Analysis of warfarin-albumin binding by HPLC with internal-surface reversed-phase silica column.

Abstract

Protein binding of warfarin with bovine serum albumin (BSA) was analyzed by HPLC with an internal-surface reversed-phase (ISRP) silica column. When a relatively large volume (2100 .mu.1) of the sample solution containing warfarin and BSA was applied directly to the ISRP column, two distinct peaks appeared separately from the protein peak. One of these had a short retention time and was ascribable to warfarin released from the strong-binding sites on BSA molecules during the process of chromatography. The other peak had a long retention time and was due to the sum of free warfarin and that released from weak-binding sites. The separation profiles of these peaks were investigated with varying injection volumes and mobile phase conditions. The binding constants for the strong site and the weak site were determined from the area of these warfarin peaks. The results were comparable to the reported values.