毛细管电泳—安培检测联用技术在药物和化妆品分析中的应用研究
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摘要
毛细管电泳(Capillary Electrophoresis,CE)又称高效毛细管电泳(HighPerformance Capillary Electrophoresis,HPCE)或毛细管电分离法(CESM),是20世纪80年代问世并且迅速发展的一种新型液相分离分析方法,是经典电泳技术和现代微柱分离技术相结合的产物。目前,毛细管电泳技术已经广泛应用于化学、生命科学、药物和环境分析等各个领域。本论文采用毛细管电泳-安培检测技术对药物和化妆品中的电活性成分进行了分析,在一定程度上拓展了毛细管电泳-安培检测技术的应用范围。本论文主要包括以下四个部分:
第一章:绪论
简要回顾了毛细管电泳的发展历史,对毛细管电泳技术的基本原理、分离模式、联用技术等进行了介绍。同时,着重阐述了毛细管电泳-安培检测技术的研究和应用现状,以及本论文的研究目的和意义。
第二章:毛细管电泳—安培检测法测定脑血栓片中的四种活性成分
脑血栓片是我国《卫生部药品标准》收录的一种中药成方制剂,用于治疗因瘀血、肝阳上亢出现之中风先兆,如肢体麻木、头晕目眩等和脑血栓形成出现的中风不语、口眼歪斜、半身不遂等症。目前,我国已经规定了该药中各组分的含量范围,但是尚未有明确方法来对其中的具体化学成分进行分析。本章节中,我们利用毛细管电泳-安培检测法(CE-AD),对脑血栓片的四种活性成分:阿魏酸、原儿茶醛、咖啡酸和原儿茶酸进行了分离和测定。分别研究了工作电极电位、运行缓冲液的pH值和浓度、分离电压和进样时间对实验的影响。所得最佳实验条件为:检测电位为+0.95 V(vs.SCE);缓冲溶液为20mM、pH=7.6的硼酸盐缓冲溶液;分离电压和进样时间分别为18kV和8s。在上述实验条件下,四种组分在15 min内实现良好分离。被测物的线性范围超过3个数量级,检测限(S/N=3)达到6.0×10~(-9) g/mL～3.0×10~(-8) g/mL。将该方法用于实际样品的分析,获得了良好的结果。加标回收率为85.2%～93.0%,RSD均小于3.3%。
第三章:毛细管电泳—安培检测法测定中药鸡血藤及其复方制剂中的活性成分
鸡血藤为传统中药,是豆科植物密花豆的干燥藤茎,用于治疗月经不调、血虚萎黄、风湿痹痛等症。其主要活性成分包括:表儿茶素、大黄素、丁香酸、香草酸、大黄酸和原儿茶酸等。本章中,我们采用毛细管电泳-安培检测法(CE-AD)测定了鸡血藤原草及其复方物制剂中的表儿茶素、大黄素、丁香酸、香草酸、大黄酸和原儿茶酸等六种生物活性成分的含量。分别研究了工作电极电位、pH值、运行缓冲液的浓度、分离电压和进样时间对实验结果的影响。所得最佳实验条件为:检测电位为+0.95 V(vs.SCE);缓冲溶液为60mM、pH=9.0的硼砂缓冲溶液;分离电压和进样时间分别为18kV和8s。在上述条件下,六种组分在26 min内达到基线分离。各组分响应在三个数量级的范围内呈良好线性关系,检测下限(S/N=3)达5.0×10~(-8) g/mL～2.0×10~(-7) g/mL。将该方法应用于中药鸡血藤及其复方制剂中这六种活性成分的分离检测,结果令人满意。
第四章:毛细管电泳—安培检测法测定化妆品中的四种防腐剂
对羟基苯甲酸酯类(俗称“尼泊金酯”),抗真菌能力强、毒性较小,是世界上广泛使用的化妆品防腐剂。但是,用量太高会引起对皮肤的不良反应。目前,欧洲经济共同体已明确规定了该类防腐剂的使用限量,我国也已出台了相关规定。在此,我们将毛细管电泳-安培检测技术应用于化妆品中四种防腐剂:对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、对羟基苯甲酸丁酯的含量测定。分别研究了工作电极电位、运行缓冲液的pH值和浓度、分离电压和进样时间等参数对实验结果的影响。所得最佳实验条件为:检测电位为+0.95 V(vs.SCE);缓冲溶液为50mM、pH=9.2的硼砂缓冲溶液;分离电压和进样时间分别为21kV和8s。在优化的实验条件下,四种物质的响应在三个数量级范围内呈良好线性,检测下限达到0.9μmol/L～1.0μmol/L。将该方法应用于化妆品中这四种防腐剂的测定,结果良好。
以上实验证明,毛细管电泳-安培检测法灵敏度高、重现性好,是一种快速、有效的分析手段,可广泛应用于实际样品中电活性成分的分析。
Capillary electrophoresis(CE),which is also called high performance capillary electrophoresis(HPCE) or capillary electric separation method(CESM),is a new separation technique which was established and quickly developed in early 1980s.It's a combination of classic electrophoresis and modern micro-column separation technology,which is a great development in analytical chemistry field.So far,CE has been widely used in many fields including chemistry,life science,pharmaceutical and biomedical analysis,environment analysis,etc,due to its advantages in high separation power,short analysis time,small sample and reagent consumption and simplicity.Capillary electrophoresis with amperometric detection(CE-AD) is an important branch of CE.It's especially effective in the analysis of electroactive analytes.In this paper,CE-AD was employed for the determination of many electroactive ingredients in pharmaceutical and cosmetic products,and the results were satisfactory.This paper could be divided into four main parts as follows.
