Adsorptive Cathodic Stripping Voltammetric Determination of Cefoperazone in Bulk Powder, Pharmaceutical Dosage Forms, and Human Urine

The electroreduction behaviour and determination of cefoperazone using a hanging mercury drop electrode were investigated. Cyclic voltammograms of cefoperazone recorded in universal Britton-Robinson buffers pH 3–6 exhibited a single irreversible cathodic peak. The process was adsorption-controlled. Britton-Robinson buffer 0.04 M pH 4.0 was selected as a supporting electrolyte for quantitative purposes by differential pulse and square wave adsorptive cathodic stripping voltammetry. The experimental voltammetric conditions were optimized using Central Composite Face design. A reduction wave was seen in the range from −0.7 to −0.8 V. These voltammetric techniques were successfully validated as per ICH guidelines and applied for the determination of cefoperazone in its single and sulbactam containing powders for injection and statistically comparable to USP-HPLC. They were further extended to determine cefoperazone in spiked human urine with no matrix effect.


Reagents and Apparatus.
Cefoperazone standard was kindly supplied by the National Institute of Drug Quality Control (Vietnam) and used without further purification. A standard stock solution of 500 g/mL cefoperazone was prepared in water and protected from light in a refrigerator. This solution was stable for at least one week as voltammetrically tested. Working standards of cefoperazone were freshly prepared just before the assay by adding appropriate amounts of stock solution with supporting electrolytes to the mark in 25 mL volumetric flasks. Supporting electrolytes (i.e., acetate 2 Journal of Analytical Methods in Chemistry pH 4.0, phosphate pH 4.0, and Britton-Robinson universal buffers) were prepared on the day the experiment was performed. Other chemicals were of analytical grade and purchased from Merck (Germany). Urine collected from healthy subjects was used for cefoperazone-spiked samples. Freshly prepared deionized doubly distilled water (Maxima Ultra Pure Water, Elga-Prima Corp, UK) was used throughout this study.
pH measurements were performed on a CyberScan pH 510 (Eutech Instruments Pte Ltd., Singapore). All voltammetric measurements were carried out at 25 ∘ C with a 797 VA Computrace (Metrohm AG, Switzerland) in connection with a Dell computer using Microsoft Windows XP and controlled by VA Computrace Software 1.3. The three-electrode system consisted of a hanging mercury drop electrode (HMDE) as working electrode, a platinum wire as auxiliary electrode, and an Ag|AgCl|KCl(3 M) as reference electrode. A medium size mercury drop was employed. Oxygen-free nitrogen gas was used for the removal of dissolved oxygen from the measured solutions for 100 s with a stirring rate of 2000 rpm. After a preconcentration step, the solutions were left quiescent for 15 s equilibrium before measurement.
The cyclic voltammetric behaviour of 0.03 g/mL cefoperazone in the Britton-Robinson universal buffers at the HMDE was studied. The experimental parameters were accumulation potential −0.3 V, accumulation time 100 s, scan rate 0.3 V/s, pulse step 0.005 V, and potential range from −0.3 to −1.2 V. The optimization process started with the selection of buffer type with voltammetric measurements of 0.03 g/mL cefoperazone. Optimization process was carried out using Modde 9.1 software (Umetrics, Sweden). All experiments were performed in a randomized order as proposed by the software so as to minimize the effect of uncontrolled factors that may introduce a bias on the response. To determine the optimal factors, the optimizer function of Modde software (i.e., Nelder Mead simplex method) was used. Unless stated otherwise, the differential pulse and square wave adsorptive cathodic stripping voltammograms (abbreviated as DP-AdCSV and SW-AdCSV, resp.,) were recorded in triplicate for each programmed run.

Sample Preparation.
A quantity of powders for injection equivalent to 500 mg cefoperazone was accurately weighed and appropriately diluted with water to obtain ca. 2.5 g/mL cefoperazone test solution.

Analysis of Samples.
The general procedure adopted for obtaining adsorptive stripping voltammograms was as follows. For pharmaceutical formulations, 0.3 mL of test solution was diluted with supporting electrolyte solution (i.e., Britton-Robinson universal buffer pH 4.0) to the mark in a 25 mL volumetric flask. For spiked human urine, accurately measured aliquots of 2.5 g/mL cefoperazone were pipetted into 25 mL volumetric flasks, subsequently added with 0.025 mL human urine, and diluted with supporting electrolyte solution to the mark. The content of these flasks was completely transferred into the voltammetric cell for measurement after 5 min mechanical shaking.

