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

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.