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期刊论文
Study on the Protein Binding of Ketoprofen Using Capillary Electrophoresis Frontal Analysis Compared with Liquid Chromatography Frontal Analysis
Journal of Chromatographic Science, Vol. 41, March 2003,-0001,():
A method of capillary electrophoresis frontal analysis (CEFA) is developed for the first time to study the binding of ketoprofen to human serum albumin (HSA) and compared with high performance liquid chromatography frontal analysis (LCFA). The separation is performed in an uncoated fused-silica capillary (60-cm × 75-μm i.d., 50-cm effective length) with a phosphate buffer (pH 7.4, ionic strength of 0.17M) as the running buffer. The applied voltage is 13 kV and the detection is set at 254 nm. A trapezoidal peak of the unbound ketoprofen appears after HSA elution in the electropherogram. The plateau height of the peak is employed to determine the unbound concentration of ketoprofen in the HSA equilibrated sample solution. The CEFA method provides the advantages of small sample injection volume and rapidity and the disadvantage of low sensitivity compared with LCFA. CEFA is applicable to the binding parameter estimation of ketoprofen to the secondary binding site; an association c:onstant (K2) of 0.24 × 106M-1 and the number for the binding site per molecule HSA of 2.54 is estimated. In contrast, LCFA measures parameters for both primary and secondary sites, which are 1.05 ×106M-l and 0.94 for K1 and n1, respectively, and 0.12 × 106M-1 and 3.16 for K2 and n2, respectively. It is found that ketoprofen binds mainly at the primary site at a molecular ratio of ketoprofen versus HSA lower than 0.75, and the binding at the secondary site occurs at a higher ratio.
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