Quantification of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in peripheral blood mononuclear cell lysate using liquid chromatography coupled with tandem mass spectrometry

For pharmacokinetic monitoring, measurement of antiretroviral agents in plasma is the gold standard. However, human immunodeficiency virus protease inhibitors (PIs) or non-nucleoside reverse transcriptase inhibitors (NNRTIs) exert their action within the infected cell. Cell-associated concentrations may therefore more adequately reflect therapy outcome. Therefore, for the quantification of nine PIs (amprenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir), 1 active PI metabolite (nelfinavir M8) and 2 NNRTIs (efavirenz and nevirapine) in lysate of peripheral blood mononuclear cells (PBMCs) an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Analytes were extracted from a PBMC pellet by means of a one-step extraction with 50% methanol containing the internal standards D6-indinavir, D5-saquinavir, 13C6-efavirenz and dibenzepine. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. The analytical run time was 10min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 1-500ng/mL in PBMC lysate for all analytes. The method was proven to be specific, accurate, precise and robust. The mean precision and accuracy was less than +/-12% at all concentration levels. Using the developed assay and a previously developed assay for these analytes in plasma, the relationship between plasma and intracellular pharmacokinetics and their relationship with therapy outcome can now be determined.