1.Preface
In this chapter,the history of capillary electrophoresis technology was reviewed, and the fundamental theory,basic separation models and detectors of CE were introduced.At the same time,we narrated the present studies and applications of CE-AD in detail,as well as the goal and significance of this paper.
2.Determination of Four Polyphenolic Active Ingredients from a Pharmaceutical Preparation by Capillary Zone Electrophoresis with Amperometric Detection
In the second chapter,the simultaneous determination of four active ingredients from a pharmaceutical preparation called "Nao Xue Shuan Tablets",including ferulic acid,protocatechuic aldehyde,caffeic acid and protocatechuic acid,was performed by capillary zone electrophoresis with amperometric detection(CZE-AD).The effects of working electrode potential,pH and concentration of running buffer,separation voltage and injection time on CZE-AD were investigated.Under the optimum conditions,the four analytes could be perfectly separated within 18 min in a 20 mM borate buffer.A 300μm diameter carbon-disc electrode had a good response at +0.95 V(versus SCE) for the four analytes.The response was linear over three orders of magnitude with detection limits(S/N=3) as low as 10~(-8) or 10~(-9) g/mL for the analytes. The assay results were satisfactory with recoveries in the range of 85.2～93.0%and RSDs less than 3.3%.
3.Determination of Active Ingredients of Spatholobus Suberectus Dunn and Its Medical Preparations by Capillary Zone Electrophoresis with Amperometric Detection
The third chapter was about the determination of active ingredients in Spatholobus Suberectus Dunn and its medicinal preparations by capillary electrophoresis with amperometric detection.A method based on CZE-AD was developed for the separation and determination of epicatechin,emodin,syringic acid, vanillic acid,rhein,and protocatechuic acid in Spatholobus Suberectus Dunn and its medicinal preparations.The effects of a working electrode potential,pH and concentration of running buffer,separation voltage,and injection time on CE-AD were investigated.Under the optimum conditions,the analytes could be separated in a 60 mM borate buffer(pH=9.0) within 26 min.A 300μm diameter carbon disk electrode had a good response at +0.95 V(vs.SCE) for all analytes.The response was linear over three orders of magnitude with detection limits(S/N=3) ranging from 5.0×10~(-8) g/mL to 2.0×10~(-7) g/mL for the analytes.The method had been successfully applied to the analysis of real samples,with satisfactory results.
4.Determination of Parabens in Cosmetic Products by Capillary Zone Electrophoresis with Amperometric Detection
In the fourth chapter,the simultaneous determination of methyl-(MP),ethyl-(EP), propyl-(PP) and butyl-paraben(BP) in cosmetic products was performed by capillary zone eletrophoresis with amperometric detection(CZE-AD).The effects of working electrode potential,pH and concentration of running buffer,separation voltage and injection time on CZE-AD were investigated.Under the optimum conditions,the four analytes could be perfectly separated within 15 min in a 50 mM sodium tetraborate buffer.A 300μm diameter carbon-disc electrode had a good response at +0.95 V (versus SCE) for the four analytes.The response was linear over three orders of magnitude with detection limits(S/N=3) as low as 1×10~(-6) mol/L to 9×10~(-7) mol/L for the analytes.The utility of this method was demonstrated by monitoring different kinds of cosmetics,and good results were obtained.
