title: Validation and Application of a Simple UHPLC-MS-MS Method for the Enantiospecific Determination of Warfarin in Human Urine.
abstract: A simple and rapid liquid chromatographic-tandem mass spectrometric method has been developed and validated for the enantiospecific determination of R- and S-warfarin in human urine. Warfarin enantiomers were extracted from urine using methyl tert-butyl ether. Chromatographic separation of warfarin enantiomers and the internal standard d5-warfarin was achieved using a Astec Chirobiotic V column with gradient mobile phase at a flow rate of 400 µL/min over 10 min. Detection was performed on a TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with a heated electrospray ionization source. Analytes were detected in negative ionization mode using selected reaction monitoring. Calibration curves were linear with a correlation coefficient of ≥0.996 for both enantiomers over a concentration range of 5-500 ng/mL. The intra- and interday accuracy and precision for both analytes were within ±9.0%. Excellent extraction efficiency and negligible matrix effects were observed. The applicability of the method was demonstrated by successful measurement of warfarin enantiomers in urine of patients with kidney disease. The method is simple, accurate and reproducible and is currently being used to support warfarin pharmacokinetic studies.
authors.affiliation: Center for Clinical Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA.
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, USA Department of Pharmacy and Therapeutics, School of Pharmacy, University of Pittsburgh, 208 Salk Pavillion, 335 Sutherland Drive, Pittsburgh, PA 15260, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Ocque
authors.affiliation: Service de Néphrologie et Centre de Recherche, Hôpital Maisonneuve-Rosemont, Montréal, QC, Canada.
authors.forename: François A
authors.initials: FA
authors.lastname: Leblond
authors.affiliation: Service de Néphrologie et Centre de Recherche, Hôpital Maisonneuve-Rosemont, Montréal, QC, Canada Département de Pharmacologie, Université de Montréal, Montréal, QC, 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, USA Department of Pharmacy and Therapeutics, School of Pharmacy, University of Pittsburgh, 208 Salk Pavillion, 335 Sutherland Drive, Pittsburgh, PA 15260, USA nolin@pitt.edu.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Nolin
mesh_terms: D000925:Anticoagulants / Q000652:urine*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014859:Warfarin / Q000737:chemistry / Q000652:urine*
chemical_list: D000925:Anticoagulants; D014859:Warfarin