毛细管电泳—电化学检测在生物胺及中草药活性成分测定中的应用研究

  本文关键词：毛细管电泳—电化学检测在生物胺及中草药活性成分测定中的应用研究，由笔耕文化传播整理发布。

        第一章绪论毛细管电泳(Capillary Electrophoresis, CE),是一种发展迅速的新型分离分析技术,它以毛细管为分离通道,以高压直流电场为驱动力,根据被分析物特性的差异实现分离分析的技术,具有分离效率高、速度快、样品用样量少、自动化程度高等优点。CE的应用得到了飞速发展,目前毛细管电泳已被广泛用于蛋白质、氨基酸、无机离子、有机化合物、药物的分离分析,涉及食品化学、药物化学、分析化学、临床化学、环境化学等诸多学科领域。绪论简要回顾了CE技术的发展历程,介绍了其原理、分离模式、进样方式和检测技术特点,并着重介绍了CE与电化学检测器联用技术,特别是安培检测(AD)技术和电容耦合非接触电导检测(C4D)技术。根据本论文的研究内容,侧重论述了CE技术在食品和中药分析中的应用。本论文分别采用CE-AD和CE-C4D技术,着重探讨了水基质中生物胺含量的测定,以及CE-AD对中草药缬草中活性成分的分析检测,为食品和中草药的质量检验和安全监督提供了一种可选择的新方法。第二章毛细管电泳-安培检测技术在缬草生物活性成分中的检测研究本实验采用毛细管电泳—安培检测法(CE-AD)同时测定了中药缬草提取物中的八种生物药学活性成分,八种化合物分别为：刺槐黄素、香叶木素、绿原酸、山奈酚、芹黄素、木樨草素、对羟基苯甲酸和咖啡酸。该方法通过线性范围、重现性、检出限和定量限等进行了方法确证。八种分析物的检出限和定量限分别达到1.0x10-8～1.2x10-7g/mL和3.3×10-8～4.0×10-7g/mL。缬草样品通过简单的萃取程序,即可采用该方法实现分析和比较,所得电化学指纹图谱可直观显示采自不同地区的中药缬草及其不同缬草部位的电化学活性成分的含量差异。研究发现,这些活性成分的含量可能会随自然条件例如土壤、气候、湿度等的改变而发生数量级的变化。该法己成功地将缬草中上述八种药学活性成分进行分离和检测,为中草药分析提供了一种可行的方法。第三章毛细管区带电泳-安培检测技术在环境水生物胺检测中的研究本实验建立了一种基于18-冠醚-6作为缓冲液添加剂的毛细管区带电泳-安培检测方法,分离测定了环境水样中的七种生物胺,包括精胺(Spm)、亚精胺(Spd)、组胺(His)、尸胺(Cad)、苯乙胺(Phe)、酪胺(Tyr)和色胺(Try)。考察了诸如缓冲液酸度和浓度、分离电压、电极电位等实验参数对分离检测的影响,得到了最佳分离条件。在优化条件下,以铜圆盘电极为工作电极,检测电位为+650mV(vs.SCE),分离电压为14kV,在180mmol/L18-冠醚-6/20mmol/L醋酸-醋酸钠的缓冲溶液(pH3.6)中,七种生物胺在29min可实现基线分离。本方法实验结果令人满意,七种生物胺的含量检出限值可达10ng/mL,峰面积(RSD≤4.8%)和迁移时间(RSD<2.4%)的重现性良好。该方法无需样品预浓缩和衍生处理,为实际水样中多种生物胺的同步定量分析提供了一个可选择的新方法。第四章毛细管区带电泳-非接触电导检测法在环境水及酒类生物胺检测中的研究本实验建立了一种基于18-冠醚-6作为缓冲液添加剂的毛细管区带电泳-电容耦合非接触电导法(CE-C4D),实现了对环境水样和酒类中的八种生物胺,包括精胺(Spm)、亚精胺(Spd)、组胺(His)、腐胺(Put)尸胺(Cad)、苯乙胺(Phe)、酪胺(Tyr)和色胺(Try)的同时分析测定。考察了诸如激发电压和激发频率、缓冲液酸度和浓度、分离电压、进样时间等实验参数对分离检测的影响,得到了最佳分离条件：激发电压60V,激发频率550KHz,分离电压为16kV,缓冲溶液为150mmol/L18-冠醚-6/500mmol/L醋酸溶液。八种生物胺在24min能够实现基线分离。被测物浓度与峰面积在3-4个数量级范围内呈良好线性关系,最低检测限范围为(S/N=3)4.43×10-8-1.49x10-7g/mL。此法已被成功用于测定环境水和酒类中生物胺的分离和检测。

