Simple and rapid high-performance liquid chromatographic method for nelfinavir, M8 nelfinavir metabolite, ritonavir and saquinavir assay in plasma

A simple reversed-phase liquid chromatographic method has been developed to determine protease inhibitors concentrations in plasma. Plasma samples (250 μl) containing protease inhibitors were prepared by a simple deproteinization (recovery: 92, 91, 91 and 90.5% for ritonavir, saquinavir, nelfinavir and M8 nelfinavir metabolite, respectively). Chromatography was accomplished using a Hypersil octadecylsilyl column (100×4.6 mm I.D.) and a mobile phase composed of acetonitrile, tetrahydrofuran and dihydrogenophosphate buffer (pH 4) (32:10:58, v/v). Ultraviolet detection at 210 nm was used. The limit of detection was 200 ng/ml for ritonavir, saquinavir, nelfinavir and M8 nelfinavir metabolite. Calibration curves were linear up to 20 000 ng/ml, with correlation coefficients better than 0.997 for all compounds. Intra- and inter-day coefficients of variation of the assay were ≤6% for all compounds. This method was used to analyse protease inhibitors plasma concentrations after oral administration within the framework of therapeutic drug monitoring and pharmacokinetic studies in AIDS patients.