Simultaneous quantitative determination of zidovudine and nevirapine in human plasma using isocratic, reverse phase high performance liquid chromatography

Purpose: To develop a sensitive and rapid reverse phase high performance liquid chromatography (HPLC) method for the measurement of the levels of zidovudine (ZVD) and nevirapine (NVP) in human plasma. Methods: Standard stock solutions for HPLC analysis were prepared by dissolving ZVD and NVP in methanol. In the HPLC measurement, sample detection was carried out at 246 nm using an ultraviolet (UV)-photo diode array (PDA) detector. Plasma sample pretreatment consisted of protein precipitation extraction with methanol. The compounds were separated using a mobile phase consisting of a pH 3.0 solution (obtained by adjusting the pH of water with orthophosphoric acid): acetonitrile (73:27 v/v) on a Phenomenex LUNA C(18), column (250x4.6 mm i.d., 5 mu m) at a flow rate of 0.9 mL min(-1). The total run time for the assay was 10.2 min. The method was validated over the range of 300-9600 ng mL(-1) and 200-6400 ng mL(-1) for ZVD and NVP, respectively. Results: The lowest limits of quantification (LLOQ) and of detection (LOD) were 300 and 63 ng mL(-1) for ZVD and 200 and 17 ng mL(-1) for NVP, respectively. The method was found to be accurate, with accuracy ranging from -10.92 to +9.57% and precise, with intra-day, inter-day as well as analyst to analyst precision of 0.68 to 9.38%. Extraction recoveries of the drugs from plasma were 91.39, 95.01, 89.51% for ZVD and 90.93, 93.26, 92.13% for NVP, for LQC (low quality control), MQC (medium quality control) and HQC (high quality control) samples, respectively. Stability data revealed that the drugs were stable in plasma under various test conditions. Conclusion: This assay can be suitably used for the determination of zidovudine (ZVD) and nevirapine (NVP) in human plasma and should be useful in HIV clinical trials and clinical therapeutic drug monitoring (TDM) programs. It would also be potentially useful in the determination of pharmacokinetic profiles and in bioequivalence studies in HIV research.