The zalcitabine (ddC) has been extensively used in the treatment of HIV patients due to its antiretroviral activity. The quality control of this active principle in medications is of outstanding importance to public health. The principal objective of the current study was the development of an alternative analytical methodology for the zalcitabine determination using a voltammetric process. The zalcitabine gives a reduction peak (at
The Food and Drug Administration has approved the AIDS drug zalcitabine, commonly known as 2′-3′-dideoxycytidine (ddC). It is the third drug licensed specifically for use in treating the human immunodeficiency virus, the cause of AIDS. Zalcitabine was approved for use in combination with the first approved AIDS drug, zidovudine (or AZT), as a treatment option for adult patients with advanced HIV infection who show signs of clinical or immunological deterioration. Early clinical trial data have shown that increases in CD4 cells (immune cells) were somewhat greater and more sustained in patients treated with the combination of zalcitabine and zidovudine than in those who received zidovudine alone as initial therapy. An increase in CD4 cells is believed to indicate that the body’s disease-fighting capability has been at least transiently enhanced. Zalcitabine is a derivative of the naturally existing deoxycytidine, made by replacing the hydroxyl group in position 3′ with a hydrogen group. It is phosphorylated in T cells and other HIV target cells into its active triphosphate form, ddCTP. This active metabolite works as a substrate for HIV reverse transcriptase, and also by incorporation into the viral DNA, hence terminating the chain elongation due to the missing hydroxyl group. Since zalcitabine is a nucleoside, reverse transcriptase inhibitor (NRTI), it possesses activity only against retroviruses [
Structures of (a) zalcitabine and (b) cytosine.
Several chromatographic methods for the determination of zalcitabine in human plasma or serum have been published [
Cyclic and differential pulse voltammograms were obtained with a Metrohm Voltammetric System, Model 757 VA Computrace. The working electrode was a hanging mercury drop electrode (HMDE), an Ag/AgCl as reference electrode, and platinum as an auxiliary electrode. All pH measurements were made with a Micronal B474 pH Meter using combined Ag/AgCl-glass electrode.
Water purified in a Milli-Q system (Millipore) was used for all dilutions and sample preparations. All chemicals were of analytical reagent grade.
Stock solutions of zalcitabine (229 mg · L−1) were prepared daily by dissolving 22.9 mg of zalcitabine in water and a diluting to 100 mL with water. Zalcitabine standard was used as received by the “Instituto Nacional de Controle de Qualidade em Saúde” (INCQS) from Brazil (lot L1, 100.2%). Capsules of medicament containing 0.750 mg of zalcitabine were dissolved with water, filtered, and transferred into a 50.0 mL volumetric flask. The solutions were stored in the dark at 4°C. The 0.2 M Clark-Lubs buffer (pH 2.0) was also prepared in water. The pH of 0.04 M Britton-Robinson buffer (pH 2.0–11.0) was adjusted by addition of hydroxide sodium solution.
10 mL of the supporting electrolyte solution (0.2 M Clark-Lubs buffer, pH 2.0) was added to the voltammetric cell and degassed with nitrogen for 2 min while being stirred. An initial potential −1.00 V was applied to the electrode and the stirring was then stopped and the voltammogram was recorded by applying a negative-going potential scan (with differential pulse mode). The scan (at 20 mV/s and 50 mV pulse amplitude) was terminated at −1.25 V. After the background differential pulse voltammograms had been obtained, aliquots of the zalcitabine (0.25 mL of the stock solution) were added. Analysis of the medicaments forms followed the same procedure. In this case, 0.50 mL of the drug solution was used and the differential pulse cycle was repeated ten times using a new mercury drop. All voltammograms were recorded at ambient temperature (25°C).
The effect of scan rate on the peak current (
Figure
Cyclic voltammogram of the zalcitabine (20 mg · L−1) in 0.2 M Clark-Lubs buffer (pH 2.0). Scan rate: 100 mV s−1.
