[Study of protein binding in ketoprofen using liquid chromatography frontal analysis in comparison with capillary...

A method of high performance liquid chromatography-frontal analysis (HPLC-FA) was developed to study the binding of ketoprofen to human serum albumin (HSA) and it was compared with high performance capillary electrophoresis-frontal analysis (HPCE-FA). The separation was performed using Pinkerton GFF II-S5-80 internal-surface reversed-phase silica column (150 mm x 4.6 mm i.d., 5 microm) at pH 7.4 in a 67 mmol/L isotonic sodium phosphate buffer at 37 degrees C. Other conditions included a flow rate of 0.2 mL/min, UV detection at a wave-length of 254 nm and an injection volume of 950 microL. A trapezoidal peak of the unbound ketoprofen appeared after HSA elution in the chromatogram. The plateau height of the peak was employed to determine the concentration of unbound ketoprofen in the HSA equilibrated sample solution. The HPLC-FA method provides the advantage of high sensitivity and however the disadvantages of large sample size and long analytical time when compared with HPCE-FA. HPLC-FA is applicable to the binding parameter estimation of ketoprofen to both primary and secondary sites, which are 0.37 x 10(6) L/mol and 1.4 for K1 (the association constant) and n (the number for the binding sites per molecule HSA), respectively, and 0.005 x 10(6) L/mol and 7.2 for K2 and n2, respectively. In contrast, HPCE-FA measures parameters for only the secondary binding sites; K2 of 0.018 x 10(6) L/mol and n2 of 2.54 can be estimated. It is found that ketoprofen binds mainly at the primary sites at a lower mole ratio of ketoprofen versus HSA, and the binding at the secondary sites occurs at a higher ratio.