The results demonstrated that capillary zone electrophoresis with amperometric detection was of high sensitivity,good repeatability and could be widely used in the rapid determination of electroactive analytes in real samples.
第一章:绪论
简要回顾了毛细管电泳的发展历史,对毛细管电泳技术的基本原理、分离模式、联用技术等进行了介绍。同时,着重阐述了毛细管电泳-安培检测技术的研究和应用现状,以及本论文的研究目的和意义。
第二章:毛细管电泳—安培检测法测定脑血栓片中的四种活性成分
脑血栓片是我国《卫生部药品标准》收录的一种中药成方制剂,用于治疗因瘀血、肝阳上亢出现之中风先兆,如肢体麻木、头晕目眩等和脑血栓形成出现的中风不语、口眼歪斜、半身不遂等症。目前,我国已经规定了该药中各组分的含量范围,但是尚未有明确方法来对其中的具体化学成分进行分析。本章节中,我们利用毛细管电泳-安培检测法(CE-AD),对脑血栓片的四种活性成分:阿魏酸、原儿茶醛、咖啡酸和原儿茶酸进行了分离和测定。分别研究了工作电极电位、运行缓冲液的pH值和浓度、分离电压和进样时间对实验的影响。所得最佳实验条件为:检测电位为+0.95 V(vs.SCE);缓冲溶液为20mM、pH=7.6的硼酸盐缓冲溶液;分离电压和进样时间分别为18kV和8s。在上述实验条件下,四种组分在15 min内实现良好分离。被测物的线性范围超过3个数量级,检测限(S/N=3)达到6.0×10~(-9) g/mL～3.0×10~(-8) g/mL。将该方法用于实际样品的分析,获得了良好的结果。加标回收率为85.2%～93.0%,RSD均小于3.3%。
第三章:毛细管电泳—安培检测法测定中药鸡血藤及其复方制剂中的活性成分
鸡血藤为传统中药,是豆科植物密花豆的干燥藤茎,用于治疗月经不调、血虚萎黄、风湿痹痛等症。其主要活性成分包括:表儿茶素、大黄素、丁香酸、香草酸、大黄酸和原儿茶酸等。本章中,我们采用毛细管电泳-安培检测法(CE-AD)测定了鸡血藤原草及其复方物制剂中的表儿茶素、大黄素、丁香酸、香草酸、大黄酸和原儿茶酸等六种生物活性成分的含量。分别研究了工作电极电位、pH值、运行缓冲液的浓度、分离电压和进样时间对实验结果的影响。所得最佳实验条件为:检测电位为+0.95 V(vs.SCE);缓冲溶液为60mM、pH=9.0的硼砂缓冲溶液;分离电压和进样时间分别为18kV和8s。在上述条件下,六种组分在26 min内达到基线分离。各组分响应在三个数量级的范围内呈良好线性关系,检测下限(S/N=3)达5.0×10~(-8) g/mL～2.0×10~(-7) g/mL。将该方法应用于中药鸡血藤及其复方制剂中这六种活性成分的分离检测,结果令人满意。
第四章:毛细管电泳—安培检测法测定化妆品中的四种防腐剂
对羟基苯甲酸酯类(俗称“尼泊金酯”),抗真菌能力强、毒性较小,是世界上广泛使用的化妆品防腐剂。但是,用量太高会引起对皮肤的不良反应。目前,欧洲经济共同体已明确规定了该类防腐剂的使用限量,我国也已出台了相关规定。在此,我们将毛细管电泳-安培检测技术应用于化妆品中四种防腐剂:对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、对羟基苯甲酸丁酯的含量测定。分别研究了工作电极电位、运行缓冲液的pH值和浓度、分离电压和进样时间等参数对实验结果的影响。所得最佳实验条件为:检测电位为+0.95 V(vs.SCE);缓冲溶液为50mM、pH=9.2的硼砂缓冲溶液;分离电压和进样时间分别为21kV和8s。在优化的实验条件下,四种物质的响应在三个数量级范围内呈良好线性,检测下限达到0.9μmol/L～1.0μmol/L。将该方法应用于化妆品中这四种防腐剂的测定,结果良好。
以上实验证明,毛细管电泳-安培检测法灵敏度高、重现性好,是一种快速、有效的分析手段,可广泛应用于实际样品中电活性成分的分析。
Capillary electrophoresis(CE),which is also called high performance capillary electrophoresis(HPCE) or capillary electric separation method(CESM),is a new separation technique which was established and quickly developed in early 1980s.It's a combination of classic electrophoresis and modern micro-column separation technology,which is a great development in analytical chemistry field.So far,CE has been widely used in many fields including chemistry,life science,pharmaceutical and biomedical analysis,environment analysis,etc,due to its advantages in high separation power,short analysis time,small sample and reagent consumption and simplicity.Capillary electrophoresis with amperometric detection(CE-AD) is an important branch of CE.It's especially effective in the analysis of electroactive analytes.In this paper,CE-AD was employed for the determination of many electroactive ingredients in pharmaceutical and cosmetic products,and the results were satisfactory.This paper could be divided into four main parts as follows.
