title: Determination of warfarin alcohols by ultra-high performance liquid chromatography-tandem mass spectrometry: application to in vitro enzyme kinetic studies.
abstract: A sensitive, accurate, and reproducible ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for determination of warfarin and its alcohol metabolites (RS/SR- and RR/SS-warfarin alcohol) in 10mM Tris-HCl incubation buffer (pH 7.4). Sample preparation involved acidification with 4% formic acid, followed by liquid-liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved using a Hypersil Gold C18 (2.1mm×100mm, 1.9μm) analytical column with gradient elution of solvent A (water containing 0.01% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The flow rate was 0.4mL/min and the total run time was 5min. Detection of analytes was performed using heated electrospray ionization (negative mode) and selected reaction monitoring. Excellent linearity was observed for all analytes over the standard curve concentration ranges of 100-10,000ng/mL for warfarin, and 0.5-250ng/mL for warfarin alcohols. The intra- and inter-day accuracy and precision for analytes were within ±10.0%. Excellent recovery and negligible matrix effects were observed. The method is robust, sensitive, accurate and reproducible, and was successfully applied to in vitro enzyme kinetic studies of warfarin.
authors.affiliation: Center for Clinical Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States; Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States.
authors.forename: Osama Y
authors.initials: OY
authors.lastname: Alshogran
authors.affiliation: Center for Clinical Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States; Department of Pharmacy and Therapeutics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Ocque
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States.
authors.forename: Jielu
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States.
authors.forename: Billy W
authors.initials: BW
authors.lastname: Day
authors.affiliation: Service de Néphrologie et Centre de Recherche, Hôpital Maisonneuve-Rosemont, Montréal, Québec, Canada.
authors.forename: François A
authors.initials: FA
authors.lastname: Leblond
authors.affiliation: Service de Néphrologie et Centre de Recherche, Hôpital Maisonneuve-Rosemont, Montréal, Québec, Canada; Département de Pharmacologie, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Vincent
authors.initials: V
authors.lastname: Pichette
authors.affiliation: Center for Clinical Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States; Department of Pharmacy and Therapeutics, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States. Electronic address: nolin@pitt.edu.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Nolin
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014859:Warfarin / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014859:Warfarin
keywords: Enzyme kinetics; LC–MS; Reduction; Warfarin; Warfarin alcohols