The influence of pH on the zalcitabine reduction process was studied by differential pulse voltammetry. The Britton-Robinson buffer solution was used in the range 2.0 to 11.5 pH and the zalcitabine reduction peak again appeared as in cyclic voltammetry only from 2.0 to 5.5 pH. Table
Effect of pH on the differential pulse peak currents and potentials for 10 mg · L−1 of the zalcitabine in a solution of Briton-Robinson or Clark-Lubs buffer. Scan rate: 20 mV/s. Amplitude: 50 mV.
pH |
|
|
---|---|---|
2.0 | 291 | 1190 |
2.5 | 307 | 1210 |
3.0 | 299 | 1230 |
3.5 | 260 | 1250 |
4.0 | 236 | 1270 |
4.5 | 222 | 1300 |
5.0 | 54 | 1360 |
5.5 | 70 | 1390 |
A new experiment was realized for a better comprehension of zalcitabine mechanism on the mercury surface. When an aqueous zalcitabine (10 mg · L−1) or cytosine solution is electrolyzed, the formation of ammonia could be observed in cases. The presence of ammonia was proved by Nessler’s reagent forming a brown coloration or precipitate. These results and the similar structure of zalcitabine with that of cytosine are indicative that the reduction of the zalcitabine mechanism could be practically the same as proposed previously for cytosine (Figure
Mechanism of cytosine reduction [
Differential pulse voltammograms obtained for solutions of increasing zalcitabine concentration, 10.0–28.0 mg · L−1 (a–e). Other conditions as in Table
Figure
The developed differential pulse voltammetric method was validated according to international guidelines for bioanalytical methods including stability of analyte, determination of specificity and selectivity, calibration curve, detection and determination limits, accuracy, and intraday precision [
For verification of zalcitabine stability, aliquots from stock solution of zalcitabine (229 mg · L−1) in 2 M HCl at 25°C, prepared two weeks previously, were added in an electrochemistry cell containing the 0.2 M Clark-Lubs buffer solution (pH 2.0) and analyzed by differential pulse voltammetry. The voltammetric results obtained were compared with those from the stock solution of zalcitabine measured immediately after its preparation.
The specificity and selectivity studies describe the extent to which a method uniquely reacts to a selected element. Seven samples were prepared, in parallel, containing a matrix (e.g., cellulose, starch glycolate sodium, magnesium stearate, hydroxypropylmethylcellulose, titanium dioxide and polyethylene glycol) with 210 mg · L−1 zalcitabine (group 1) while seven samples containing purified water with 210 mg · L−1 zalcitabine (group 2) were analyzed. The results obtained were compared using
The major sources of interferences would likely be coexisting ions and organic surfactants. The presence of these species could result in either new reduction peaks or the overlap with the zalcitabine peak thus interfering with the measurement. If this is the case, then selectivity studies must be performed in order to investigate the effect of potentially interfering ions and compounds. The presence of zidovudine (AZT) and lamivudine (3TC) compounds were investigated as possible interferent in the zalcitabine (100 mg · L−1 in pharmaceutical form) determination. The presence of lamivudine (3TC) enhanced the zalcitabine peak. The zidovudine (AZT) yields a well-defined reduction peak at −0.96 V (far zalcitabine peak) and lamivudine (3TC) at −1.16 V (near zalcitabine peak) versus Ag/AgCl during forward cathodic scan. The effect of Fe3+, Cu2+, Zn2+, Li+, Pb2+, Cd2+, Ni2+, Co2+, and Cr3+ ions in the free zalcitabine solution was also verified. Concentrations of 100 mg · L−1, Fe3+, Cu2+, Zn2+, Li+, Pb2+, Cd2+, Ni2+, Co2+, and Cr3+ did not interfere with the determination of 23 mg · L−1 zalcitabine.
Another interest in studying the presence of ions in the development of this methodology for the determination of zalcitabine was to verify the possibility of complexing zalcitabine with these ions in order to further separate its voltammetric peak potential for better resolution in analytical determinations simultaneously with other drugs antiretrovirals, but no signs of formation of these complexes were detected.