1.Preface
In this chapter,the history of capillary electrophoresis technology was reviewed, and the fundamental theory,basic separation models and detectors of CE were introduced.At the same time,we narrated the present studies and applications of CE-AD in detail,as well as the goal and significance of this paper.
2.Determination of Four Polyphenolic Active Ingredients from a Pharmaceutical Preparation by Capillary Zone Electrophoresis with Amperometric Detection
In the second chapter,the simultaneous determination of four active ingredients from a pharmaceutical preparation called "Nao Xue Shuan Tablets",including ferulic acid,protocatechuic aldehyde,caffeic acid and protocatechuic acid,was performed by capillary zone electrophoresis with amperometric detection(CZE-AD).The effects of working electrode potential,pH and concentration of running buffer,separation voltage and injection time on CZE-AD were investigated.Under the optimum conditions,the four analytes could be perfectly separated within 18 min in a 20 mM borate buffer.A 300μm diameter carbon-disc electrode had a good response at +0.95 V(versus SCE) for the four analytes.The response was linear over three orders of magnitude with detection limits(S/N=3) as low as 10~(-8) or 10~(-9) g/mL for the analytes. The assay results were satisfactory with recoveries in the range of 85.2～93.0%and RSDs less than 3.3%.
3.Determination of Active Ingredients of Spatholobus Suberectus Dunn and Its Medical Preparations by Capillary Zone Electrophoresis with Amperometric Detection
The third chapter was about the determination of active ingredients in Spatholobus Suberectus Dunn and its medicinal preparations by capillary electrophoresis with amperometric detection.A method based on CZE-AD was developed for the separation and determination of epicatechin,emodin,syringic acid, vanillic acid,rhein,and protocatechuic acid in Spatholobus Suberectus Dunn and its medicinal preparations.The effects of a working electrode potential,pH and concentration of running buffer,separation voltage,and injection time on CE-AD were investigated.Under the optimum conditions,the analytes could be separated in a 60 mM borate buffer(pH=9.0) within 26 min.A 300μm diameter carbon disk electrode had a good response at +0.95 V(vs.SCE) for all analytes.The response was linear over three orders of magnitude with detection limits(S/N=3) ranging from 5.0×10~(-8) g/mL to 2.0×10~(-7) g/mL for the analytes.The method had been successfully applied to the analysis of real samples,with satisfactory results.
4.Determination of Parabens in Cosmetic Products by Capillary Zone Electrophoresis with Amperometric Detection
In the fourth chapter,the simultaneous determination of methyl-(MP),ethyl-(EP), propyl-(PP) and butyl-paraben(BP) in cosmetic products was performed by capillary zone eletrophoresis with amperometric detection(CZE-AD).The effects of working electrode potential,pH and concentration of running buffer,separation voltage and injection time on CZE-AD were investigated.Under the optimum conditions,the four analytes could be perfectly separated within 15 min in a 50 mM sodium tetraborate buffer.A 300μm diameter carbon-disc electrode had a good response at +0.95 V (versus SCE) for the four analytes.The response was linear over three orders of magnitude with detection limits(S/N=3) as low as 1×10~(-6) mol/L to 9×10~(-7) mol/L for the analytes.The utility of this method was demonstrated by monitoring different kinds of cosmetics,and good results were obtained.
The results demonstrated that capillary zone electrophoresis with amperometric detection was of high sensitivity,good repeatability and could be widely used in the rapid determination of electroactive analytes in real samples.
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