| High-performance capillary electrophoresis (HPCE) and high-performance liquid chromatography (HPLC) were applied to determine free drug concentration in drug-protein mixture without disturbing the equilibrium. The purpose was to provide simple, robust, easy to implement and feasible approaches for drug-protein binding study in new drug design and therapeutic drug monitoring (TDM).A simple HPCE method based on the principle of frontal analysis (FA) was developed for the determination of the free concentrations of TDM drugs in 40umol.L-1 human serum albumin (HSA) equilibrated mixtures in an uncoated fused silica capillary under the following conditions: running buffer of 67mmol.L-1 phosphate (pH 7.4); UV detector at wavelength 214nm; hydrostatic injection mode (0.11m high) and the injection time of 80s, applied voltage of-lOkV. The investigated drugs included ketoprofen, chloramphenicol, ofloxacin, theophylline, amitriptyline hydrochloride and clozapine, which display a range of different properties with respect to structure, hydrophocity, charge at physiological pH and electrophoretic mobility. CE-FA was also used to investigate the effect of 1 8-methyl norethindron on ketoprofen-HSA interaction and the results indicated that 18 -methyl norethindrone can not displace ketoprofen from its primary binding site while ketoprofen could be displaced by high concentration 1 8-methyl norethindrone from its second binding site.Capillary electrophoresis in the FA mode was developed to determine the unbound clozapine concentration in HSA solution, human plasma, rabbit serum and plasma in in vitro experiment. Samples were injected directly into an uncoatedfused-silica capillary and separation was accomplished within 11 min without samplepretreatment. The most suitable running buffer to separate unbound clozapine from endogenous substances was 67mmol.L-1 phosphate buffer of pH 7.4 containing 1mmolL.L-1 EDTA and 0.5mol.L-1 glycine. The unbound clozapine concentration was directly measured from the height of the frontal peak. The methodology was comparable with good correlation to the conventional ultraflltration method. It was found that clozapine was bound in human plasma, rabbit plasma and serum strongly, while weakly bound to HSA. The present method enables determination of unbound drug concentration in multiple equilibria system with sample volume of nl level, and would be useful especially for the study on protein binding in biological samples available in minute quantity.Theophylline-HSA interaction was studied using affinity capillary electrophoresis (ACE) method. The experiments were carried out by adding theophylline in varying concentrations to the buffer (20mmol.L-1 phosphate buffer pH 7.4, containing 1mmol.L-1 EDTA and 0.5mol.L-1 glycine), and 30umol.L-1 HSA solution (containing 5% acetone) was injected to an uncoated fused silica capillary with hydrostatic injection mode and applied voltage of -12kV. The binding constant obtained was consistent with literature value. The free concentrations of the drug in 40umol.L-1 HSA equilibrated mixtures were determined using capillary electrophoresis-ligand separation method. The injection time and applied voltage are 2s and -10kV, respectively. Other conditions are the same as in CE-FA method. The investigated drugs included ketoprofen, imipramine hydrochloride, lidocaine hydrochloride, amitriptyline hydrochloride and clozapine. The concentrations of free drugs agreed well with those determined by the conventional ultraflltration method. It was found that amitriptyline hydrochloride and clozapine hardly bound appreciably to HSA.The mechanisms, application scope, advantages and disadvantages of aforementioned three high performance-capillary electrophoresis modes were discussed in detail in this work. The type of qualitative and quantitative information obtained, application conditions and application scope of different modes werespecific, and frequently, it is the nature of the system being evaluated that determines the optimal experimen...
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