Sensitive and rapid method for the simultaneous quantification of the HIV-protease inhibitors indinavir, nelfinavir, ritonavir, and saquinavir in human plasma by reversed-phase liquid chromatography

A rapid, sensitive, and specific liquid chromatography method for the simultaneous determination of four protease inhibitors (indinavir, nelfinavir, ritonavir, and saquinavir) in human plasma is described. After a liquid-liquid extraction with terbutyl methyl ether and a sequential washing of the reconstituted sample with hexane, protease inhibitors are separated on a phenyl column using a simple binary mobile phase of ammonium acetate buffer:acetonitrile (48:52) (pH = 7.5) with an ultraviolet detection at 260 nm. The standard curves are linear in the range 0.025-1 mug/mL for saquinavir. 0.1-4 mug/mL for indinavir and nelfinavir, and 0.25-10 mug/mL for ritonavir, with an average recovery ranging from 79% to 99%, and with both low interday and intraday coefficients of variation (<15%). This assay is simple, rapid (15-minute interval between runs). and useful for therapeutic monitoring of the protease inhibitors on a routine basis.