Simultaneous HPLC Assay for Quantification of Indinavir, Nelfinavir, Ritonavir, and Saquinavir in Human Plasma

Abstract

HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed. Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 microL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 x 3 mm i.d.) at 40 degrees C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma. The extraction recovery was 87-92%. Within-run accuracy for quality-control samples was 6-8%, with CVs of 2-8%. Limits of quantification were 40-50 microg/L for indinavir, nelfinavir, and ritonavir, and 20 microg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r(2) >0.94) between methods. Larger variations at concentrations >4000 microg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined. A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications.