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

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® MAX extraction cartridges. The chiral separation of warfarin and the internal standard p-chlorowarfarin enantiomers was performed on an Astec Chirobiotic™ 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 (λexcit = 320 nm, λemiss = 415 nm). The limit of detection was found to be 0.121 μmol/L of (S)-warfarin and 0.109 μmol/L of (R)-warfarin. The range of applicability and linearity was estimated from 0.25 to 100 μmol/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.