毛细管电泳—电化学检测在疾病监测和中药分析中的应用研究
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摘要
第一章绪论
毛细管电泳(Capillary Electrophoresis, CE)是近二十年来发展较快的分析分离技术之一,具有分离效率高、分析速度快、所需样品少等特点,在生物化学、药物化学、食品化学、环境化学等许多领域有着广阔的应用前景。在绪论中回顾了CE发展的历史、现状以及研究进展,对CE的基本原理和特点进行了简单介绍,重点对毛细管电泳-电化学检测(CE-ED)技术进行了较为全面的介绍。作为一种新型高效的分离分析技术,CE-ED在21世纪仍然面临着极大的机遇和挑战,尤其是分析仪器和分析方法有待进一步的改进和完善以提高灵敏度和重现性。本论文采用CE-ED技术,测定了多种病患者生物样品中的疾病标志物和传统中药蜘蛛香中的活性成分,探讨了CE-ED技术在临床疾病的初步诊断和中药分析中的应用。
第二章毛细管电泳-电化学检测法测定尿样和水样中的丙酮醛和乙二醛
本文首次将两种非电活性标志物丙酮醛(methylglyoxal, MGo)和乙二醛(glyoxal, Go)与电活性物质2-硫代巴比妥酸衍生,得到其衍生物具有电活性,首次采用CE-ED法测定尿样和环境水样中的丙酮醛和乙二醛,优化了衍生实验和CE-ED的条件。MGo和Go分别在1.0～2000.0μg L-1和2.0～2000μgL-1浓度范围内呈良好线性,相关系数分别为0.9996和0.9995,检出限分别为0.2μgL-1和0.5μgL-1,定量限分别为1.0μgL-1和2.0μgL-1。实际样品中MGo和Go的回收率范围为90.9%～101.3%,相对标准偏差不超过2.2%,表明实验的精确度良好。实验结果表明CE-ED是一种能够对实际样品中的MGo和Go进行定量分析的方法,且既可靠又灵敏。
第三章毛细管电泳-电化学检测法测定病人尿样中儿茶酚胺及其代谢物
本实验采用CE-ED法同时分离测定了多种慢性病患者尿样中的儿茶酚胺类物质及其代谢物,肾上腺素(epinephrine, E)、去甲肾上腺素(norepinephrine, NE)、多巴胺(dopamine, DA)、变肾上腺素(又称3-甲氧基肾上腺素metanephrine, M)、高香草酸(homovanillic acid, HVA)和香草基杏仁酸(vanillymandelic acid, VMA)的浓度,考察了运行缓冲液的酸度和浓度、分离电压、氧化电位和进样时间等实验参数对分离检测的影响。在最优化条件下,以300μm碳圆盘电极为检测电极,检测电位为+0.95 V(vs.SCE),80 mmol／L硼砂缓冲运行液(pH 9.66)中,上述各组分在25 min内可实现基线分离。各组分浓度与峰电流在三个数量级内呈良好线性,检测下限(S／N=3)分别为1～5μg L-1。该方法已成功运用于肾病综合症患者尿样中儿茶酚胺类物质及其代谢物的测定,结果令人满意。
第四章毛细管电泳-电化学检测法测定蜘蛛香不同部位中的活性成分
本实验采用CE-ED法同时分离测定了蜘蛛香叶、茎和根部的8种活性成分,预处理只需简单的乙醇萃取。在最优化条件下,以16 kV为分离电压,在50mmol／L硼砂的运行缓冲液(pH 9.2)中,各组分可在23 min内实现基线分离。8种分析物浓度与峰电流在超过两个数量级的范围内呈良好线性,检测下限(S／N=3)达1.7×10-7～1.8×10-8g／mL。同时,基于各组分的电泳图谱和电化学特性,本实验还研究比较了8种分析物在蜘蛛香的不同部位中的含量差异,结果令人满意。
1. Preface
Capillary electrophoresis (CE) has been greatly developed and improved since its introduction. Now it has been widely applied in analytical chemistry, biological chemistry, food chemistry, environmental chemistry and so on, due to its speed, efficiency, reproducibility, ultra-small sample volume and ease of clearing up the contaminants. The introduction section deals with the basic principle, development history, actuality, trend and primary characteristics of CE. Capillary electrophoresis with electrochemical detection (CE-ED), as a new highly efficient technique, was rapidly developed last century. However, it also faces a lot of opportunities and challenges. Instrument needs further improvement in both reproducibility and sensitivity.
The goal of this thesis is to explore some applications in traditional medicine analysis and biochemical analysis by CE-ED.
