Capillary electrophoresis/frontal analysis for microanalysis of enantioselective protein binding of a basic drug

A new HPCE method was developed for the enantioselective determination of the unbound concentration of a basic drug under plasma protein binding equilibrium. The racemic basic drug verapamil (VER) and human serum albumin mixed solution was used as a model sample solution. The sample solution was introduced into a fused-silica capillary hydrodynamically or electrokinetically. During the electrophoresis following hydrodynamic injection, the unbound drug zone migrated apart from the sample zone and was separated into two zonal peaks with a plateau due to enantiomers by a chiral selector (trimethyl-beta-cyclodextrin) dissolved in the acidic running buffer solution (pH 2.5). By the electrokinetic introduction of the same sample solution from the anodic end, only the unbound drug entered the capillary and was separated into the enantiomers, which also gave the zonal peaks with plateau. The unbound concentration of each enantiomer was determined from the plateau peak height. The results obtained by the different methods for sample introduction agreed well with those determined by conventional ultrafiltration-chiral HPLC, which was employed as a reference method. The unbound concentration of (S)-VER was 1.7 times higher than that of the antipode. The sample size used in the present method was approximately 200 nL, which is about one-thousandth of that in the reference method. The electrokinetic introduction gave a better peak shape than the hydrodynamic introduction.