New high-performance liquid chromatography method for the determination of (R)-warfarin and (S)-warfarin using chiral separation on a glycopeptide-based stationary phase

摘要

Warfarin is a well-known anticoagulant agent that occurs in two enantiomers. (R)-(+)-warfarin and (S)(-)-warfarin. A new liquid chromatography method for the determination of both enantiomers was developed, validated and applied in in vitro studies with the aim of evaluating the accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line. OptiMEM cell cultivation medium samples and cellular lysates were purified using Waters Oasis (R) MAX extraction cartridges. The chiral separation of warfarin and the internal standard p-chlorowarfarin enantiomers was performed on an Astec Chirobiotic (TM) V2 column at a flow rate of 1.2 mL/min. The mobile phase was composed of 31% acetonitrile, 5% of methanol and 64% of ammonium acetate buffer (10 mmol/L, pH 4.1). The enantiomers were quantified using a fluorescence detector (lambda(excit) = 320 nm, lambda(emiss) = 415 nm). The limit of detection was found to be 0.121 mu mol/L of (S)-warfarin and 0.109 mu mol/L of (R)-warfarin. The range of applicability and linearity was estimated from 0.25 to 100 mu mol/L. The precision ranged from 1.3% to 12.2% of the relative standard deviation, and the accuracy reached acceptable values from 95.5% to 108.4%. The new bioanalytical method confirmed the same accumulation of (R)-warfarin and (S)-warfarin in the hepatoma HepG2 cell line.