摘要
毛细管电泳电致化学发光技术,是近十年来发展最快的一种新型分离分析技术,它集成了毛细管电泳的高效分离能力和电化学发光检测的高灵敏度。目前电致化学发光分析中应用最为广泛的发光试剂是Ru(bpy)_3~(2+) ,它由于具有水溶性好、化学性质稳定、氧化还原可逆等特点倍受人们青睐。在CE-ECL技术中,工作电极表面性能对Ru(bpy)_3~(2+)的发光效率影响极大,尤其在复杂试样的分离分析中工作电极的抗毒化能力更是决定了检测的可靠性。PB-Eu修饰电极能够催化联吡啶钌的氧化过程,且自身具有良好的稳定性,藉此可改善CE-ECL方法的检测灵敏度及重现性。
本工作的主要内容是利用PB-Eu修饰电极为CE-ECL工作电极,以大环内酯类药物,麻黄碱类药物为研究对象,研究了该电极上被分析物的分离分析特性,实现了药物药剂和中药中几种有效成份含量的测定,并发展了CE-ECL测定药物-蛋白结合常数的分析方法。
论文的主要研究内容包括以下四部分:
第一章综述
简述了毛细管电泳的发展状况及前途,对毛细管电泳的原理、分离模式、检测器种类做了简单介绍;概述了CE-ECL技术在药物、环境及生命物质分析中的应用;介绍了新型功能材料在联吡啶钌发光体系中的应用情况,并着重介绍了新型材料在ECL技术中的应用,对该分析体系发展中存在的问题进行了探讨并对其发展方向进行了展望。
第二章毛细管电泳电致化学发光法测定琥乙红霉素
铕离子掺杂普鲁士蓝化学修饰铂电极对联吡啶钌的电催化氧化作用可显著增强联吡啶钌/琥乙红霉素体系的电致化学发光强度,据此建立了毛细管电泳电致化学发光检测琥乙红霉素的分析新方法。在优化条件下,体系发光强度与琥乙红霉素的浓度在1 ?g/mL～100 ?g/mL(R=0.9993)之间呈良好线性关系,检测限为0.25 ?g/mL (S/N=3)。对浓度为10 ?g/mL的琥乙红霉素标准溶液连续测定6次,其发光强度和迁移时间的RSD分别为2.26 %和0.81 %。本法快速、灵敏、准确,且分析试样无需复杂处理可直接用于药剂及人尿中琥乙红霉素含量的测定。
第三章毛细管电泳电致化学发光法测定阿奇霉素、罗红霉素和克林霉素及其与人血清白蛋白结合常数
以PB-Eu化学修饰电极为工作电极,建立了毛细管电泳电致化学发光法同时灵敏快速分离和检测阿奇霉素、罗红霉素和克林霉素含量及其与蛋白结合常数的新方法。考察了检测电位,缓冲介质及酸度,分离缓冲液种类,添加剂等条件对电泳分离及检测结果的影响。在优化条件下,三种药物在5分钟内即可达到基线分离,其峰面积与样品浓度对阿奇霉素在0.025-2.50μg/mL之间,罗红霉素在0.50-100μg/mL之间和克林霉素在0.10-100μg/mL之间呈良好线性关系。对浓度为1.00μg/mL的混合样进行6次平行测定,其峰高和迁移时间的RSD分别在0.53-1.8%和0.53-0.57%之间。将本方法用于测定药剂中三种药物含量的测定,加标回收率在94.6%~104.6%之间。并测得三种药物与蛋白结合常数分别为阿奇霉素3.55×103 L/mol,罗红霉素1.44×104 L/mol,克林霉素1.67×104 L/mol。
第四章毛细管电泳电致化学发光法测定麻黄中的麻黄碱、伪麻黄碱和甲基麻黄碱
以PB-Eu化学修饰电极为工作电极,离子液体为拆分添加剂,首次建立了毛细管电泳电致化学发光法同时分离和检测麻黄碱、伪麻黄碱和甲基麻黄碱的新方法。考察了检测电位,缓冲介质及酸度,分离缓冲液种类,添加剂等条件对电泳分离及检测结果的影响。在优化条件下,三种药物在8分钟内可达到基线分离,其峰面积与样品浓度对甲基麻黄碱在0.025~10μg/mL之间,麻黄碱在0.025~25μg/mL之间和伪麻黄碱在0.05~10μg/mL之间呈良好的两段线性关系。对浓度为1.00μg/mL的混合样进行6次平行测定,其峰面积和迁移时间的RSD分别在3.95% ~ 4.30%和0.14% ~ 0.94%之间。将本方法用于测定药剂和中药中三种物质含量的测定,回收率在104.3%~111.2%之间。
Capillary electrophoresis (CE) with electrochemiluminescence detection (ECL) is one of the most fast developed analysis and separation technology, it is combined the high efficiency of separation of capillary electrophoresis with the high sensitivity of ECL detection. At present, Tris(2,2’-bipyridyl)ruthenium(II) (Ru(bpy)_3~(2+)) is the most widely used electrochemiluminescence reagents in CE-ECL. It has become the most favorite reagents because of its good water soluble, excellent chemical stability and reversible redox. However, the working electrode surface condition has great impact on the electrochemiluminescence efficiency of Ru(bpy)_3~(2+) in CE-ECL. In particular, in the analysis and separation of complicated samples the work electrode conditions is crucial for the determination. PB-Eu modified electrode possesses good stability, and has excellent catalytic oxidation activity on Ru(bpy)_3~(2+), so it can improve sensitivity and reproducibility of CE-ECL.
