电动微分析系统及其在药物分析中的应用研究
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摘要
本文应用毛细管电泳领域相关理论与技术对毛细管区带电泳、微流控芯片电泳和整体毛细管电色谱在药物分析中的应用进行了研究。
1毛细管电泳理论研究及其在药物分析中的应用
1．1推导了一个毛细管电泳峰分离度的表达式t_s=(W_(inj)+W_(det))/(Δv-v_d)。由上式可得出一定操作条件下，所需毛细管柱的最短柱长为t_sv_1。该式对一般的色谱分析和毛细管电色谱的条件优化也具有指导意义。
1．2指出了毛细管电渗流产生与实验之间存在的矛盾，提出了一个关于电渗流产生机制的假设：在电场作用下，由于双电层扩散层对缓冲液中同号离子的“电润滑”作用和对缓冲液中异号离子的“电阻滞”作用，使缓冲液中的正负离子相向运动产生的对本体缓冲液的推动力平衡被打破，从而产生与扩散层离子泳动方向一致的“净推动力”，使电渗流产生。同时，毛细管内壁对缓冲液的一般阻滞作用对电渗流的大小也有重要影响。利用该假设对电渗流平头流型的成因、毛细管内径、温度、缓冲液浓度和有机改性剂等对电渗流的影响重新进行了解释，这也有助于不同种类毛细管电色谱整体柱柱床的设计和分析条件的优化。
1．3对影响毛细管电泳重现性的因素与控制方法进行了总结和讨论，阐明了对分析过程的综合控制有助于结果重现性的改善。
1．4采用毛细管区带电泳法对曲克芦丁制剂中三羟乙基芦丁的含量，板蓝根注射液中胞苷、腺苷、鸟苷和尿苷的含量进行了测定。同时采用毛细管电泳—质谱／质谱方法对曲克芦丁及其主要杂质进行了鉴定。
2微流控芯片检测系统的建立及其在药物分析中的应用
本文研制了一台正交结构的微流控芯片激光诱导荧光检测器，结构简单，体积较小，对微流控芯片的尺寸与通道结构适应性强，操作灵活方便，并可采用不同波长的激光与滤光片来选择不同的测定条件。利用自组建的显微成像系统对芯片的弯道效应进行了观察。提出了芯片电泳组分进样时间与其迁移时间的关系式t_(inj)=(l_(inj)·E_(sep))/(l_(sep)·E_(inj))·t_m。以维生素B_2为实际样品，激发波长473nm，检测波长520hm，考察了芯片通道结构的设计、芯片通道的处理过程、分析条件的优化和结果重现性等。还对安非他明类毒品的芯片电泳分离分析进行了初步考察。由于药物大多具有紫外吸收，本文同时设计了一套芯片紫外检测系统，采用通道尾部接合石英毛细管的杂交芯片，对联磺甲氧苄啶片中的三组分、冬虫夏草中的三种核苷类成分进行了分析。结果表明微流控芯片电泳分析在药物分析中具有一定的实用性，有进一步深入研究的价值。
3毛细管电色谱基本理论及其在药物分析中的应用
3．1在组分的电泳因素基本不影响其色谱行为的前提下，推导出新的组分保留因子表达式k_(CEC)=k′-μ_(ep)/(μ_(eo)+μ_(ep))(Ⅰ)及k_(CEC)=k′-(μ_(ep)-μ_(ep)~0)/(μ_(eo)+μ_(ep))(Ⅱ)，两者互为补充。同时对毛细管电色谱文献中组分保留因子的两种表达式k_(CEC)=k′+k′μ_(ep)/μ_(eo)+μ_(ep)/μ_(eo)(Ⅲ)及k_(CEC)=(k′-μ_(ep)/μ_(eo))/(1+μ_(ep)/μ_(eo))(Ⅳ)进行了讨论，指出了表达式(Ⅲ)推导中引用组分电泳迁移距离的错误。当μ_(eo)=0时，(Ⅱ)式能够弥补表达式(Ⅰ)和(Ⅳ)式的不足，有效反映组分的μ_(ep)和k′对k_(CEC)的影响。(Ⅰ)和(Ⅱ)式能够使组分保留因子始终反映出组分色谱与电泳行为的综合作用。
3．2制备了四种毛细管电色谱整体柱：分别以BMA和LMA为单体，均含电渗流引发剂AMPS的两种整体柱与分别以BMA和LMA为单体均不含AMPS的两种整体柱。结果表明，采用阴离子表面活性剂对不含电渗流引发剂的两种电色谱整体柱进行表面修饰，电渗流产生状况一致，分离性能相似。而含电渗流引发剂的LMA柱电渗流小且不稳定，组分峰形差。电渗流引发剂似乎只适用于短碳链固定相的情况。
3．3采用含电渗流引发剂的BMA柱对联磺甲氧苄啶片中的三组分进行了分离，对曲克芦丁片中曲克芦丁的含量、冬虫夏草中胞苷和腺苷的含量进行了测定。结果表明整体毛细管电色谱法对于中药和西药的分析都具有很大的应用潜力。虽柱效较低，但方法的重现性和耐用性等优于相应的毛细管区带电泳。整体毛细管电色谱在药物分析中的应用前景广阔。
Theories of capillary zone electrophoresis, microfluidic chip analysis and monolithic capillary electrochromatography and their applications in pharmaceutical analysis were investigated and demonstrated in the paper.1 Theoretical studies on CE and the applications of CE in pharmaceutical analysis1.1 An expression that permits the determination of the resolution froman electropherogram was firstly formulated. The least length of a capillary column was t_sv_1. The expression should be useful in the optimization of common chromatographicanalysis and CEC.1.2 The conflicts between the motivity of EOF and the experimental phenomena in CE were described, and a hypothesis on the mechanism of EOF development was presented for the first time: under the applied voltage, the diffuse layer of the EDL acted as a stationary phase and had a chromatographic function to the ions in buffer, the co-ions in buffer which had the same charge