Validation of a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the simultaneous determination of existing and new antiretroviral compounds

Clinical pharmacokinetic studies of antiretrovitals require accurate and precise measurement of plasma drug concentrations Here we describe a simple. fast and sensitive HPLC-MS/MS method for determination oft he commonly used protease inhibitors (PI) amprenavir, atazanavir, daiunavir, lopinavir, ritonavir, saquinavir and the non-nucleoside reverse transcriptase inhibitor(NNRTI) nevirapine, as well as the more recent antiretrovirals, the CCRS antagonist maraviroc and the "second generation" NNRTI etravirine and rilpivirine. An internal standard (quinoxalone: QX) was added to plasma aliquots (100 mu l) prior to protein precipitation with acetonitrile (500 mu l) followed by centrifugation and addition of 0.05% formic acid (200 mu l) to the supernatant Chromatographic separation was achieved using a gradient (acetonitrile and 0.05% formic acid) mobile phase on a reverse-phase C(18) column Detection was via selective reaction monitoring (SRM) operating in positive ionization mode on a triple-quadrupole mass spectrometer All compounds eluted within a 5 min run time. Calibration curves wet e validated over concentration ranges reflecting therapeutic concentrations observed in HIV-infected patients from pharmacokinetic data reported in the literature Correlation coefficients (r(2)) exceeded 0 998 Inter- and intra-assay variation ranged between 1% and 10% and % recovery exceeded 90% for all analytes The method described is being successfully applied to measure plasma antiretroviral concentrations from samples obtained from clinical pharmacokinetic studies (C) 2010 Elsevier B V. All rights reserved