High-performance capillary electrophoresis/frontal analysis for the study of protein binding of a basic drug.

A simple high-performance capillary electrophoresis (HPCE) method based on the principle of frontal analysis was applied to the determination of the concentration of unbound basic drug in protein binding equilibrium. A small volume of sample solution (approximately 80 nL) containing 113-340 microM of verapamil (VER) and 100-550 microM of human serum albumin was introduced into the fused silica capillary (effective length, 22 cm; 50-microns i.d.) by suction. Because the silanol groups on the inner surface of the capillary were bound with linear polyacrylamide through Si-C bonds, electroosmotic flow was not generated even at pH 7.4 with an applied voltage of +10 kV. The unbound drug bearing positive charge migrated electrophoretically from the drug-protein mixed zone toward the detection end, whereas human serum albumin did not co-migrate because of its negative charge. The bound drug migrated after it was released from the protein. As a result of an 80-nL injection of the sample solution, VER was eluted as a zonal peak with a plateau region. The VER concentration calculated from the plateau height agreed well with the unbound VER concentration determined by the conventional ultrafiltration-HPLC method, with good reproducibility (CV, < 6.23%, n = 15). The present HPCE/FA system was applied to the Scatchard analysis of VER and alpha 1-acid glycoprotein binding, and the estimated binding parameters agreed well with literature values.