A validated LC-MS/MS method for the simultaneous determination of BMS-791325, a hepatitis C virus NS5B RNA polymerase inhibitor, and its metabolite in plasma

BMS-791325 is a hepatitis C virus (HCV) non-structural protein 5B (NS5B) RNA polymerase inhibitor that is being developed for the treatment of HCV infection. A rugged and accurate LC-MS/MS method was developed and validated for the quantitation of BMS-791325 and its metabolite, BMS-794712, in rat and dog plasma. This method utilized stable-isotope labeled [D6]-BMS-791325 and [13CD3]-BMS-794712 as internal standards. The samples were extracted using liquid-liquid extraction with n-butyl-chloride. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column (2.1 mm × 50 mm, 3.0 μm). Analytes and their stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curves, which ranged from 5.00 to 2000 ng/mL for BMS-791325 and from 1.00 to 400 ng/mL for BMS-794712, were fitted to a 1/x2 weighted linear regression model. For both species, the intra-assay precision was within ±4.3% CV, the inter-assay precision was within ±6.2% CV, and the assay accuracy was within ±10.8% of the nominal values for BMS-791325 and BMS-794712. The validated method was successfully applied to support pre-clinical toxicokinetic studies.