Sensitive liquid chromatographic assay for amprenavir, a human immunodeficiency virus protease inhibitor, in human plasma, cerebrospinal fluid and semen

A sensitive bio-analytical assay for amprenavir, a human immunodeficiency virus protease inhibitor, based on reversed-phase liquid chromatography and fluorescence detection, is reported. The analyte is extracted from the matrix, plasma, cerebrospinal fluid (CSF) or semen, with chloroform using propyl-p-hydroxybenzoate as an internal standard. After centrifugation, evaporation of the organic phase and reconstitution in the eluent, the sample is injected into the chromatograph. The analyte is detected spectrofluorometrically at 270 and 340 nm for excitation and emission, respectively. The method has been validated in the 1-1000 ng/ml range for a 50-microl volume of plasma and in the 0.5-50 ng/ml range for a 100-microl volume of CSF and semen. The lower limit of quantification was 0.5 ng/ml in CSF and 1 ng/ml in both plasma and semen. Precision and accuracy both meet the current requirements for a bio-analytical assay and are <15% in the validated ranges. The assay was successfully used to obtain a concentration-time curve of amprenavir in plasma.