of the ions in the diffuse layer were "lubricated", while the counter-ions in buffer were "retarded". So, the balance of the push forces to the bulk buffer in two directions was broken and a net push force produced, and thus formed the motivity of EOF in the direction of the electrophoresis of the ions in the diffuse layer. The inhibition of the capillary inner wall to the bulk solution was also played an important role in the process of EOF. The flat profile mechanism and how the capillary inside diameter, buffer concentration, temperature and added modifiers, etc. influenced EOF were re-explained according to the assumption, and this will be helpful for the design of different kinds of monolithic capillary electrochromatographic bed and the optimization of its operational condition.1.3 Factors that influenced the reproducibility in CE and its controls were summarized and discussed. The reproducibility of the results could be improved when the analytical process was synthetically controlled.1.4 The content of troxerutin in troxerutin preparations and the contents of Cytidine, Adenosine, Guanosine and Undine in Banlangen injection were determined by CE. Troxerutin and its impurities were also identified by CE-MS/MS.2 The establishment of microfluidic chip analytical systems and their applications in pharmaceutical analysis
A small-scale LIF detector with simple orthogonal structure for the chip analysis was developed. It was seasoned with chips of different size and channel network. The position of the detection could be adjusted conveniently. The replacement of the laser and the filter with different wavelength could adapt to the corresponding condition. The turn-induced broadening named turn effect was observed through a set of homemade micro=imagining system. The expression t_(inj)=(l_(inj)·E_(sep))/(l_(sep)·E_(inj))·t_m, which described the relationship between the injection time and the migration time of a component in chip analysis, was given. Vitamin B_2 was analyzed with excited wavelength 473nm and emission wavelength 520nm. The design of the channel structure, the treatment of the channel, the operational condition optimizing and the reproduction of the results were investigated. The primary separation of amphetamine kind drugs on microchip was also carried out. A set of UV detection system for chip analysis was also established since many medicines had UV absorbanee. Three components in sulfamethoxazole, sulfadiazine and trimethoprim tablets and three nueleotides in aweto were analyzed by the designed UV system, using the hybridized chip with a length of fused silica capillary was conglutinated to the channel end. The results demonstrated the feasibility of chip analysis for pharmaceutical analysis. Chip analysis is of value for further investigation.
