| Capillary electrophoresis has been widely used in the analysis of single cell, protein, nuclei and pharmaceutical efffective components for it's extremely high efficiency, high speed, small sample and reagent consumption, low cost and simplicity. Laser induced fluorescence detection has high sensitivity and can be adapted to different separation modes of capillary electrophoresis. The combination of capillary electrophoresis and laser-induced fluorescence not only provide powerful separation efficiency but also over come the poor sensitivity caused by short diameter of capillary. It has become an important tool in the pharmaceutical analysis of vivo and life science. Analytes that can not be dissolved in aqueous solution or can not be separated in aqueous system could be separated by nonaqueous capillary electrophoresis with satisfactory separation effect. However, the derivatives comes from aqueous system hardly can be analyzed by nonaqueous capillary electrophoresis directly. Although the electrical break down can be reduced when the derivatives comes from aqueous system were diluted with nonaqueous running buffer solution, it still occurs very often and the performance could not be continued any more.Based on the previous literatures, the following major innovation works for the separation and determination of pharmaceutical effective components using capillary electrophoresis were carried out in this dissertation:(1) The derivatization reaction conditions of four hydrophobic amino acids in nonaqueous system were investigated and a new nonaqueous capillary electrophoresis with laser-induced fluorescence detection method was established for separation and determination of them.(2) The derivatization reaction conditions of ephedrine and pseudoephedrine in nonaqueous system were studied and a new method was developed for the assay of them by nonaqueous capillary electrophoresis with laser-induced fluorescence detection.(3) A micellar capillary electrokinetic chromatography with laser-induced fluorescence methodwas developed for quantification of epinephrine and dopamine in traditional Chinese medicines and the derivatization reaction conditions were invstigated.(4) Based on the above research, a microemulsion capillary electrophoresis with laser-induced fluorescence detection method was established for determination of epinephrine and dopamine in traditional Chinese medicines and rheumatic heart disease patient's plasma.(5) A new nonaqueous capillary electrophoresis with diode arrary ultraviolet detection method was developed for separation and determination of pseudoephedrine, dextromethorphan, diphenhydramine and chlorpheniramine in cold medicines.(6) The approach of rapid quantification of berberine in Tianshen capsule was investigated using nonaqueous capillary electrophoresis.This dissertation consists of seven chapters.Chapter 1 The approach and application of capillary electrophoresis with laser-induced fluorescence detection were reviewed. The separation principle and modes of capillary electrophoresis and the theory and characters of laser-induced fluorescence were introduced firstly. Secondly, application of capillary electrophoresis with laser-induced-fluorescence detection in the area of natural products, biological fluids, life science and pharmaceutical analysis were reviewed. At last, the problems and shortcomings of capillary electrophoresis with laser-induced fluorescence were summarized. In addition, the future hot research areas of the novel techniquewere pointed.Chapter 2 A method of nonaqueous capillary electrophoresis was established for the separation and determination of alanine, phenylalanine, isoleucine and valine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-l, 3-diazol in nonaqueous media. The derivatization and separation conditions were optimized. The optimum derivatization conditions were 70 min for reaction time, 55 °C for reaction temperature and 20 mM ammunium acetate in methanol for derivatization buffer. The most suitable running buffer was composed of 60 mM ammonium acetate, 10 % acetontrile in methanol, 20 kV applied voltage and 20 °C capillary temperature. The method was applied to determine four compounds in two compound amino acid injections, except phenylalanine, the recoveries for the other three compounds ranging between 90 %-l 16 % and 88%-l 14%, respectively.Chapter 3 A nonaqueous capillary electrophoresis with laser-induced fluorescence detection method was developed for the quantification of ephedrine and pseudoephedrine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-l, 3-diazol in nonaqueous media. The derivatization and separation conditions were optimized. The detection limits for ephedrine and pseudoephedrine were 0.014 mg/L and 0.011 mg/L, respectively. The method was applied to the analysis of ephedrine and pseudoephedrine in four preparations with recoveries in the range of 93.9%-105.1%.Chapter 4 A micellar electrokinetic chromatography method with laser-inducedfluorescence detection to analyze epinephrine and dopamine after derivatizated with 4-chloro-7-nitrobenzo-2-oxa-l, 3-diazol was established. The derivatization and separation conditions were optimized. The detection limits and quantification limits for epinephrine and dopamine were 0.0038 mg/L, 0.013 mg/L and 0.065 mg/L, 0.020 mg/L, respectively. The method was applied to the analysis of the two compounds in two Chinese medicines with recoveries in the range of 92.6 %-108.7 %.Chapter 5 Based on the chapter 4, a microemulsion electrokinetic chromatography method with laser-inducedfluorescence detection to analyze epinephrine and tlopamine after derivatizated with 4-chloro-7-nitrobenzo-2-oxa-l, 3-diazol was established. The separation conditions were optimized. The detection limits for epinephrine and dopamine were 0.23 ng/mL and 1.90 ng/mL, respectively. The method was applied to the analysis of the two compounds in Chinese medicines and rheumatic heart disease patient plasma with recoveries in the range of 95.8 %-105.6 %.Chapter 6 A nonaqueous capillary electrophoresis method was developed for the identification and determination of four basic nitrogenous compounds, i.e. pseudoephedrine, dextromethorphan, diphenhydramine and chlorpheniramine. The separation conditions were optimized. The method was successfully applied to determine the four compounds in five cold medicines, the recoveries of the four constituents ranging between 91 % and 109 %.Chapter 7 A rapid nonaqueous capillary electrophoresis method has been developed for the separation and determination of berberine in a new kind of Chinese Medicine Preparation. Theseparation conditions were optimized. The optimum running buffer composed of 40 mM ammonium acetate, 2 % (v/v) acetic acid and 40 % (v/v) acetonitrile in methanol. The applied voltage was 25 kV. The analytes were detected by UV at 245 nm. In addition to, a high performance liquid chromatographic method was also established for the determination of berberin in Tianshen Capsule and the results were compared with that analyzed by nonaqueous capillary electrophoresis. |
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