Validation of a method for the determination of (R)-warfarin and (S)-warfarin in human plasma using LC with UV...

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 microl of acetonitrile. A 50-microl aliquot was injected onto the HPLC system. Separation was achieved on a beta-cyclodextrin column (250 x 4.6 mm, 5 microm) 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 beta-cyclodextrin precolumn (50 x 4.6 mm, 5 microm) 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.