High-throughput simultaneous determination of the HIV protease inhibitors indinavir and L-756423 in human plasma using semi-automated 96-well solid phase extraction and LC-MS/MS

A method for the simultaneous determination of the HIV protease inhibitors indinavir and L-756313. in human plasma has been developed. Plasma samples (0.5 ml) were extracted using a 3M Empore(TM) 96-well plate in the mixed ph ase cation exchange (MPC) format. The extraction method was automated throu gh the application of both the Packard 204DT and TOMTEC Quadra 96 work stat ions, and the resulting extracts were analyzed using a PE-Sciex API-3000 LC -MS/MS with a heated nebulizer interface (500 degreesC). The assay was line ar in the concentration range 1-2500 ng/ml for indinavir and 5-2500 ng/ml f ur L-756423 when 0.5-ml aliquots of plasma were extracted. Recoveries of in dinavir and L-756423 were greater than 76 and 80%, respectively, over the c alibration curve range when using the described sample preparation method. Within-batch precision and accuracy for the quantitation of indinavir over the range 1-2500 ng/ml were 5.4% R.S.D. or less and within 4.0%. respective ly. Within-batch precision and accuracy for the quantitation of L-756423 ov er the range 5-2500 ng/ml were 5.3% R.S.D. or less and within 3.4%, respect ively. Interbatch variability for the analysis of indinavir QC samples at l ow (3 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) were 3.2, 2.9, and 1 .9%, respectively. Interbatch variability for the analysis of L-756323 QC s amples at low (15 ng/ml), middle (250 ng/ml) and high (2250 ng/ml) concentr ation were 2.0, 2.5, and 3.3%, respectively. The validated assay was used i n support of human clinical trials, (C) 2000 Elsevier Science B.V. All righ ts reserved.