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.
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 nevirapine 4-carboxylic acid (4-CANVP), in baboon serum was developed and validated. Nevirapine, 2-OHNVP, 4-CANVP, and the internal standard, hesperetin, were extracted from baboon serum with ethyl acetate. Components in the extract were separated on a 50 x 2.1 mm Aquasil C(18) 5 microm 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 baboon serum, 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.
Liu
Zhongfa
Z
College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA. liu.550@osu.edu
Fan-Havard
Patty
P
Xie
Zhiliang
Z
Ren
Chen
C
Chan
Kenneth K
KK
D000818:Animals; D019380:Anti-HIV Agents / Q000097:blood / Q000493:pharmacokinetics; D001274:Atmospheric Pressure; D001774:Blood Chemical Analysis / Q000379:methods*; D002264:Carboxylic Acids / Q000097:blood* / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008657:Metabolic Clearance Rate; D019829:Nevirapine / Q000097:blood* / Q000493:pharmacokinetics*; D010215:Papio; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D019380:Anti-HIV Agents; D002264:Carboxylic Acids; D019829:Nevirapine