A liquid chromatography/atmospheric pressure ionization tandem mass spectrometry quantitation method for nevirapine and its two oxidative metabolites, 2‐hydroxynevirapine and nevirapine 4‐carboxylic acid, and pharmacokinetics in baboons

2007
A rapid highly sensitive and specific electrospray ionization (ESI) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantification of nevirapine(NVP) and its two metabolites, 2-hydroxynevirapine (2-OHNVP) and nevirapine4-carboxylic acid (4-CANVP), in baboonserum was developed and validated. Nevirapine, 2-OHNVP, 4-CANVP, and the internal standard, hesperetin, were extracted from baboonserum with ethyl acetate. Components in the extract were separated on a 50 × 2.1 mm Aquasil C18 5 µm stainless steel column by isocratic elution with 40% acetonitrile/0.1% formic acid at a flow rate of 0.2 mL/min. The liquid flow was passed through a pre-source splitter and 5% of the eluant was introduced into the atmospheric pressure ionization (API) source. The components were analyzed in the multiple-reaction monitoring (MRM) mode as the precursor/product ion pair of m/z 267.2/226.2 for NVP, 283.0/161.2 for 2-OHNVP, 297.2/279.2 for 4-CANVP, and 303.2/177.2 for hesperetin. Linear calibration curves were obtained in the range of 1–1000 ng/mL for NVP and 2-OHNVP and 5–1000 ng/mL for 4-CANVP, using 0.2 mL baboonserum, respectively. The within-day and between-day precisions were <10% for NVP and 2-OHNVP, and <11.5% for 4-CANVP. Due to the similar structures and fragmentation patterns of 2-OHNVP and 3-OHNVP, it is not expected that the LC/MS/MS can differentiate 2-OHNVP and 3-OHNVP and they were assayed as a composite. The method was applied to a single-dose escalation study of NVP in non-pregnant baboons(Papio anubis) to characterize the pharmacokinetics of NVP, 2-OHNVP plus 3-OHNVP, and 4-CANVP, and to determine the appropriate dose necessary to achieve comparable peak serum concentration of NVP as reported in healthy human adults. Copyright © 2007 John Wiley & Sons, Ltd.
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