Characteristics of the Electrode Process.
A well-defined single peak was yielded in the cathodic direction in the pH range 3-6 ( Figure 1). The peak current obtained without accumulation was substantially smaller than that obtained after a preconcentration step (data not shown). It strongly indicates that cefoperazone adsorbs readily onto the surface of HMDE, and a considerable increase in sensitivity can be gained by adsorptive stripping voltammetry. The peak potential ( ) shifted to more negative values and broadened upon the increase of medium pH revealing the involvement of protons in the electrode reaction, and that the proton-transfer reaction precedes the electron transfer [22]. No oxidation peak was observed in the positive scanning half-cycle confirming the irreversible nature of the electrode process. This irreversible cathodic peak disappeared at pH ≤ 2 and ≥7 (data not shown).
With reference to most cephalosporins [23,24], the electrochemical activity of cefoperazone could be attributed to the reduction of the 3-4 double bond of the cephem nucleus activated by the [(1-methyl-1Htetrazol-5-yl)sulphanyl]methyl group present at C 3 (Scheme 1).
The value of (product of symmetry transfer coefficient and number of electrons transferred in the ratedetermining step) of 0.603 was estimated from slope value of the obtained versus ln V plot at pH 4.0 (i.e., = −0.0213 ln V − 0.9267; 2 = 0.961, = 4) using the following equation: For the number of electrons transferred in the ratedetermining step for the electroreduction of the double bond of the analyte equals 2, the value of the transfer coefficient (0.301) was estimated. This estimated value indicates the symmetry of the energy barrier in such an irreversible reduction process [25]. Scheme 1: Proposed mechanism of cefoperazone reduction in acidic medium at the mercury electrode.
The number of protons ( ) involved in the rate-determining step was consequently estimated to be 0.55 (ca. 1) from the slope value of the versus pH plot (i.e., = −0.0537pH − 0.7592; 2 = 0,999, = 4) using the following relation: Given the fact that two electrons ( = 2) and one proton ( = 1) are involved in the rate-determining step of the electro-reduction process of a cefoperazone molecule as explained above, the proposed scheme was reasonably justified. This is different from the attribution of cefoperazone irreversible electrode reaction by Hammam and coworkers to the reduction of an electroactive group in the sidechain on C-7 [18].
On the other hand, linear plot of versus scan rate V was obtained at pH 4.0 with slope value of 884.3 nA ⋅ V −1 s ( 2 = 0.999, = 4) revealing that the reduction process of cefoperazone at the HMDE is controlled by adsorption. The slope value (1.022 nA V −1 s) of log versus log V plot ( 2 = 0.999, = 4) is very close to the expected theoretical value 1.0 for an ideal reaction of surface species. This indicates again the strong adsorptive behavior of cefoperazone onto the mercury electrode surface [26].

Optimal Parameters and Experimental Conditions.
Although DP-AdCSV and SW-AdCSV measurements of 0.03 g/mL cefoperazone recorded in Britton-Robinson, acetate, phosphate buffers pH 4.0 showed a well-defined sharp peak, the peak most intensified in Britton-Robinson medium. This is in agreement with previously reported data [19]. Accordingly, this type of buffer was chosen as a supporting electrolyte for further investigation on voltammetric parameters, which most influenced the peak current magnitude on preliminary measurements.
The optimization process was done using a Central Composite Face experiment design with 6 variables as presented It is remarked that a minimum peak current of 0.03 g/ mL cefoperazone reached when the values of variables were maximum (i.e., accumulation time (120 s); pulse amplitude (0.1 V); voltage step (0.02 and 0.007 V for DP-AdCSV and SW-AdCSV, resp.); frequency (80 Hz) for SW-AdCSV) and minimum (i.e., pulse time (0.02 s) and voltage step time (0.05 s) for DP-AdCSV) as representatively shown in Figures  2 and 3.
The relationship between peak current and voltammetric variables under study could be mathematically described as follows. According to the 2 / 2 diagnostic tool (>0.9), the fitted interaction models for both techniques were excellent.
where 1 is accumulation time; 2 is voltage step; 3 is pulse amplitude; 4 is frequency; 5 is pulse time; 6 is voltage step time ( * indicates statistically insignificant, > 0.05); 2 is goodness of fit; 2 : goodness of prediction.

