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 1.2mL/min. The mobile phase was composed of 31% acetonitrile, 5% of methanol and 64% of ammonium acetate buffer (10mmol/L, pH 4.1). The enantiomers were quantified using a fluorescence detector (lambda(excit)=320nm, lambda(emiss)=415nm). The limit of detection was found to be 0.121micromol/L of (S)-warfarin and 0.109micromol/L of (R)-warfarin. The range of applicability and linearity was estimated from 0.25 to 100micromol/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.
Malakova
Jana
J
Institute of Clinical Biochemistry and Diagnostics, Charles University in Prague, Faculty of Medicine and University Hospital Hradec Králové, Sokolská 581, CZ-50005 Hradec Králové, Czech Republic. Malakovaj@lfhk.cuni.cz
Pavek
Petr
P
Svecova
Lucie
L
Jokesova
Iveta
I
Zivny
Pavel
P
Palicka
Vladimir
V
D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D006020:Glycopeptides / Q000737:chemistry*; D006801:Humans; D016018:Least-Squares Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D014859:Warfarin / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification
D006020:Glycopeptides; D014859:Warfarin