Quantitation of five nevirapine oxidative metabolites in human plasma using liquid chromatography-tandem mass spectrometry

摘要

A multiple-reaction-monitoring LC/MS/MS method for the analysis of nevirapine oxidative metabolites, 2-hydroxynevirapine, 3-hydroxynevirapine, 8-hydroxynevirapine, 12-hydroxynevirapine, and 4-carboxynevirapine, in human plasma was developed and validated. The metabolites were isolated from 50 mu L heparinized plasma by enzymatic hydrolysis of the glucuronide conjugates to the free metabolite followed by protein precipitation with acetonitrile. Peaks were quantitated at 3.03 min for the 4-carboxynevirapine metabolite, at 3.72, 4.27, 5.27, and 5.73 min for the positional 2-hydroxynevirapine, 12-hydroxynevirapine, 3-hydroxynevirapine, and 8-hydroxynevirapine metabolites, respectively, and 2.30 min for the internal standard, pirenzepine. The assay was accurate and precise based on assay validation controls over the nominal range of 0.010-1.0 mg/L. The average accuracy at the lowest concentration quality control (QC) sample was 16% (difference from theoretical value) for 8-hydroxynevirapine, all others were closer to their known respective standards. Within- and between-day precisions were within 12% for quality control samples for all five metabolites. Repetitive thawing and freezing did not have an effect on any metabolite through a minimum of three cycles. Thawed samples, remaining in plasma for 4 It before extraction, were within 5% of theoretical value. Stability of the extracted samples on the autosampler at room temperature was evaluated for 48 h and was observed to be within 12% of a fresh analytical sample for 2-hydroxynevirapine and 3-hydroxynevirapine; other metabolites were within 6% of theoretical value. The utility of the analytical method was demonstrated using trough steady-state plasma samples collected from 48 patients in a hepatic impairment study. (C) 2007 Elsevier B.V. All rights reserved.