Validation and Application.
Under the optimized conditions, the proposed voltammetric techniques (DP-AdCSV and SW-AdCSV) were validated according to ICH guidelines [27].
A linear correlation between the voltammetric peak intensity and drug concentration was obtained over the range 0.01-0.06 g/mL (Figures 4(a) and 4(b) and Table 2).
The accuracy and intermediate precision of the described stripping voltammetric techniques were examined for 0.01-0.06 g/mL (Table 3). RSD < 2% and recovery percentages of 99-101% mean that these techniques were accurate and precise. Obviously, the voltammetric determination of cefoperazone was not markedly influenced in the presence of equimolar concentration of sulbactam. In other words, sulbactam does not possess any electrochemical signal in the potential range from −0.3 to −1.   In our study, USP-HPLC was used as a reference method [28] for quantitatively analyzing cefoperazone in powders for injection. The statistical comparison of voltammetric and liquid chromatographic techniques was done by ANOVA (multiple group mean test) and Bartlett's test for equal variances at a 95% confidence level (Table 4). No statistically significant differences were observed between these chromatographic and voltammetric techniques ( > 0.05) suggesting that they had comparable accuracy and precision. In addition, the influence of small variation (5%) of some variables (e.g.,  For the determination of cefoperazone in spiked human urine, the above-mentioned optimal experimental conditions were also applied. Results show that the analysis was specific as no reduction wave was seen in the range from −0.7 to −0.8 V with pure urine water-diluted by 1000 times (blank) (Figures 5(a) and 5(b)). Supposing that urine levels in excess of 32 g/mL are maintained for at least 12 h [29], the calibration curve for this analysis was constructed in the corresponding range 0.01-0.07 g/mL ( Table 2). As a consequence of this linear range, the sensitivity (Lower Limit of Quantification (LLOQ)) was 0.01 g/mL (ca. 1.5 × 10 −8 M) for both DP-AdCSV and SW-AdCSV techniques. This is comparable to LOQ values reported in other voltammetric studies on cefoperazone [19,20]. Analyte peak was identifiable, discrete, and reproducible at this concentration (RSD < 20% and recovered within 80-120%). The precision (RSD < 15%) and accuracy (85-115%) were obtained from the back calculation of other calibration standards. It is noteworthy that the urine matrix diminished the peak currents of cefoperazone voltammograms by 10-30% as compared to the corresponding ones in the Britton-Robinson buffer pH 4.0.
This analysis was also intraday-and interday-validated with four spiked quality control (QC) samples ( Table 5). The relative analytical recovery (comparing the measured concentration with actual added ones) of these techniques was within the range 85-115% with RSD <15%. The determination of cefoperazone in spiked urine samples was further studied in the presence of sulbactam. According to human pharmacokinetic data, 75% of a 500 mg sulbactam parenteral dose is excreted unchanged in urine over the range 0.015-0.15 g/mL [30]. The relative recovery of cefoperazone was shown to be 87.9-108.0% for both voltammetric techniques within the sulbactam concentration range specified. Clearly, it is evidenced that DP-AdCSV and SW-AdCSV determination of cefoperazone in human urine satisfactorily meets the requirement for assay methods for biological samples [31].

Conclusion
The mechanism of electro-reduction of cefoperazone at the HMDE was elucidated. Using Briton-Robinson buffer 0.04 M pH 4.0 as a supporting electrolyte, DP-AdCSV and SW-AdCSV techniques were developed for the determination of cefoperazone in bulk powder, pharmaceutical dosage forms, and spiked human urine without any prerequisite extraction, separation, and adsorption steps. Voltammetric data were statistically comparable to US pharmacopoeia HPLC meaning that the determination of cefoperazone was accurate and precise. Moreover, the sensitivity and reliability of the determination of cefoperazone in spiked human urine sample were also acceptable. It is recommended that these validated techniques could be used for cefoperazone quality control and pharmacokinetics studies.