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Simultaneous Determination of Warfarin Enantiomers and Its Metabolite in Human Plasma by Column-Switching High-Performance Liquid Chromatography With Chiral Separation

Uno, Tsukasa PhD*; Niioka, Takenori BS; Hayakari, Makoto PhD; Sugawara, Kazunobu PhD†‡; Tateishi, Tomonori MD, PhD*

Therapeutic Drug Monitoring: June 2007 - Volume 29 - Issue 3 - p 333-339
doi: 10.1097/FTD.0b013e31805c956e
Original Article
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A simple and sensitive column-switching high-performance liquid chromatographic method for the simultaneous determination of warfarin enantiomers and their metabolites, 7-hydroxywarfarin enantiomers, in human plasma is described. Warfarin enantiomers, 7-hydroxywarfarin enantiomers, and an internal standard, diclofenac sodium, were extracted from 1 mL of a plasma sample using diethyl ether-chloroform (80:20, v/v). The extract was injected onto column I (TSK precolumn BSA-C8, 5 μm, 10 mm × 4.6 mm inside diameter) for cleanup and column II (Chiralcel OD-RH analytical column, 150 mm × 4.6 mm inside diameter) coupled with a guard column (Chiralcel OD-RH guard column, 10 mm ×4.6 mm inside diameter) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (84:16 v/v, pH 2.0) for clean-up and phosphate buffer-acetonitrile (45:55 v/v, pH 2.0) for separation. The peaks were monitored with an ultraviolet detector set at a wavelength of 312 nm, and total time for chromatographic separation was approximately 25 minutes. The validated concentration ranges of this method were 3 to 1000 ng/mL for (R)- and (S)-warfarin and 3 to 200 ng/mL for (R)- and (S)-7-hydroxywarfarin. Intra- and interday coefficients of variation were less than 4.4% and 4.9% for (R)-warfarin and 4.8% and 4.0% for (S)-warfarin, and 5.1% and 4.2% for (R)-7-hydroxywarfarin and 5.8% and 5.0% for (S)-7-hydroxywarfarin at the different concentrations. The limit of quantification was 3 ng/mL for both warfarin and 7-hydroxywarfarin enantiomers. This method was suitable for therapeutic drug monitoring of warfarin enantiomers and was applied in a pharmacokinetic study requiring the simultaneous determination of warfarin enantiomers and its metabolite, 7-hydroxywarfarin enantiomers, in human volunteers.

From the *Department of Clinical Pharmacology, Hirosaki University School of Medicine, Hirosaki, Japan; †Department of Pharmacy, Hirosaki University Hospital, Hirosaki, Japan; and ‡Department of Clinical Pharmacy, Aomori University School of Pharmacy, Aomori, Japan.

Received for publication September 4, 2006; accepted September 26, 2007.

Correspondence: Tsukasa Uno, PhD, Department of Clinical Pharmacology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan (e-mail: uno-hki@umin.ac.jp).

© 2007 Lippincott Williams & Wilkins, Inc.