Protein binding study of clozapine by capillary electrophoresis in the frontal analysis mode

We have used capillary electrophoresis in the frontal analysis mode (CE-FA) to determine the unbound concentration of clozapine in human serum albumin (HSA), human plasma, rabbit serum and plasma sample. The unbound clozapine concentration was directly measured from the height of the frontal peak. Samples were injected directly into an uncoated fused silica capillary (0.65 m (LC) x 75 microm i.d.; LE = 0.35 m) and separation was accomplished within 11 min without extensive sample pretreatment. The most suitable running buffer to separate unbound clozapine peak from the other peaks due to endogenous substances was found to contain 1 mmol1(-1) EDTA, 0.5 mol1(-1) glycine, and 67 mmol1(-1) phosphate with pH 7.4. The concentrations of unbound clozapine agreed well with those determined by the conventional ultrafiltration method. The methodology is validated and good correlation and precision are obtained. It was found that clozapine is strongly bound to protein in human plasma, rabbit plasma, and serum, while hardly bound to HSA. The present method enables the determination of the unbound drug concentration in multiple equilibrium system with less than ultra-micro injection volume, and would be hence especially useful for the protein binding study in biological samples that are only available in minute quantities.