Determination of an in vivo metabolite of a human immunodeficiency virus protease inhibitor in human plasma by high-performance liquid chromatography with tandem mass spectrometry

A method for the determination of a metabolite of the human immunodeficiency virus protease inhibitor indinavir, in human plasma is described. Isolation of the analyte and the internal standard from plasma was achieved via liquid-liquid extraction with a mixture of isopropanol-chloroform (5:95, v/v). The analytes were chromatographed under reversed-phase conditions on a Waters Symmetry C-8 column. A Sciex API III+ tandem mass spectrometer equipped with a heated nebulizer was used as a detector and was operated in the positive ion mode. Multiple reaction monitoring using the precursor-product ion combinations of mit 523.4-->273.4 and 512.4-->345.2 was used to quantify analyte and internal standard, respectively. The method was validated in the concentration range of 5-500 ng/ml plasma with adequate assay precision and accuracy. The assay was used to analyze samples collected during drug interaction studies of indinavir.