2. A novel capillary electrophoretic method for determining methylglyxoal and glyoxal in urine and water samples
Two non-electroactive biomarkers methylglyoxal and glyoxal in urine and environmental water samples were determined for the first time by capillary electrophoresis with amperometric detection (CE-AD) after derivatising with an electroactive compound 2-thiobarbituric acid. Experiment conditions of derivatization and CE-AD detection were optimized. The calibration curve was linear over a concentration range from 1.0 to 2000.0μg L-1 for MGo and 2.0 to 2000.0μg L-1 for Go with a correlation coefficient of 0.9996 and 0.9995, respectively. The detection limit of MGo and Go were 0.2μg L-1and 0.5μg L-1, and quantitation limit were 1.0μg L-1 and 2.0μg L-1, respectively. Recoveries varied from 90.9% to 101.3% and precisions, calculated as relative standard deviation (RSD), were better than 2.0%. The proposed CE-AD method does not require sophisticated analytical instruments and provides a reliable and sensitive quantitative evaluation of MGo and Go in real sample matrices.
3. Determination of Catecholamines and their metabolites in urine samples by Capillary Electrophoresis with Electrochemical Detection
Capillary electrophoresis with electrochemical detection (CE-ED) has been employed for the separation and determination of catecholamines and their metabolites in urine samples of several patients. The effects of several factors such as the acidity and concentration of running buffer, the separation voltage, the applied potential and the injection time on CE-ED were investigated. Good linear relationship was established between peak current and concentration of analytes over three orders of magnitude with detection limits (S/N=3) ranged from 1 to 5μg L-1 for all analytes. This proposed method has been successfully applied for the study on the diagnosis of Parkinson's disease, hypertension, diabetes, nephrotic syndrome and cancer (Non-Hodgkins Lymphoma, NHL), and the assay results were satisfactory.
4. Determination of polyphenols in Valeriana wallichii DC by capillary electrophoresis with electrochemical detection
A high performance capillary electrophoresis with electrochemical detection (CE-ED) method has been developed for the analysis of eight bioactive ingredients in the leaf, stem and root of Valeriana wallichii DC after a relatively simple extraction procedure with ethanol. Under the optimum conditions, eight components can be well separated or nearly baseline separated (apigenin and luteolin peaks) within 23 min at the separation voltage of 16 kV in a 50 mM borax running buffer (pH 9.2).Notably, excellent linearity was obtained over two orders of magnitude with detection limits (S/N=3) ranged from 1.7×10-7 to 1.8×10-8 g/mL for all eight analytes. This method was also used for the comparison of the bioactive ingredient disparity in different parts of Valeriana wallichii DC based on their electropherograms or "electrochemical characteristic profiles", and the assay results were satisfactory.
毛细管电泳(Capillary Electrophoresis, CE)是近二十年来发展较快的分析分离技术之一,具有分离效率高、分析速度快、所需样品少等特点,在生物化学、药物化学、食品化学、环境化学等许多领域有着广阔的应用前景。在绪论中回顾了CE发展的历史、现状以及研究进展,对CE的基本原理和特点进行了简单介绍,重点对毛细管电泳-电化学检测(CE-ED)技术进行了较为全面的介绍。作为一种新型高效的分离分析技术,CE-ED在21世纪仍然面临着极大的机遇和挑战,尤其是分析仪器和分析方法有待进一步的改进和完善以提高灵敏度和重现性。本论文采用CE-ED技术,测定了多种病患者生物样品中的疾病标志物和传统中药蜘蛛香中的活性成分,探讨了CE-ED技术在临床疾病的初步诊断和中药分析中的应用。
第二章毛细管电泳-电化学检测法测定尿样和水样中的丙酮醛和乙二醛
本文首次将两种非电活性标志物丙酮醛(methylglyoxal, MGo)和乙二醛(glyoxal, Go)与电活性物质2-硫代巴比妥酸衍生,得到其衍生物具有电活性,首次采用CE-ED法测定尿样和环境水样中的丙酮醛和乙二醛,优化了衍生实验和CE-ED的条件。MGo和Go分别在1.0～2000.0μg L-1和2.0～2000μgL-1浓度范围内呈良好线性,相关系数分别为0.9996和0.9995,检出限分别为0.2μgL-1和0.5μgL-1,定量限分别为1.0μgL-1和2.0μgL-1。实际样品中MGo和Go的回收率范围为90.9%～101.3%,相对标准偏差不超过2.2%,表明实验的精确度良好。实验结果表明CE-ED是一种能够对实际样品中的MGo和Go进行定量分析的方法,且既可靠又灵敏。
第三章毛细管电泳-电化学检测法测定病人尿样中儿茶酚胺及其代谢物
本实验采用CE-ED法同时分离测定了多种慢性病患者尿样中的儿茶酚胺类物质及其代谢物,肾上腺素(epinephrine, E)、去甲肾上腺素(norepinephrine, NE)、多巴胺(dopamine, DA)、变肾上腺素(又称3-甲氧基肾上腺素metanephrine, M)、高香草酸(homovanillic acid, HVA)和香草基杏仁酸(vanillymandelic acid, VMA)的浓度,考察了运行缓冲液的酸度和浓度、分离电压、氧化电位和进样时间等实验参数对分离检测的影响。在最优化条件下,以300μm碳圆盘电极为检测电极,检测电位为+0.95 V(vs.SCE),80 mmol／L硼砂缓冲运行液(pH 9.66)中,上述各组分在25 min内可实现基线分离。各组分浓度与峰电流在三个数量级内呈良好线性,检测下限(S／N=3)分别为1～5μg L-1。该方法已成功运用于肾病综合症患者尿样中儿茶酚胺类物质及其代谢物的测定,结果令人满意。
