Validation of high-performance liquid chromatography methods for determination of zidovudine, stavudine, lamivudine and indinavir in human plasma.

OBJECTIVE Simple methods for the determination of zidovudine (AZT), stavudine (d4T), lamivudine (3TC) and indinavir (INV) in human plasma by reversed-phase liquid chromatography (HPLC) with UV detection were described and validated. METHOD Solid-liquid extraction procedures were applied to the samples prior to analysis. Chromatography was performed on a C-18 analytical columns and the retention time ranged from 6.8 to 8.0 min for stavudine, 7.5 to 9.0 min for lamivudine, 11.2 to 11.9 min for zidovudine and indinavir. Four methods were validated for specificity, inter-and intra-assay precision and accuracy, absolute recovery and stability. RESULTS Analytical curve ranged from 10-1600 ng/ml for stavudine, 50-3200 ng/ml for lamivudine, 0.05-5.0 microg/ml for zidovudine and 0.1-10.0 microg/ml for indinavir. Analytes stability during sampling processing and storage were established. Extraction recoveries are higher than 89% for all formulations. CONCLUSIONS These methods proved to be simple, accurate and precise, and are currently in use in our laboratory for the quantitative analysis of antiretrovirals products in plasma, and for further pharmacokinetics and bioequivalence studies.