Simultaneous Determination of Lamivudine, Zidovudine and Abacavir in Tablet Dosage Forms by RP HPLC Method

: A simple, accurate and reproducible RP-HPLC method has been developed for the simultaneous determination of lamivudine, zidovudine and abacavir in tablet dosage forms. Chromatography was carried out on a HiQ Sil C 18 V column using a mobile phase consisting of 0.01 M potassium dihydrogen ortho -phosphate (pH 3.0) and methanol (55:45 v/v) at a flow rate of 0.8 mL/min. The detection was made at 272 nm and stavudine was used as the internal standard for this study. The retention times for lamivudine, abacavir and zidovudine were found to be 3.8, 6.3, 8.1 min. respectively. The calibration curves were linear over the range 5-250 µg/mL for both zidovudine and abacavir and 5-140 µg/mL for lamivudine. The proposed method was validated as per ICH and USP guidelines and it was found suitable for the routine quality control analysis of the drugs in tablet dosage forms.


Introduction
Antiretroviral drugs like nucleoside reverse transcriptase inhibitors, non nucleoside reverse transcriptase inhibitors, and protease inhibitors are essential in the management of HIV infection. The synthetic nucleoside reverse transcriptase inhibitor analogues abacavir, lamivudine and zidovudine form one of the fixed dosage combinations used in the effective management of HIV [1][2] . Abacavir, chemically known as [(1R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl}-2-cyclopentene]-1-methanol, is a carbocyclic synthetic analogue. Its active metabolite carbovir triphosphate, an analogue of deoxyguanosine-5'-triphosphate (d GTP), inhibits the activity of HIV-1 reverse transcriptase both by competing with the natural substrate dGTP and by its incorporation into viral DNA. The active triphosphate metabolites zidovudine (3azido-3-deoxythymidine) and lamivudine (4-amino-1-((2R,5S-2-(hydroxymethyl)-1,3oxathiolan-5-yl) pyrimidin-2-(1H)-one) act against HIV by inhibition of reverse transcriptase via DNA chain termination after incorporation of the nucleotide analogue. A literature survey reveals the report of a few analytical methods for the determination of these drugs individually in serum samples and in their dosage forms. Methods for the simultaneous determination of lamivudine and zidovudine in biological samples and in pharmaceutical preparations were also reported 3-6 . A reported method for the determination of the lamivudine, zidovudine and abacavir in tablets, human serum and in drug dissolution studies employs a complex buffer system (methanol, water, phosphate buffer) 7 . The authors now propose a simple, precise and accurate method for the simultaneous determination of lamivudine, ziduvudine and abacavir in tablet dosage forms employing a mobile phase consisting of 0.01 M potassium dihydrogen ortho-phosphate (pH 3.0) and methanol to achieve good peak shapes with appreciable resolution.

Chromatographic conditions
A Jasco HPLC instrument equipped with a HiQ Sil C18 V analytical column (250 X 4.6 mm; 5µ), a PU 2080 pump, a Rheodyne 7125 sample injector with a 20 µL loop and a UV-2075 detector was employed for this analysis.
A mobile phase consisting of a mixture of 0.01 M potassium dihydrogen orthophosphate (PH 3.0) and methanol in a ratio of 45:55 (v/v) was prepared and filtered through a 0.45µ membrane filter and degassed by helium sparging before use.

Drug samples
Reference standard samples of abacavir, zidovudine and lamivudine and internal standard stavudine were procured from Aurobindo Pharma Ltd., Hyderabad. A commercial sample trizivir tablets (Glaxo-smithkline) containing zidovudine (300 mg), lamivudine (150 mg) and abacavir (300 mg) were purchased from local market.

Drug and internal standard solutions
Stock solutions of lamivudine, zidovudine and abacavir were prepared by dissolving 25 mg of each drug in separate 25 mL volumetric flasks in methanol with sonication for about 15 min. From the individual stock solutions working standard solutions were prepared in a concentration range of 5-300 µg/mL for both zidovudine and abacavir and 5-200 µg/mL for lamivudine.
A stock solution of internal standard, stavudine, was prepared by dissolving 25 mg of the drug in a 25 mL volumetric flask in methanol with sonication for about 15 min. From this a working standard solution of 20 µg/mL was prepared with mobile phase.

