Abstract
A sensitive and selective chiral high-performance liquid chromatography (HPLC) method was developed for the determination of (R)-warfarin and (S)-warfarin in human plasma. (R)- and (S)-warfarin and the internal standard (oxybenzone) were extracted from human plasma that had been made acidic with 1 N sulfuric acid into ethyl ether. The ethyl ether layer was removed and evaporated, and the residue was reconstituted in 200 μl of acetonitrile. A 50-μl aliquot was injected onto the HPLC system. Separation was achieved on a β-cyclodextrin column (250×4.6 mm, 5 μm) with a mobile phase composed of acetonitrile:glacial acetic acid:triethylamine (1000:3:2.5, v/v/v). Detection was by ultraviolet absorbance at 320 nm. Late-eluting peaks were diverted from the analytical column by using a β-cyclodextrin precolumn (50×4.6 mm, 5 μm) and a column switching device. The retention times of (R)- and (S)-warfarin and the internal standard were approximately 7.7, 6.9 and 4.0 min, respectively. The run time was 15 min. The assay was linear in concentration ranges of 12.5–2500 ng/ml for (R)- and (S)-warfarin in human plasma. The analysis of quality control samples for (R)- and (S)-warfarin (25.0, 400 and 2000 ng/ml) demonstrated excellent precision with relative standard deviations (R.S.D.) for (R)-warfarin of 10.9, 2.8, and 2.8%, respectively ( n=18), and for (S)-warfarin of 7.0, 2.4 and 2.6%, respectively ( n=18). The method was accurate with all overall ( n=18) mean concentrations being less than 6.0% from nominal at all quality control sample concentrations.
Published Version
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