The measurement of warfarin enantiomers in serum using coupled achiral/chiral, high-performance liquid chromatography (HPLC).

An assay for the serum concentration of the enantiomers of warfarin, R-warfarin and S-warfarin, has been developed using a bovine serum albumin chiral stationary phase (BSA-CSP) coupled to a Pinkerton internal-surface reverse-phase (ISRP) achiral column. The ISRP column is used to separate R,S-warfarin from the serum components and warfarin metabolites and to quantitate the total R,S-warfarin concentration. The eluent containing R,S-warfarin is then selectively transferred to the BSA-CSP, where the enantiomers are stereochemically resolved (alpha = 1.19) and the enantiomeric composition is determined. This system is sensitive and accurate, does not require extensive precolumn manipulations, and can be automated for use in large-scale clinical studies.