Determination of protein-drug binding constants by pressure-assisted capillary electrophoresis (PACE)/frontal analysis (FA).

A simple method for the determination of binding constants of drugs to human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) was developed by pressured-assisted capillary electrophoresis (PACE) based on the principle of frontal analysis (FA). The free drug concentration was measured from the height of the frontal peak and calculated based on the external drug standard in the absence of protein. With a known concentration of total drug, the percentage of drug bound to HSA or AGP was then determined. The binding constants of drug to HSA or AGP were obtained from non-linear curve fitting of the percentage of bound drug as a function of total protein concentration or total drug concentration. The sample was prepared by mixing known concentrations of drug and protein in phosphate buffered saline (PBS) and equilibrated for 30 min. A large volume of sample solution (approximately 80 nl) was injected at 1.0 psi for 40 s into the fused silica capillary, which was filled with PBS buffer. Due to the difference in charge/size ratio, the free drug was separated from the protein/protein-drug complex when 15-25 kV voltage and 0.5-1.5 psi air pressure were applied. External air pressure was used to improve the throughput, prevent protein loss, and achieve a better drug plateau. By modifying experimental conditions, a wide range of binding constants could be measured. This PACE/FA method works well for basic, neutral, and weakly acidic compounds.