitivity of the proposed SWAdSV method as expressed by low values of LOD and LOQ, the method was applied for the pharmacokinetic study of PAN sodium in rabbit plasma for, to our knowledge, the first time. The concentration-time curve in rabbits following the I.P. administration of pantazol ® injection containing PAN sodium at a dose of 0.55 mg kg −1 (n = 6) is shown in figure 9. The drug concentration in rabbit plasma decreased gradually (T max = 30 min) after an I.P. injection in this study.

Reaction mechanism
The oxidation mechanism proposed scheme 1 is based on the electrochemical data described and supported by the PAN sodium structure with two systems that are in resonance separated by a methylene group. The oxidation step in the ortho position will be favoured via resonance by the stabilization of the radical formed. Preferential attack is in the aromatic ring where the extent of conjugation is greater making removal of an electron easier. One electron is removed followed by deprotonation to produce a cation radical which reacts with water and leads to the formation of quinone species [41].
6. Comparison of the proposed method with other reported methods Table 6 summarizes the analytical performances of our proposed and the literature methods in terms of linearity LOQ and LOD. Our method has the lowest LOD for PAN sodium compared with the literature methods. These results could indicate that the poly (BCG)/PGE method would be an attractive alternative choice for determining of the cited drug in pure form pharmaceutical formulations and biological fluids.

Conclusion
In summary, a simple and fast electrochemical method was used for determination of PAN sodium by electro-polymerization of BCG on pencil graphite electrode. It showed excellent electro-catalytic activity towards the oxidation of the studied drug. The sensor exhibited wide linear range, low detection limit and high selectivity. Moreover, it was used for determination of PAN sodium in pharmaceutical formulations and during pharmacokinetic studies. Therefore, the proposed method provides a promising platform for determination of the cited drug in the field of electro-analytical chemistry.
Ethics. The experimental protocol was approved by the Institutional Animal Ethics Committee Assiut University for the use of experimental animals and all animal studies were carried out according to the Guide for Care and Use of Laboratory Animals.