Simultaneous quantitative determination of amprenavir and indinavir in human plasma by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for thr determination of the HIV protease inhibitors amprenavir (Agenerase(infinity)) and indinavir (Crixivan(infinity)) in human plasma is described using a mobile phase consisting of 0.05 M phosphate buffer (adjusted to pH 5.5) - Milli-Q water - acetonitrile (120: 1,080: 800. v/v/v). A solid-phase extraction using C18 extraction columns (Discovery columns 100 mg. 1 ml Supelco) and a liquid-liquid extraction with 0.5 ml hydrogenocarbonate/carbonate buffer (adjusted to pH 10.6) and 6ml methyl ter-butyl ether have been compared. The liquid-liquid extraction has been chosen to be easier and cheaper. The method has been validated over the range of 60 to 3,000 ng/ml for amprenavir and 20 to 3,000 ng/ml for indinavir using a 0.5 ml sample volume. The specificity linearity accuracy and precision have been studied. The limit of detection was respectively for amprenavir and indinavir 15 and 4 ng. and the limit of quantification was 60 and 20 ng/ml. Stability tests under various conditions were performed. This assay can readily he used in 3 hospital laboratory for the routine monitoring of plasma concentrations of amprenavir in HIV-infected patient. The trough plasma concentrations average has been determined in patients treated by amprenavir and indinavir for seven months.