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.
Cefoperazone is a third-generation cephalosporin antibiotic effective in treating
In the literature, the determination of cefoperazone has been achieved by high performance liquid chromatography (HPLC) [
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
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
A quantity of powders for injection equivalent to 500 mg cefoperazone was accurately weighed and appropriately diluted with water to obtain ca. 2.5
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
A well-defined single peak was yielded in the cathodic direction in the pH range 3–6 (Figure
Cyclic voltammograms of 0.03
With reference to most cephalosporins [
Proposed mechanism of cefoperazone reduction in acidic medium at the mercury electrode.
The value of
The number of protons (
Given the fact that two electrons (
On the other hand, linear plot of
Although DP-AdCSV and SW-AdCSV measurements of 0.03
The optimization process was done using a Central Composite Face experiment design with 6 variables as presented in Table
Variable ranges used in the optimization process of voltammetric measurements of 0.03
Variable | DP-AdCSV | SW-AdCSV |
---|---|---|
Accumulation time | 100→120 s | |
Pulse amplitude | 0.05→0.1 V | |
Pulse time | 0.02→0.04 s | |
Frequency | 60→80 Hz | |
Voltage step | 0.01→0.02 V | 0.005→0.007 V |
Voltage step time | 0.05→0.06 s |
It is remarked that a minimum peak current of 0.03
Response surface plots for the influence of deposition time and pulse amplitude (pulse time 0.02 s, voltage step 0.02 V, and voltage step time 0.05 s) (a) and pulse time and voltage step (deposition time 120 s, pulse amplitude 0.1 V, and voltage step time 0.05 s) (b) on DP-AdCSV peak current of 0.03
Response surface plots for the influence of deposition time and pulse amplitude (frequency 80 Hz, voltage step 0.007 V) (a) and frequency and voltage step (deposition time 120 s, pulse amplitude 0.1 V) (b) on SW-AdCSV peak current of 0.03
The relationship between peak current and voltammetric variables under study could be mathematically described as follows. According to the
For DP-AdCSV;
Under the optimized conditions, the proposed voltammetric techniques (DP-AdCSV and SW-AdCSV) were validated according to ICH guidelines [
A linear correlation between the voltammetric peak intensity and drug concentration was obtained over the range 0.01–0.06
Linearity statistical analysis for the proposed voltammetric techniques.
Parameter | Pharmaceutical formulation | Spiked human urine | ||
---|---|---|---|---|
DP-AdCSV | SW-AdCSV | DP-AdCSV | SW-AdCSV | |
Concentration range ( |
0.01–0.06 | 0.01–0.06 | 0.01–0.07 | 0.01–0.07 |
Number of data points: |
6 | 6 | 7 | 7 |
Coefficient of determination: |
0.999 | 0.999 | 0.999 | 0.999 |
Slope (nA/ |
−5902 | −2338 | −3046 | −1133 |
Intercept (nA) | −53 | −26 | −76 | −25 |
SD of the residuals: |
0.8 | 0.8 | 1.5 | 0.9 |
SD of the slope: |
20.1 | 18.1 | 28.5 | 17.1 |
SD of the intercept: |
0.8 | 0.7 | 1.2 | 0.8 |
DP-AdCSV (a) and SW-AdCSV (b) measurements of 0.01–0.06
The accuracy and intermediate precision of the described stripping voltammetric techniques were examined for 0.01–0.06
Accuracy and intermediate precision analysis for the proposed voltammetric techniques.
Number | Cefoperazone 0.03 µg/mL | Cefoperazone + sulbactam (0.03 µg/mL) | ||
---|---|---|---|---|
DP-AdCSV | SW-AdCSV | DP-AdCSV | SW-AdCSV | |
1 | 101.4 | 101.2 | 99.4 | 97.3 |
2 | 99.5 | 97.9 | 101.2 | 98.4 |
3 | 99.1 | 100.8 | 101.6 | 98.9 |
4 | 99.6 | 99.9 | 100.1 | 100 |
5 | 100.4 | 100.4 | 100.3 | 100.6 |
6 | 101.1 | 98.3 | 101.2 | 101.6 |
| ||||
Mean | 100.2 | 99.8 | 100.6 | 99.5 |
SD | 0.9 | 1.4 | 0.8 | 1.5 |
RSD (%) | 0.9 | 1.4 | 0.8 | 1.5 |
In our study, USP-HPLC was used as a reference method [
Assay results for the determination of cefoperazone in powders for injection by the proposed voltammetric techniques and USP-HPLC.
Pharmaceutical formulation | Recovery |
||
---|---|---|---|
HPLC | DP-AdCSV | SW-AdCSV | |
Cefoperazone 500 or 1000 mg* | |||
Bacamp |
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Cefobid |
|
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Neoaxon* |
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|
|
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Cefoperazone 500 or 1000 mg** + sulbactam 500 or 1000 mg** | |||
Cefactam** |
|
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Sulperazon |
|
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Jincetam |
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|
|
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
DP-AdCSV (a) and SW-AdCSV (b) measurements of 0.01–0.07
This analysis was also intraday- and interday-validated with four spiked quality control (QC) samples (Table
Accuracy and precision data for the determination of cefoperazone in spiked human urine by the proposed voltammetric techniques (
Actual conc. (µg/mL) | Experimental conc. (mean ± SD µg/mL) | Precision as RSD (%) | Accuracy (%) | |
---|---|---|---|---|
DP-AdCSV | ||||
Intraday | 0.01 |
|
1.0 | 97.2 |
0.03 |
|
1.0 | 97.9 | |
0.04 |
|
0.5 | 101.2 | |
0.06 |
|
0.8 | 100. 9 | |
Interday | 0.01 |
|
3.3 | 92.3 |
0.03 |
|
2.6 | 100.7 | |
0.04 |
|
2.7 | 102.1 | |
0.06 |
|
0.5 | 100.2 | |
| ||||
SW-AdCSV | ||||
Intraday | 0.01 |
|
2.1 | 94.8 |
0.03 |
|
1.6 | 101.6 | |
0.04 |
|
0.5 | 102.0 | |
0.06 |
|
1.8 | 101.0 | |
Interday | 0.01 |
|
2.1 | 96.7 |
0.03 |
|
0.7 | 101.1 | |
0.04 |
|
0.7 | 101.3 | |
0.06 |
|
0.8 | 100.3 |
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.
This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant no. 104.07-2012.58.