Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
39, Number 10, October 2001, pp. 137-141
Study on the Protein Binding of Ketoprofen Using Capillary Electrophoresis
Frontal Analysis Compared with Liquid Chromatography Frontal Analysis
Famei Li*, Dawei Zhou, and Xingjie Guo
Department of Analytical Chemistry, Shenyang Pharmaceutical University, 103
Wenhua Road, Shenyang 110016, P.R. China
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 constant (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–1 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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