title: Comparison of enzyme kinetics of warfarin analyzed by LC-MS/MS QTrap and differential mobility spectrometry.
abstract: Warfarin is an anticoagulant used in the treatment of thrombosis and thromboembolism. It is given as a racemic mixture of R and S enantiomers. These two enantiomers show differences in metabolism by CYPs: S-warfarin undergoes 7 hydroxylation by CYP2C9 and R-warfarin by CYP3A4 to form 10 hydroxy warfarin. In addition, warfarin is acted upon by different CYPs to form the minor metabolites 3'-hydroxy, 4'-hydroxy, 6-hydroxy, and 8-hydroxy warfarin. For analysis, separation of these metabolites is necessary since all have the same m/z ratio and similar fragmentation pattern. Enzyme kinetics for the formation of all of the six hydroxylated metabolites of warfarin from human liver microsomes were determined using an LC-MS/MS QTrap and LC-MS/MS with a differential mobility spectrometry (DMS) (SelexION™) interface to compare the kinetic parameters. These two methods were chosen to compare their selectivity and sensitivity. Substrate curves for 3'-OH, 4'-OH, 6-OH, 7-OH, 8-OH and 10-OH warfarin formation were generated to determine the kinetic parameters (Km and Vmax) in human liver microsomal preparations. The limit of quantitation (LOQ) for all the six hydroxylated metabolites of warfarin were in the range of 1-3nM using an LC-MS/MS QTrap method which had a run time of 22min. In contrast, the LOQ for all the six hydroxylated metabolites using DMS interface technology was 100nM with a run time of 2.8min. We compare these two MS methods and discuss the kinetics of metabolite formation for the metabolites generated from racemic warfarin. In addition, we show inhibition of major metabolic pathways of warfarin by sulfaphenazole and ketoconazole which are known specific inhibitors of CYP2C9 and CYP3A4 respectively.
authors.affiliation: Department of Pharmaceutical Sciences, MCPHS University, 179 Longwood Ave., Boston, MA 02115, USA; Department of Drug Metabolism and Pharmacokinetics, Biogen, 14 Cambridge Center, Cambridge, MA 02140, USA. Electronic address: naveed.shaik@my.mcphs.edu.
authors.forename: Abdul Naveed
authors.initials: AN
authors.lastname: Shaik
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Biogen, 14 Cambridge Center, Cambridge, MA 02140, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Grater
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Biogen, 14 Cambridge Center, Cambridge, MA 02140, USA.
authors.forename: Mukesh
authors.initials: M
authors.lastname: Lulla
authors.affiliation: Department of Pharmaceutical Sciences, MCPHS University, 179 Longwood Ave., Boston, MA 02115, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Williams
authors.affiliation: Development Center for Biotechnology, Taipei 221, Taiwan.
authors.forename: Lawrence L
authors.initials: LL
authors.lastname: Gan
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics, Biogen, 14 Cambridge Center, Cambridge, MA 02140, USA.
authors.forename: Tonika
authors.initials: T
authors.lastname: Bohnert
authors.affiliation: Department of Pharmaceutical Sciences, MCPHS University, 179 Longwood Ave., Boston, MA 02115, USA.
authors.forename: Barbara W
authors.initials: BW
authors.lastname: LeDuc
mesh_terms: D000925:Anticoagulants / Q000493:pharmacokinetics*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D065729:Cytochrome P-450 CYP2C9 / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014859:Warfarin / Q000493:pharmacokinetics*
chemical_list: D000925:Anticoagulants; D014859:Warfarin; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A
keywords: Differential mobility spectroscopy (DMS); Human liver microsomes; Hydroxywarfarin; Ketoconazole; LC–MS/MS; Method validation; QTrap; Substrate kinetics; Sulfaphenazole; Warfarin