Study And Applications Of High Sensitive Analysis Methods With Capillary Electrophoresis

Studies on the interaction between protein and drug molecules are of great importance in theoretical and practical aspects. The commonly used methods for research are fluorescence spectra, UV-vis spectra, CD and FT-IR specta and molecular modelling. The technique of laser light scattering has been widely used in the investigations of biomacromolecule solutions, which can provide some more important parameters such as molar mass, root-mean-square radius of gyration, the second virial coefficients, diffusion coefficients and hydrodynamic radius. Therefore, it is very sutiable for the study of state behavior of biomacromolecule solutions. Capillary electrophoresis (CE) has been widely used in the field of biology, chemistry, medicine, environmental and food due to the advantages of multiple separation modes, higher separation power, short analysis time, consuming small volumes of sample and reagents, easy to control etc. Laser induced fluorescence (LIF) detection has high sensitivity and can be adapted to different separation modes of CE. So it has become an important detection technique in the field of drug analysis and life science. In this dissertation, new separation and analysis systems were brought forward based on CE-LIF detection and laser light scattering research.Based on the previous literatures, the following major innovation works were carried out in this dissertation:(1) The derivatization reaction conditions of ephedrine and pseudoephedrine with sensitive derivation reagent in aqueous system were studied and two new methods were developed for the assay of them by microemulsion electrokinetic chromatography (MEEKC) and micellar electrokinetic chromatography (MEKC) with LIF detection. The sensitivity and analysis times were greatly improved compared with the previous reports;(2) A new method of CZE with indirect LIF detection was developed for the simultaneous determination of six coumarin compounds (esculin, esculetin, isofraxidin, genistein, naringin and sophoricoside) with fluorescein as the probe. The proposed method enlarges the application range of LIF detector, and provides new approach for the analysis of certain compounds difficult to derivatize;(3) Based on the above research, an MEKC with indirect LIF detection method for the simultaneous determination of adenine and guanine in DNA extracts from fungus, maize and soybean was established;(4) A new method for the investigation of the complexes formed between human serum albumin (HSA) and ampicillin sodium under the simulated physiology conditions using laser light scattering technique was developed. This method offered a new way for the study of pharmacokinetic and the interaction between drug molecules and protein.This dissertation consists of six chapters.Chapter 1 The approach and application of capillary electrophoresis with laser-induced fluorescence detection were reviewed. This part mainly focused on the applications of direct LIF through chemical reaction, the theory and the developmentof indirect LIF.Chapter 2 An MEEKC with LIF detection method for the separation and quantification of ephedrine (E) and pseudoephedrine (PE) after derivatizated by 5-(4,6-dichloro-s-triazin-2-ylarnino) fluorescein (DTAF) was proposed. The baseline separation of the two alkaloids could be obtained in less than 11 min under the optimum conditions. Good linear relationship for E and PE was obtained, and the detection limits for E and PE were 2.39×10^-3ng mL^-1, 1.09×l0^-3ng mL^-1, respectively. The method was successfully applied to the analysis the alkaloids in ehpedra herb and its preparations.Chapter 3 An MEKC with LIF detection for the analysis of ephedrine (E) and pseudoephedrine (PE) after derivatizated with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein was developed. The baseline separation was achieved within 8 min under the optimum conditions. The proposed method surpassed other chromatographic alternatives in terms of limit of detection at least 10³ folds with good reproducebility. Chapter 4 A capillary zone electrophoresis (CZE) with indirect LIF detection method is developed for the simultaneous determination of six coumarin compounds (esculin, esculetin, isofraxidin, genistein, naringin and sophoricoside).This proposed method was applied to the analysis of the courmin compounds in herb plants, and also would extend the application range of LIF detection.Chapter 5 A new method for the simultaneous determination of adenine and guanine in DNA extracts using MEKC with indirect LIF detection was proposed. This developed method was simple and rapid, and it has been applied to the analysisof the two purines in DNA extracts with recoveries in the range of 85.3 - 110.2%.Chapter 6 The complexes formed by the interaction of human serum albumin and ampicillin sodium in aqueous solutions were investigated under the simulated physiology conditions using laser light scattering technique. The results of static light scattering have suggest the molecular weight of the protein/drug complexes, z-average root-mean-square radius of gyration and the second virial coefficients. Dynamic light scattering provides information on diffusion coefficient and particle distributions of protein/drug complexes under different conditions.