Coupling of acetonitrile deproteinization and salting-out extraction with acetonitrile stacking in chiral capillary electrophoresis for the determination of warfarin enantiomers

Concurrent sample clean-up and enhancement in detection sensitivity for chiral capillary electrophoresis was demonstrated based on the coupling of salting-out extraction with acetonitrile stacking and the use of dimethyl-beta-cyclodextrin as the chiral selector for the sensitive and enantioselective separation of warfarin enantiomers in urine samples. By optimizing the pH of salting-out extraction, warfarin enantiomers can be efficiently extracted from the aqueous sample solution into a smaller volume organic solvent (acetonitrile) phase. The pressure injection of the enriched acetonitrile phase (containing ca. 1% NaCl) into the CE capillary at 10% capillary volume resulted in additional concentration of the warfarin enantiomers. The limit of detection for both warfarin enantiomers was as low as 1.5 ng/mL in urine sample. Our results show that the novel strategy offers improved sensitivity compared to conventional CE analysis, reaching a combined enrichment factor higher than 1000. Calibration curves of warfarin enantiomers in urine samples were found to be linear between 10 and 1000 ng/mL, and intra- and inter-day precision (N = 9) for both warfarin enantiomers in terms of migration time and peak area were found to be within the range of 0.1–0.8% and 1.0–6.7%, respectively. The recovery of warfarin enantiomers from urine was ca. 90%.