Determination of free drug in protein binding equilibrium by high-performance frontal analysis using internal-surface reversed-phase silica support.

Abstract

An internal-surface reversed-phase silica column was employed in the frontal analysis method to determine free drug concentration in warfarin (Wf)-bovine serum albumin (BSA) mixed solution and in indomethacin(Im)-BSA mixed solution. When a 4-ml portion of aqueous solution containing 2-30 microM Im and 28 microM BSA and a 10-ml portion of aqueous solution containing 0.5-175 microM Wf and 28 microM BSA were applied, the elution curves reached a plateau level corresponding to both free drug and drug-BSA complex (beta-plateau), followed by another plateau due to free drug alone (gamma-plateau). The drug concentration at the gamma-plateau agreed well with the free drug concentration determined after ultrafiltration of the same solution. The gamma-plateau was observed even when the applied volume was reduced to a level which was insufficient to produce the beta-plateau. The injection of a 400-microliters portion of the 0.5-100 microM Im and 28 microM BSA mixed solution and a 90-microliters portion of 50 microM Im and 550 microM BSA mixed solution onto the ISRP silica column gave a clear gamma-plateau. Compared to the conventional frontal analysis, the present method can determine a wide range of drug levels with much smaller injection volume. This method is applicable to plasma. By a single frontal analysis, both free and total concentrations of carbamazepine in plasma were determined simultaneously.