【作者】 王卫平；

【导师】 胡之德；

【作者基本信息】 兰州大学 ， 分析化学， 2007， 博士

【摘要】 研究蛋白质与药物分子的相互作用具有重要的理论和实际意义，常用的方法有荧光光谱法、紫外-可见光谱法、圆二色谱法、红外光谱法以及分子模拟等。激光散射技术在生物大分子溶液性质研究方面的应用越来越广泛，它可以提供大分子的重均分子量、均方旋转半径、第二维里系数、平动扩散系数及平均流体力学半径等重要参数，这对于研究生物大分子的溶液状态行为特别适用。毛细管电泳具有分离模式多、分离效率高、分析速度快、试剂和样品用量少、易于调控等优点，已经在在生物、化学、医药、环保、食品等领域中有了广泛的应用。激光诱导荧光检测器具有很高的灵敏度，可以适应毛细管电泳不同模式的检测，已成为药物分析和生命科学领域的一种重要的分析检测技术。因此，本文用毛细管电泳-激光诱导荧光检测和激光散射法进行研究并提出了分离分析的新体系。该论文在综述前人工作的基础上，开展了如下未见文献报道的创新性的研究工作：（1）使用灵敏的衍生试剂对麻黄碱和伪麻黄碱在水体系中的衍生反应条件进行了系统研究，建立了微乳电动色谱-激光诱导荧光检测法和胶束电动色谱—激光诱导荧光检测法测定麻黄碱和伪麻黄碱的灵敏分析新方法，与以前的报道相比，灵敏度和分析时间均有很大改善；（2）使用荧光素钠作为背景荧光试剂，建立了同时分析测定六种黄酮类化合物（秦皮甲素、秦皮乙素、异秦皮定、染料木素、柚皮苷和槐角苷）的毛细管区带电泳—间接激光诱导荧光检测新方法，扩大了激光诱导荧光检测的应用范围，对难衍生化合物的分离分析提供了一种新思路和新途径；（3）以荧光素钠作为背景荧光试剂，建立了一种用于同时测定食品（蘑菇、玉米和大豆）提取DNA中的腺嘌呤和鸟嘌呤含量的胶束电动色谱—间接激光诱导荧光检测新方法；（4）用动态和静态激光散射法研究了模拟生理条件下人血清白蛋白与氨比西林钠盐相互作用所形成的复合物，为药物分子与蛋白质的相互作用、药代动力学等的研究提供了一种新思路。论文共分为六章第一章对毛细管电泳-激光诱导荧光检测技术的原理及其应用研究进行了综述。主要是近年来通过化学反应衍生标记的直接激光诱导荧光检测的应用，及间接激光诱导荧光检测的原理和应用进展。第二章以5-（4，6-三氯-s-三嗪基-2-氨基）荧光素作为衍生试剂，建立了一种胶束电动色谱-激光诱导荧光检测法分析麻黄碱和伪麻黄碱的新方法。在最佳条件下，8分钟内可实现基线分离，灵敏度高，重现性好，与其他色谱法相比，检测限至少提高了10³倍。第三章以5-（4，6-三氯-s-三嗪基-2-氨基）荧光素为衍生试剂，建立了一种微乳电动色谱-激光诱导荧光检测法分析麻黄碱和伪麻黄碱的新方法。在最优条件下，分析物可在11分钟内实现基线分离。分析物的峰高与浓度之间存在较好的线性关系，麻黄碱和伪麻黄碱的检测限分别为2.39×10^-3和3.85×10^-3μg 1^-1。将该法成功用于麻黄及其制剂中两种生物碱的分析。第四章建立了一种毛细管区带电泳—间接激光诱导荧光检测法同时测定6种黄酮类化合物（秦皮甲素、秦皮乙素、异秦皮定、染料木素、柚皮苷和槐角苷）的新方法，扩大了LIF检测的应用范围。将该方法用于含黄酮类化合物的植物分析，得到了94.7-102.1％的回收率。第五章建立了一种用于同时测定DNA中腺嘌呤和鸟嘌呤含量的胶束电动色谱—间接激光诱导荧光检测新方法。该方法简单、快速，可用于食品样品DNA提取物中两种嘌呤碱的分析，得到的回收率范围在85.3-110.2％之间。第六章用动态和静态激光散射法研究了在模拟生理条件下人血清白蛋白与氨比西林钠盐相互作用所形成的复合物。静态激光散射结果给出了蛋白质和药物分子所形成复合物的分子量、均方旋转半径和第二维里系数；动态激光散射给出了不同pH条件下体系的扩散系数和粒径分布信息。更多还原

【Abstract】 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.更多还原