Sensitive determination of warfarin and its metabolic enantiomers in body fluids via capillary electrophoresis combined with ultrasound-assisted dispersive liquid-liquid microextraction and online sample stacking

Abstract

A sensitive technique for the measurement of warfarin and its metabolic enantiomers in human fluids such as urine and serum technique was developed by applying ultrasound-assisted dispersive liquid-liquid microextraction linked with poly(ethylene oxide)-mediated stacking in capillary electrophoresis. The parameters that influence extraction and stacking performance-the type of the extraction and dispersive solvents and their volume, extraction time, salt additives, sample matrix, solution pH, and the composition of background electrolyte stacking-were carefully studied and optimized based on the considering of obtaining the best detection sensitivity. Under the optimal extraction (30 μL C2H2Cl4 and 200 μL tetrahydrofuran in 1 mL of sample solution) and separation (0.5% PEO, 200 mM Tris-borate, pH 8.5, and 5 mM dimethyl-β-cyclodextrin) conditions, the enrichment factors of D-/L-warfarins and D-/L-7-OH warfarins covered from 1758 to 1859 and their limits of detection ranged from 0.34 to 0.38 nM. Calibration-related results exhibited acceptable linearity with the coefficient of determination higher than 0.99; the relative standard deviations of the peak area were determined to 4.1%–6.3% (n = 3). The recovery of four separated analytes spiked in samples of urine and serum was found to be 93%–110% and 95%–109%, respectively. We revealed that ultrasound-assisted dispersive liquid-liquid microextraction with poly(ethylene oxide)-mediated stacking in capillary electrophoresis could be a prompt and practical method for quantifying the levels of warfarin and its metabolic enantiomers in real-world samples, especially in biological fluids.