Simultaneous determination of the antiretroviral agents: amprenavir, lopinavir, ritonavir, saquinavir and efavirenz in human peripheral blood mononuclear cells by high-performance liquid chromatography–mass spectrometry

A selective and accurate assay for the simultaneous quantitation of four protease inhibitors (PIs) (amprenavir (APV), lopinavir (LPV), ritonavir (RTV) and saquinavir (SQV)) and a non-nucleoside reverse transcriptase inhibitor (NNRTI) (efavirenz, EFV) in human peripheral blood mononuclear cells using high-performance liquid chromatography–mass chromatography (LC/MS) has been developed and validated. After liquid–liquid extraction, the antiretroviral agents were separated within 15 min. The calibration curves of each drug showed a good linearity in a range of concentration between 2 and 200 ng/3 × 106 cells for amprenavir, lopinavir, efavirenz, 1.60 and 128 ng/3 × 106 cells for ritonavir and saquinavir. Mean intra- and inter-assay coefficients of variation over the ranges of the standard curves were less than 15% and mean extraction recoveries ranged 88.7–112.1%. The limits of quantification were 2 ng/3 × 106 cells for amprenavir, lopinavir, efavirenz, 1 ng/3 × 106 cells for ritonavir and 1.6 ng/3 × 106 cells for saquinavir. This novel LC/MS assay, which provides an excellent method for simultaneous intra-cellular determination of amprenavir, lopinavir, ritonavir, saquinavir and efavirenz in human peripheral blood mononuclear cells, could be successfully applied for therapeutic drug monitoring and pharmacokinetic studies.