In this thesis, we study the analysis and separation characteristics of macrolide antibiotics and Ephedrine on CE-ECL by using PB-Eu modified platinum electrode as a working electrode. The contents of macrolide antibiotics in drugs and Ephedrine in Chinese herb medicines are detected. This work has enlarged the method for the determination of binding constants between drug and human serum albumin.
This paper including the following four parts:
Chapter one: Review.
A brief introduction for the current situation, the future trend, the principle, separation mode and detectors of capillary electrophoresis are given; Furthermore, the application of CE-ECL technology in medicine, environment and life analysis are summarized; Last, the application of new functional materials in Ru(bpy)_3~(2+) luminescence system and particular in ECL determined technology is reviewed. The problems existed in this system has been discussed and the development directions are prospected.
Chapter two: Determination of erythromycin ethyisuccinate(EES) by capillary electrophoresis coupled with chemically modified electrode electrochemiluminescence detector
A Eu-PB modified platinum electrode is used as working electrode of CE-ECL detector, it shows excellent electrocatalytic activity for the oxidation of Ru(bpy)_3~(2+). Therefore, the ECL intensity of Ru(bpy)_3~(2+) could be markedly enhanced for measuring EES. A simple and novel method for the determination of EES in pharmaceutical preparations has been developed. Parameters affecting separation and detection are also optimized. Good linear relationships between the peak area and the concentration of EES is observed in the range of 1μg/mL to 100μg/mL and detection limit is 0.25μg/mL (S/N=3).When the concentration of EES was 10.0μg/mL, the RSD value of migration time and peak height were 2.2% and 0.81% for six repeating detection, respectively. This method is fast, sensitive, accurate and sample can be injected for anaylsis without complicated pretreatment. It has been applied to the determination of EES in erythromycin ethyisuccinate granules drug and human urine sample.
Chapter Three: Simultaneous separation and determination of azithromycin roxithromycin and clindamycin by CZE coupled with end-column PB-Eu modified electrode electrochemiluminescence detection and application for the determination the binding constants between three compounds and human serum albumin
Based on PB-Eu chemically modified electrode as a work electrode, a fast and sensitive method for the simultaneous determination of AZM, Rox, CLI and the binding constants between the three compounds with human serum albumin has been developed. The effects factors such as the detection potential, the kinds of buffer solution, acidity and concentrations of separating buffer and additive on CE-ECL technology are investigated. Under the optimum conditions, three drugs are separated well in five minutes and the peak area and concentration of samples is good linear relationships for AZM 0.025-2.50μg/mL, for Rox 0.50-100μg/mL and for CLI 0.10-100μg/mL. When the three analytes concentration are 1.00μg/mL, the RSD of peak height and migration time are in the range of 0.53-1.8%, 0.53-0.57%, respectively (n=6). The method has been successfully applied to the determination of three analytes in pharmaceutical preparations with the recoveries from 94.6% to 104.6% .The binding constants between three drugs and human serum albumin are obtained, the binding constants for AZM, Rox and CLI are 3.55×103 L/mol,1.44×104 L/mol and 1.67×104 L/mol, respectively.
Chapter four: Simultaneous separation and determination of Ephedrine, Pseudoephedrine, Methylephedrine by CZE coupled with end-column PB-Eu modified electrode electrochemiluminescence detection
Based on PB-Eu chemically modified electrode as a work electrode, ion liquid as an additive, a novel and sensitive method for the simultaneous determination of ME, E, PE has been developed in the first time. The effect factors such as the detection potential, the kinds of buffer solution, acidity and concentration of separating buffer, on CE-ECL technology are also investigated. Under the optimum conditions, three drugs are separated well in eight minutes and the peak area is linear with the analytes concentration for ME 0.025~10μg/mL, for E 0.025~25μg/mL and for PE 0.05~10μg/mL. With the concentration of 1.0μg/mL ME, E and PE, the RSD of peak area and migration time are in the rang of 3.95%~4.30%,0.14%~0.94%, respectively(n=6).The method has been used for the determination of the three analytes in pharmaceutical preparations and in Chinese herbal Ma-Huang with the recoveries 104.3%~111.2%.
引文
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