Rapid and Simultaneous Determination of Efavirenz, 8-Hydroxyefavirenz, and 8,14-Dihydroxyefavirenz Using LC–MS–MS in Human Plasma and Application to Pharmacokinetics in Healthy Volunteers

摘要

We developed a rapid and sensitive method for determining efavirenz, 8-hydroxyefavirenz, and 8,14-dihydroxyefavirenz in human plasma simultaneously using liquid chromatography–tandem mass spectrometry (LC–MS–MS). Three compounds and ritonavir, an internal standard, were extracted from plasma using ethyl acetate in the presence of 0.1 M sodium carbonate after incubation of β-glucuronidase (500 U). After drying the organic layer, the residue was reconstituted in mobile phase (acetonitrile:20 mM ammonium acetate, 90:10, v/v) and injected onto a reversed-phase C₁₈ column. The isocratic mobile phase was eluted at 0.2 mL min−1. The ion transitions monitored in multiple reaction-monitoring mode were m/z 314 → 244, 330 → 258, 346 → 262, and 721 → 296 for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The retention time is 1.93, 1.70, 1.52, and 1.82 min for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The coefficients of variation of the assay precision were less than 10.7%, and the accuracy was 90–111%. The lower limits of quantification (LLOQ) were 5 ng mL−1 for efavirenz and 8-hydroxyefavirenz. This method was used to measure the plasma concentrations of efavirenz and its metabolites from healthy volunteers after a single 600 mg oral dose of efavirenz. This analytical method is a very rapid, sensitive, and accurate to determine the pharmacokinetic profiles of efavirenz including its metabolites.