Calibration curve
Separate standard calibration curves were constructed for each drug. Different volumes of stock solutions were accurately transferred into 10 mL volumetric flasks to prepare 5-300 µg/mL concentration range for each component. Six replicate solutions in the above range were prepared for each concentration. Suitable aliquot of internal standard solution was added to get a final concentration of 20 µg/mL in all solutions. The calibration lines were constructed by plotting the analyte to internal standard peak area ratio against the concentration. The linear regression parameters of each drug are presented in Table 1. The results are highly reproducible with a correlation coefficient greater than 0.998.

Table 1. Regression characteristics of the proposed HPLC method
Estimation of the drugs from tablet dosage forms Ten tablets of trizivir, containing 300 mg each of abacavir and zidovudine and 150 mg of lamivudine were weighed and finely powdered. A quantity of the powder equivalent to one tablet content was accurately weighed, transferred into a 100 mL volumetric flask and dissolved in the mobile phase by sonication for about 15 min. This solution was filtered through a 0.45µ filter paper. From the filtrate different aliquots were taken in separate 10 mL volumetric flasks. These solutions were spiked with a suitable volume of the internal standard solution, such that the concentration of the internal standard in each solution was 20 µg/mL. The contents of the flasks were made up to the volume with mobile phase and mixed well. Each of these solutions (20 µL) was then injected five times into the column. The mean peak area ratios of the drug to the internal standard of five such determinations for each drug were calculated and the drug contents in the tablets were quantified using the respective regression equations obtained for the reference samples.

Results and Discussion
The present study was carried out to develop a sensitive, precise and accurate HPLC method for the simultaneous determination of lamivudine, zidovudine and abacavir in pharmaceutical dosage forms. In order to effect analysis of the component peaks under isocratic conditions, mixtures of methanol with potassium dihydrogen ortho-phosphate buffer in different combinations were tested as mobile phase on a Hi Q Sil C 18 stationary phase. A binary mixture of methanol and 0.01 M potassium dihydrogen ortho-phosphate (pH 3.0) in a ratio of 70:30 (v/v) was proved to be the most suitable of all the combinations since the chromatographic peaks obtained were better defined and resolved and almost free from tailing. The retention times obtained for lamivudine, abacavir, zidovudine and the stavudine (internal standard) were 3.8, 6.3, 8.1 and 4.6 respectively. A Typical chromatogram showing the separation is given in Figure 1.
The method was validated by determining the linearity, sensitivity, precision and accuracy for each analyte. Six point calibration curve constructed with the working standard dilutions was found to be linear (r 2 >0.998) for each of the analytes in the concentration range of 5-250 µg/mL for zidovudine and abacavir sulphate and in a range of 5-140 µg/mL for lamivudine. The results are presented in Table 1.
Both precision and accuracy were determined with standard quality control samples prepared in triplicates at different concentration levels covering the linearity range. The repeatability and reproducibility are reported as % RSD in Table 2 and minimum variation in the % RSD indicate the present method is precise.
The accuracy of the proposed method was assessed by adding known amount (25, 50 and 75 µg/mL) of the drug to a drug solution of known concentration and subjecting the samples to the proposed HPLC method. All solutions were prepared and analysed in triplicate. The above procedure is adopted for all the three drugs and a high recovery values obtained (   Table 2. Precision of the proposed method. The recovery values of the three drugs from the tablet dosage forms by the developed method range from 99.4 to 100.4 percent ( Table 4). The method specificity was assessed by studying the chromatograms obtained from a mixture of the drugs and the common excipients. The method was found to be specific as none of the excipients interfered with the analytes of interest. Hence, the method was found to be suitable for analysing the commercial antiretroviral formulations.
System suitability parameters were studied with six replicates of standard sample solutions and the parameters are presented in Table 5.

Conclusion
The proposed RP HPLC method is rapid, precise and accurate and can be used for the routine quality control analysis for the simultaneous determination of the above drugs in their tablet dosage forms.