Although voltammetry is well known for benefiting from a wide linear concentration range, this parameter was evaluated by checking the linearity from a set of standard zalcitabine solutions at five levels of concentrations. The differential pulse voltammograms from −1.00 to −1.25 V for each standard solution were obtained with the increasing addition of five aliquots (4.50 mg · L−1) of zalcitabine concentration (other conditions: Clark-Lubs buffer, pH 2.0) as supporting electrolyte, a scan rate of 20 mV s−1 and a pulse amplitude of 50 mV. The linearity results were analyzed by linear regression and the homoscedasticity by the Cochran test. Using the same conditions the detection and determination limits of zalcitabine in pharmaceutical form were experimentally determined as the lower concentration detected or measured (results in Table
Results obtained from validation of the voltammetric method.
Validation parameters | Results |
---|---|
Selectivity | Satisfactory (in accordance with the |
Linearity | 10.0–28.0 mg/L |
Detection limit | 2.08 mg/L |
Quantification limit | 4.12 mg/L |
Repeatability | RSD = 0.82% |
Intraday precision (different days) | Satisfactory (in accordance with the Cochran, |
Accuracy (recovery) | 100.95% |
Robustness | Only to purge time variations (110 or 130 seconds) |
Repeatability was calculated using the current peak of seven successive measurements from independent sets of standard zalcitabine solution. These results were obtained in the same laboratory by the same operator using the same equipment in a short interval of time. The lower relative standard deviations obtained could also be attributed to the use of a new drop of a reproducible area of a mercury electrode in each run (results in Table
Intra-day precision of the method was evaluated by assaying seven replicate samples of zalcitabine (150 mg · L−1) by the same operator using the same equipment in a long interval of time (two days). The results were available using the Cochran test (to check the variance homogeneity in each day),
The accuracy was determined by the addition of zalcitabine standard solution at three different concentrations. The used concentrations were near the quantification limit and at medium and maximum points of the calibration curve. The mean of the recovery was calculated using the results obtained from the multiple determinations. After method development and validation were realized, the following aspects of robustness were investigated: the effect of pH (1.5, 2.0, and 2.5) and purge time (110, 120, and 130 seconds). The “
The optimized voltammetric method was then used for the determination of zalcitabine in medicament. Twenty aliquots from this sample were, in parallel, analyzed by voltammetry. These results are shown in Table
Results obtained in the medicament.
Medicament | Measured quantities (mg/cap.) | Declared quantities (mg/cap.) | Standard deviation | Relative standard deviation (%) | Quantities obtained (%) |
---|---|---|---|---|---|
Zalcitabine | 0.758 | 0.750 | 0.006216 | 0.82 | 101.07 |
The differential pulse voltammetry proved to be a satisfactory method for the determination of zalcitabine in an acid medium (pH 2.0). Accordingly, the reduction zalcitabine peak potential appears at −1.18 V using drops of mercury as the working electrode. The application of this technique for the analysis of pharmaceutical formulations containing this substance produced statistically valid results, indicating that the voltammetric method may be an alternative in these analytical procedures. Various parameters studied, such as selectivity, linearity, precision, LOD, LOQ, accuracy, and robustness, showed also satisfactory results. In our laboratory we have been investigating the utilization of preadsorptive voltammetric techniques to the improvement of sensitivity and selectivity in the zalcitabine metabolites determination. The same voltammetric procedure also can be extended to many other medicaments used in the treatment of human immunodeficiency virus (HIV) infection: (i) the nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine, didanosine, stavudine, lamivudine, abacavir, and emtricitabine; (ii) the nucleotide reverse transcriptase inhibitor (NtRTI) tenofovir disoproxil fumarate; (iii) the nonnucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, delavirdine, and efavirenz; (iv) the protease inhibitors saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, and atazanavir [
The authors gratefully acknowledge the Fiocruz, CNPq, and CAPES of the Government of Brazil and PUC-Rio for their support of this work.