Antiretrovirals: Simultaneous determination of five protease inhibitors and three nonnucleoside transcriptase inhibitors in human plasma by a rapid high-performance liquid chromatography-mass spectrometry assay

An analytical technique using liquid chromatography (LQ coupled with electr ospray-mass spectrometry (ESI-MS) has been developed for the simultaneous d etermination of five protease inhibitors (PIs): saquinavir, indinavir, rito navir, nelfinavir, and amprenavir; and three non-nucleoside reverse transcr iptase inhibitors (NNRTIs): nevirapine, delavirdine, and efavirenz, in huma n plasma. This assay allows the elution and identification of these drugs i n a single run (10 minutes) using a linear gradient with water and acetonit rile. The procedure involves liquid-liquid extraction. High-performance liq uid chromatography (HPLC) separation was achieved on a C18 reversed-phase c olumn, with a linear gradient elution followed by mass spectrometry detecti on. The calibration curves, obtained by automatic process peak area integra tion, show a good linearity in a range of concentrations between 20 and 10, 000 ng/mL (40-10,000 ng/mL for efavirenz). The limit of detection was appro ximately 10 ng/mL for seven drugs (25 ng/mL for efavirenz). The coefficient s of variation (CV) were always less than 15% for both intraday and interda y precision for each compound. The recovery of the eight drugs ranged from 88.5% to 100%. This novel LC/ESI-MS assay provides an excellent method for simultaneous quantitative monitoring of different components of the highly active antiretroviral treatments (HAARTs) in patients treated simultaneousl y with PIs and NNRTIs, and it has been successfully applied to therapeutic drug monitoring and pharmacokinetic studies.