An efficient HPLC method for the quantitative determination of atazanavir in human plasma suitable for bioequivalence and pharmacokinetic studies in healthy human subjects

Abstract

An efficient HPLC method for the determination of atazanavir in human plasma has been developed and validated. A relatively simple mobile phase consisting of acetonitrile–ammonium formate buffer (pH 3; 10 mM) (45:55, v/v) was pumped at a low flow rate of 0.3 ml/min through a reverse phase Phenomenex ® Luna C18 (2) (5 μm, 150 mm × 2.0 mm i.d.) column maintained at 30 °C. Diazepam was used as an internal standard and the eluent was monitored at 210 nm. The major advantage of this method over previously reported procedures is that the narrow-bore HPLC column used resulted in relatively short retention times for the internal standard (6.8 min) and atazanavir (8.3 min) with excellent peak resolution and associated reduction in solvent usage. Sample preparation involved liquid–liquid extraction using 400 μl plasma treated with sodium carbonate (2 M) and extracted with a mixed organic solvent consisting of ethyl acetate–n-hexane (50:50, v/v). The organic layer was removed and evaporated to dryness under nitrogen. Samples were reconstituted in mobile phase (100 μl) and 20 μl was injected onto the column. The procedures were validated according to international standards with good reproducibility and linear response with correlation coefficients ( r) consistently ≥0.999. The intra- and inter-day accuracies were 97.1 ± 5.04 and 98.0 ± 11.3 respectively at the LLOQ and between 101 ± 4.48% and 104 ± 2.09% for the QC samples. The intra- and inter-day precision were ≤11.6% RSD at the LLOQ and ≤6.78% RSD across the entire QC concentration range. Mean recovery based on high, medium and low quality control standards ranged between 94.4 ± 1.07% and 100 ± 2.22%. Plasma samples were evaluated under short-term (ambient temperature for 6 h) and long-term (−10 ± 2 °C for 2 months) storage conditions and were found to be stable. The method described is efficient and has the necessary accuracy and precision for the rapid quantitative determination of atazanavir in human plasma and is thus highly suitable for use in pharmacokinetic/bioavailability/bioequivalence studies in healthy human subjects.