Use of a human serum albumin-based high-performance liquid chromatography chiral stationary phase for the investigation of protein binding: detection of the allosteric interaction between warfarin and benzodiazepine binding sites.

The interaction between the benzodiazepine and the warfarin binding sites in human serum albumin (HSA) has been investigated using an HSA-based HPLC chiral stationary phase (HSA-CSP). (R)-Warfarin and (S)-warfarin were added to the mobile phase and racemic mixtures of oxazepam, lorazepam, and their hemisuccinic derivatives were injected onto the HSA-CSP. The presence of (R)-warfarin in the mobile phase did not significantly affect the chromatographic retention (expressed as capacity factor, k') of the investigated benzodiazepine hemisuccinate derivatives. The presence of (S)-warfarin did not significantly affect the k' of oxazepam and oxazepam hemisuccinate, but resulted in a dramatic increase in the k' of (S)-lorazepam hemisuccinate and also improved the enantiomeric resolution of lorazepam. These results confirm the existence of an allosteric interaction between the benzodiazepine binding site and the warfarin binding site. Furthermore, the study indicates that chromatography on the silica-immobilized HSA can detect interactions between binding sites on the protein. This can be of great importance in the determination of drug-drug interactions.