Development and validation of a high-performance liquid chromatographic method for the quantitation of warfarin enantiomers in human plasma.

An HPLC method for the quantitation of warfarin enantiomers in human plasma has been developed and validated. Baseline separation of S- and R-warfarin was achieved on a silica-bonded beta-cyclodextrin column with a mobile phase of acetonitrile-acetic acid-triethylamine (1000:3:2.5, v/v/v). The detection was performed at 320 nm. The established linearity range was 12.5-2500 ng ml-1 (r > 0.99). The limit of quantitation was 12.5 ng ml-1 for each enantiomer. Inter-day precision and accuracy of 12.5 ng ml-1 standards were 12.1% relative standard deviation (RSD) and +0.67% bias for S-warfarin and 9.7% RSD and +10.8% bias for R-warfarin. The low quality control samples at 37.5 ng ml-1 showed 6.9% RSD and 0.0% bias for S-warfarin, 7.2% RSD and +0.5% bias for R-warfarin. S-Naproxen was used as internal standard. Potential metabolites of warfarin were well resolved from S- and R-warfarin, the internal standard (S-naproxen) and each other. The run time was 25 min. The silica-bonded beta-cyclodextrin column showed excellent stability; over 1000 samples were injected without significant loss of performance. The column variability test showed that the method can be applied on several batches of beta-cyclodextrin columns but not all the beta-cyclodextrin columns were suitable for this method.