第四章毛细管电泳-电化学检测法测定蜘蛛香不同部位中的活性成分
本实验采用CE-ED法同时分离测定了蜘蛛香叶、茎和根部的8种活性成分,预处理只需简单的乙醇萃取。在最优化条件下,以16 kV为分离电压,在50mmol／L硼砂的运行缓冲液(pH 9.2)中,各组分可在23 min内实现基线分离。8种分析物浓度与峰电流在超过两个数量级的范围内呈良好线性,检测下限(S／N=3)达1.7×10-7～1.8×10-8g／mL。同时,基于各组分的电泳图谱和电化学特性,本实验还研究比较了8种分析物在蜘蛛香的不同部位中的含量差异,结果令人满意。
1. Preface
Capillary electrophoresis (CE) has been greatly developed and improved since its introduction. Now it has been widely applied in analytical chemistry, biological chemistry, food chemistry, environmental chemistry and so on, due to its speed, efficiency, reproducibility, ultra-small sample volume and ease of clearing up the contaminants. The introduction section deals with the basic principle, development history, actuality, trend and primary characteristics of CE. Capillary electrophoresis with electrochemical detection (CE-ED), as a new highly efficient technique, was rapidly developed last century. However, it also faces a lot of opportunities and challenges. Instrument needs further improvement in both reproducibility and sensitivity.
The goal of this thesis is to explore some applications in traditional medicine analysis and biochemical analysis by CE-ED.
2. A novel capillary electrophoretic method for determining methylglyxoal and glyoxal in urine and water samples
Two non-electroactive biomarkers methylglyoxal and glyoxal in urine and environmental water samples were determined for the first time by capillary electrophoresis with amperometric detection (CE-AD) after derivatising with an electroactive compound 2-thiobarbituric acid. Experiment conditions of derivatization and CE-AD detection were optimized. The calibration curve was linear over a concentration range from 1.0 to 2000.0μg L-1 for MGo and 2.0 to 2000.0μg L-1 for Go with a correlation coefficient of 0.9996 and 0.9995, respectively. The detection limit of MGo and Go were 0.2μg L-1and 0.5μg L-1, and quantitation limit were 1.0μg L-1 and 2.0μg L-1, respectively. Recoveries varied from 90.9% to 101.3% and precisions, calculated as relative standard deviation (RSD), were better than 2.0%. The proposed CE-AD method does not require sophisticated analytical instruments and provides a reliable and sensitive quantitative evaluation of MGo and Go in real sample matrices.
3. Determination of Catecholamines and their metabolites in urine samples by Capillary Electrophoresis with Electrochemical Detection
Capillary electrophoresis with electrochemical detection (CE-ED) has been employed for the separation and determination of catecholamines and their metabolites in urine samples of several patients. The effects of several factors such as the acidity and concentration of running buffer, the separation voltage, the applied potential and the injection time on CE-ED were investigated. Good linear relationship was established between peak current and concentration of analytes over three orders of magnitude with detection limits (S/N=3) ranged from 1 to 5μg L-1 for all analytes. This proposed method has been successfully applied for the study on the diagnosis of Parkinson's disease, hypertension, diabetes, nephrotic syndrome and cancer (Non-Hodgkins Lymphoma, NHL), and the assay results were satisfactory.
4. Determination of polyphenols in Valeriana wallichii DC by capillary electrophoresis with electrochemical detection
A high performance capillary electrophoresis with electrochemical detection (CE-ED) method has been developed for the analysis of eight bioactive ingredients in the leaf, stem and root of Valeriana wallichii DC after a relatively simple extraction procedure with ethanol. Under the optimum conditions, eight components can be well separated or nearly baseline separated (apigenin and luteolin peaks) within 23 min at the separation voltage of 16 kV in a 50 mM borax running buffer (pH 9.2).Notably, excellent linearity was obtained over two orders of magnitude with detection limits (S/N=3) ranged from 1.7×10-7 to 1.8×10-8 g/mL for all eight analytes. This method was also used for the comparison of the bioactive ingredient disparity in different parts of Valeriana wallichii DC based on their electropherograms or "electrochemical characteristic profiles", and the assay results were satisfactory.
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