Simultaneous determination of the HIV-protease inhibitors indinavir, nelfinavir, saquinavir and ritonavir in human plasma by reversed-phase high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method has been develop ed for the simultaneous determination of the four licensed HIV-protease inh ibitors indinavir, nelfinavir, saquinavir and ritonavir. An aliquot of 500 mu l plasma, spiked with internal standard, was extracted with 0.5 ml 0.1 M NH4OH and 5 ml methyl tert-butyl ether. After evaporating, the residue was dissolved in eluent consisting of acetonitrile-50 mM phosphate buffer, pH 5.63 (40:60, v/v). Subsequently, the eluent was washed with hexane. Chromat ography was performed using a C-18 reversed-phase column and gradient eluti on with a linear increase of acetonitrile from 36 to 66%. Ultraviolet detec tion at 215 nm was used. Linearity of the method was obtained in the concen tration range of 45-30 000 ng/ml for all four analytes. The method was vali dated extensively and stability tests under various conditions were perform ed. The assay is now in use to analyse plasma samples from patients treated with (combinations of) HIV-protease inhibitors. (C) 1999 Elsevier Science B.V. All rights reserved.