Plasma Protein Binding of Warfarin: Methodological Considerations

Recent theoretical work has suggested that radiochemical impurities can significantly alter the binding results for highly protein-bound drugs. We compared protein binding of warfarin by ultrafiltration and equilibrium dialysis with 98% radiochemically pure [14C]warfarin. Ultrafiltration and equilibrium dialysis were performed at 37°C and pH 7.45 on the plasma of patients receiving chronic warfarin therapy. Binding to plasma from seven patients was measured in duplicate by both a nonspecific radioisotopic technique and a specific HPLC technique. The nonspecific technique gave percentage of free warfarin values of 1.84 ± 0.11 (mean ± SD) and 1.59 ± 0.14 for ultrafiltration and equilibrium dialysis, respectively. The HPLC procedure yielded a percentage of free warfarin by ultrafiltration of 0.969 ± 0.203 and a value of 0.690 ± 0.095 by equilibrium dialysis (p < 0.05). The HPLC procedure for protein binding was performed on plasma samples from 12 additional patients and yielded a percentage of free warfarin of 1.01 ± 0.69 by ultrafiltration and 0.44 ± 0.34 by equilibrium dialysis (p < 0.05). It can be concluded that radiochemical impurities may lead to significant overestimation of the percentage of free warfarin. Ultrafiltration yielded a higher percentage of free warfarin than did equilibrium dialysis, but the ability to distinguish binding differences among patients was similar.