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Capillary Electrophoresis and Its Application on Stereoselective Binding Study

Author: YangFeng
Tutor: ZhangZhuJun
School: Southwestern University
Course: Analytical Chemistry
Keywords: capillary electrophoresis frontal analysis enantiomeric separation protein binding laser-induced fluorescence
CLC: O658.9
Type: Master's thesis
Year: 2007
Downloads: 195
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Abstract


Capillary electrophoresis （CE） has manifested its versatility and potential in the separation of chiral drugs, because of its high efficiency, rapid analysis, low cost, wide range of separation modes and very low consumption of buffer and samples etc.The separation of enantiomers of drugs is an important subject of research especially in the pharmaceutical field. Because of the similar physicochemical properties of two enantiomers, the separation becomes more difficult. When chiral drugs are taken orally or injected into biological body, they are transferred to target organ, the determination of enantiomer concentration in biological fluid and the measurement of binding constant of drug with serum albumin have a great significance in the pharmacology researches and stereoselective phannacokinetic analysis of chiral drugs. Unbound drug concentrations show better correlation to the pharmacological activity than the total drug concentration. Serum protein binding has a significant effect on the phannacokinetic and pharmacodynamic properties of a drug.In this paper, capillary electrophoresis was combined with frontal analysis. An efficient and sensitive analysis method of the simendan’s stereoselective binding study was established. Moreover, a high performance capillary electrophoresis （HPCE） method for determination of pazufloxacin mesilate in human plasma and urine was developed. Eventually, a work based on capillary electrophoresis with laser-induced fluorescence was reporteded for the determination of the natural L-dopa in peasecod.The thesis is divided into two parts. Part one describes capillary electrophoresis in details and reviews recent development and application in separation of enantiomers of drugs and their protein binding. Part two includes three aspects:One: An efficient and sensitive analysis method of the simendan’s stereoselective binding study was established. The enantiomeric seperation was successfully applied with capillary electrophoresis using carboxymethyl-β-cyclodextrin （CM-β-CD） as chiral selector. The samples were prepared by mixing known concentrations of drug and BSA in phosphate buffered saline （PBS, pH7.4） and equilibrated at room temperature for 30 min, a large volume of sample solution was injected at 0.5 p.s.i. for 40sec. into the fused silica capillary （58.5cm×75μm I.D.）, which was filled with PBS buffer containing CM-β-CD, the drug was eluted as a zonal peak with a plateau region under condition of PBS buffer with 25kV at 25℃.Two: A capillary electrophoresis method for determination of pazufloxacin mesilate in human plasma and urine was developed. The determination was performed on a fused-silica capillary of 39cm×50μm （i.d.） , of which effective length is 28.5cm. 75mmol/L phosphate buffer （pH 6.0） was used as running buffer. Samples were injected electrically （12s,10kV） into the capillary. The applied voltage was 20kV and the detection was set at 247nm. Internal standard was ofloxacin. The linearity range was from1.0×10-8to7.0×10-7g/mL （r=0.9996）. The detection limit was 5.0×10-9g/mL. The method is efficient, reproducible and feasible for determination of pazufloxacin.Three: A high sensitive method of capillary electrophoresis coupled with laser-induced fluorescence （LIF） detection and selected fluorescein isothiocyanate （FITC） as the derivatization reagent for the determination of L-dopa in peasecod with the detective limit of 7.8×10-10g/mL. We also determined L-dopa in human blood after taking peasecod and verified that eating 100 g peasecod approximately equals to taking one synthetical L-dopa tablet with the quality of 0.2g. In our method, the sensitivity is nearly 1000 times higher than that of other CE methods. We have also determined L-dopa in rabbit blood by microdialysis sampling and high-performance liquid chromatography with chemiluminescence （HPLC-CL） detection.

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