    1. PrefaceCapillary electrophoresis (CE) is a new and rapidly developing separation technology which is in buffer-filled, narrow-bore capillaries, with a high-voltage power supply providing electric field; separation by electrophoresis relies on differences of characteristics of charged species. It has the characteristics of high efficiency, high speed, ultra-small sample volume, and high degree of automation. Nowadays, CE has been used to separate protein, ammo acid, inorganic ions, organic compounds, separation and determination of pharmaceutical compounds. It has been widely applied in food chemistry, medicine chemistry, analytical chemistry, clinical chemistry, environmental chemistry, and so on. In this part, the history of CE development has been retrieved, and the principle, the separation modes, the instrumental system, the technological characteristics of CE, in particular the method of capillary chromatography with amperometric detection (CE-AD) and capillary chromatography with capacitively coupled contactless conductivity detection (CE-C4D) have been introduced. According to the research area of this thesis, the application of CE in food and traditional Chinese medicine analysis has been reviewed. In this thesis,CE-AD and CE-C D has been used separately in the determination of biogenic amines in water matrix. CE has also been applied for the study on the valeriana medicinal plants. This method would find wide application in the fields of food and traditional Chinese medicine analysis.2. Study on electrochemical profiles of valeriana medicinal plants by capillary electrophoresisA method based on amperometric detection with capillary electrophoresis has been employed for the separation and determination of pharmacologically active ingredients in extracts of Valeriana medicinal plants, including acacetin, diosmetin, chlorogenic acid, kaempferol, apigenin, luteolin, p-hydroxybenzoic and caffeic acids. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ), etc. The LODs and LOQs of eight compounds were found to be in the range from1.0×10-8to1.2×10-7and3.3×10-8to4.0×10-7g/mL,respectively. The proposed method was successfully applied for the analyses and comparison of bioactive components in Valerian samples after a relatively simple extraction procedure, and the resultant "electrochemical profiles" can intuitively demonstrate the content diversity of each electrochemically active ingredient in Valerian samples from different places and plant parts. It was found the content of bioactive ingredients may vary by an order of magnitude depending on natural conditions, e.g. soil, climate, humidity etc. This method has been successfully used in the analysis of pharmacologically active ingredients of Valeriana medicinal plants, which provided an alternative for the analysis of traditional Chinese medicine.3. Determination of biogenic amines in water matrix by zone electrophoresis with amperometric detectionA high-performance18-crown-6modified capillary zone electrophoresis with amperometric detection (CZE-AD)method is developed for the fast determination of seven biogenic amines (BAs), including spermine (Spm), spermidine (Spd), histamine (His), cadaverine (Cad), β-phenylethylamine (Phe), tyramine (Tyr) and tryptamine (Try). The effects of several important factors such as the acidity and concentration of running buffer, separation voltage, injection time and detection potential were investigated to acquire the optimum conditions. A copper disc electrode was used as the working electrode positioned carefully opposite the outlet of capillary at potential of+650mV (vs SCE). Under the optimum conditions, seven BAs could be well separated within29min at the separation voltage of14kV in a180mmol/L18-crown-6/20mmol/L acetate buffer solution (pH3.6). Extrapolated limits of detection for seven BAs were low to10ng/mL for the standard mixture. Repeatability of the peak current (<4.8%) and migration time (≤2.4%) of analytes for the method were achieved. The main advantages of the proposed CZE-AD method are no preconcentration and derivatization procedure, which provides an alternative method for the quantitative multi-analysis of BAs in real water matrix.4. Determination of biogenic amines in water matrix and wines by zone electrophoresis with capacitively coupled contactless conductivity detectionA high performance capillary zone electrophoresis with capacitively coupled contactless conductivity detection (CZE-C4D) method has been developed for the separation and sensitive determination of the eight biogenic amines, including Spermine (Spm), spermidine (Spd), histamine (His), putrescine(Put), cadaver (Cad), β-phenylethylamine (Phe), tyramine (Tyr) and tryptamine (Try).The effects of several important factors such as excitation voltage and frequency, the acidity and concentration of running buffer, separation voltage, and injection time were investigated to acquire the optimum conditions. Under the optimum conditions, BAs can be well separated within24min at the separation voltage of16kV in a150mmol/L18-crown-6/500mmol/L acetic acid solution and excitation voltage and frequency of60V and550KHz. Good linear relationships were established between the concentration and peak area of analytes over3-4orders of magnitude, the detection limits ranged from4.43×10-8～1.49×10-7g/mL. This proposed procedure has been successfully used for the analysis of water and wine samples.

        毛细管电泳—电化学检测在生物胺及中草药活性成分测定中的应用研究

摘要6-8ABSTRACT8-10第一章 绪论12-46    第一节 毛细管电泳概述12-25    第二节 毛细管电泳电化学检测技术25-28    第三节 毛细管电泳在食品、中草药分析中的应用28-38    第四节 本论文的研究目的和意义38-40    参考文献40-46第二章 毛细管电泳-安培检测技术在缬草生物活性成分中的检测研究46-60    1 引言46-48    2 实验部分48    3 结果与讨论48-57    4 结论57-58    参考文献58-60第三章 毛细管区带电泳-安培检测技术在环境水生物胺检测中的研究60-72    1 引言60-61    2 实验部分61-63    3 结果与讨论63-69    4 结论69-70    参考文献70-72第四章 毛细管区带电泳-非接触电导检测法在环境水及酒类生物胺检测中的研究72-82    1 引言72-73    2 实验部分73-74    3 结果与讨论74-80    4 结论80-81    参考文献81-82附录：硕士期间论文发表情况82-83致谢83

  本文关键词：毛细管电泳—电化学检测在生物胺及中草药活性成分测定中的应用研究，由笔耕文化传播整理发布。