HIGH-PERFORMANCE CAPILLARY ELECTROPHORESIS FRONTAL ANALYSIS FOR THE STUDY OF PROTEIN-BINDING OF A BASIC DRUG

A simple high-performance capillary electrophoresis (HPCE) method base d on the principle of frontal analysis was applied to the determinatio n of the concentration of unbound basic drug in protein binding equili brium. A small volume of sample solution (similar to 80 nL) containing 113-340 mu M of verapamil (VER) and 100-550 mu M of human serum album in was introduced into the fused silica capillary (effective length, 2 2 cm; 50-mu m i.d.) by suction. Because the silanol groups on the inne r surface of the capillary were bound with linear polyacrylamide throu gh Si-C bonds, electroosmotic flow was not generated even at pH 7.4 wi th an applied voltage of + 10 kV. The unbound drug bearing positive ch arge migrated electrophoretically from the drug-protein mixed zone tow ard the detection end, whereas human serum albumin did not co-migrate because of its negative charge. The bound drug migrated after it was r eleased from the protein. As a result of an 80-nL injection of the sam ple solution, VER was eluted as a zonal peak with a plateau region. Th e VER concentration calculated from the plateau height agreed well wit h the unbound VER concentration determined by the conventional ultrafi ltration-HPLC method, with good reproducibility (CV, < 6.23%, n = 15). The present HPCE/FA system was applied to the Scatchard analysis of V ER and alpha(1)-acid glycoprotein binding, and the estimated binding p arameters agreed well with literature values.