A Sensitive and Specific Liquid Chromatography/Tandem Mass Spectrometry Method for Quantification of Nevirapine and Its Five Metabolites and Their Pharmacokinetics in Baboons

A highly sensitive and specific LC-MS/MS assay was developed and validated to quantify nevirapine(NVP) and its five metabolites(2-, 3-, 8-, 12-hydroxyl NVP [OHNVP] and 4-carboxyl NVP [CANVP]) simultaneously in baboonserum and the assay was used to characterize their pharmacokinetic studies of an oral-dose escalation study in baboon. The lower limit of quantification (LLOQ) for NVP and its four hydroxyl nevirapine metabolitesis 1.0 ng/mL, at least 10-fold higher than reported methods. The LLOQ for 4-CANVP is 5.0 ng/mL. The between-run and within-run precisions and accuracies at the following quality control concentrations (1, 5, 50 and 500 ng/mL) were evaluated in baboonserum with less than 14% variation and 93% to 114% accuracies (n=6) except LLOQ for 2-OHNVP, which had an accuracy of 115.8% for between-run validation. The pharmacokinetics of NVP and its five metabolitesin non-pregnant baboonsby single dose escalation study were also profiled. The major metabolitesdetected were 4-CANVP and 12-OHNVP. 3-OHNVP and 2-OHNVP were the minor metaboliteswith only a trace amount of 2-OHNVP detected in some PK samples. No 8-OHNVP was observed in all of the PK samples. In addition, the fragmentation for the four hydroxyl metaboliteisomers was also discussed.