Determination of protein binding by in vitro charcoal adsorption

Certain compounds such as SC-52151 have extensive nonspecific adsorption to the ultrafiltration devices or to dialysis membranes and therefore can not be measured by the conventional ultrafiltration or equilibrium dialysis methods. A new method based on charcoal adsorption was developed to overcome this difficulty. Unlike many conventional methods, which are based on the separation of free drug from bound drug under equilibrium conditions, the new method is operated under nonequilibrium conditions and involves measuring the time course of decline of the percentage of bound drug remaining in plasma while the free drug is being removed by charcoal adsorption. Theoretical aspects of the method and the data processing procedure are presented. SC-98A, a compound with minimal nonspecific adsorption to the ultrafiltration membrane, was used to demonstrate the applicability of this method against the ultrafiltration method. Using this method, the protein binding of SC-52151 in human plasma at 1.0 microgram/ml was determined to be in the range of 91.4-97.7% at room temperature.