3 Theoretical study on CEC and the applications of CEC in pharmaceutical analysis
3.1 On the assumption that component electrophoresis factor basically did not have parametric interaction with its chromatographic behavior in CEC, two new retention factor expressions k_(CEC)=k′-μ_(ep)/(μ_(eo)+μ_(ep))(Ⅰ)and k_(CEC)=k′-(μ_(ep)-μ_(ep)~0)/(μ_(eo)+μ_(ep))(Ⅱ) was deduced, and they can complement each other. Two expressions of component retention factor in literatures k_(CEC)=k′+k′μ_(ep)/μ_(eo)+μ_(ep)/μ_(eo)(Ⅲ) and k_(CEC)=(k′-μ_(ep)/μ_(eo))/(1+μ_(ep)/μ_(eo))(Ⅳ) were diseussed. Wrong citation of the component electrophoresis distance expression in the deduction of expression (Ⅲ) was pointed out. The expression (Ⅱ) could make up the limitation of above expressions, especially whenμ_(eo)=0. Expressions (Ⅰ)and (Ⅱ)could reflect the integrated effect of component chromatographic and electrophoresis behaviors.
3.2 Four kinds of MLCEC column with or without AMPS as an EOF initiator using BMA and LMA, respectively, were prepared. The performance of the EOF on the four columns was investigated. The results indicated that the performances of the two columns without AMPS were consistent when modified using anionic surfactant. The EOF on the LMA column with AMPS was smaller and not stable causing poor peak shape. EOF initiator seems only applicable to the short chain stationary phase.
3.3 The separation of three components in sulfamethoxazole, sulfadiazine and trimethoprim tablets and the determination of two nucleotides in aweto, and troxerutin in troxerutin tablets were performed by the BMA column with AMPS. The reproducibility and the ruggedness of MLCEC were better than those of CE though with lower column efficiency. The results indicated that M.LCEC had the broad prospects in pharmaceutical analysis.
1毛细管电泳理论研究及其在药物分析中的应用
1．1推导了一个毛细管电泳峰分离度的表达式t_s=(W_(inj)+W_(det))/(Δv-v_d)。由上式可得出一定操作条件下，所需毛细管柱的最短柱长为t_sv_1。该式对一般的色谱分析和毛细管电色谱的条件优化也具有指导意义。
1．2指出了毛细管电渗流产生与实验之间存在的矛盾，提出了一个关于电渗流产生机制的假设：在电场作用下，由于双电层扩散层对缓冲液中同号离子的“电润滑”作用和对缓冲液中异号离子的“电阻滞”作用，使缓冲液中的正负离子相向运动产生的对本体缓冲液的推动力平衡被打破，从而产生与扩散层离子泳动方向一致的“净推动力”，使电渗流产生。同时，毛细管内壁对缓冲液的一般阻滞作用对电渗流的大小也有重要影响。利用该假设对电渗流平头流型的成因、毛细管内径、温度、缓冲液浓度和有机改性剂等对电渗流的影响重新进行了解释，这也有助于不同种类毛细管电色谱整体柱柱床的设计和分析条件的优化。
1．3对影响毛细管电泳重现性的因素与控制方法进行了总结和讨论，阐明了对分析过程的综合控制有助于结果重现性的改善。
1．4采用毛细管区带电泳法对曲克芦丁制剂中三羟乙基芦丁的含量，板蓝根注射液中胞苷、腺苷、鸟苷和尿苷的含量进行了测定。同时采用毛细管电泳—质谱／质谱方法对曲克芦丁及其主要杂质进行了鉴定。
2微流控芯片检测系统的建立及其在药物分析中的应用
本文研制了一台正交结构的微流控芯片激光诱导荧光检测器，结构简单，体积较小，对微流控芯片的尺寸与通道结构适应性强，操作灵活方便，并可采用不同波长的激光与滤光片来选择不同的测定条件。利用自组建的显微成像系统对芯片的弯道效应进行了观察。提出了芯片电泳组分进样时间与其迁移时间的关系式t_(inj)=(l_(inj)·E_(sep))/(l_(sep)·E_(inj))·t_m。以维生素B_2为实际样品，激发波长473nm，检测波长520hm，考察了芯片通道结构的设计、芯片通道的处理过程、分析条件的优化和结果重现性等。还对安非他明类毒品的芯片电泳分离分析进行了初步考察。由于药物大多具有紫外吸收，本文同时设计了一套芯片紫外检测系统，采用通道尾部接合石英毛细管的杂交芯片，对联磺甲氧苄啶片中的三组分、冬虫夏草中的三种核苷类成分进行了分析。结果表明微流控芯片电泳分析在药物分析中具有一定的实用性，有进一步深入研究的价值。
3毛细管电色谱基本理论及其在药物分析中的应用
3．1在组分的电泳因素基本不影响其色谱行为的前提下，推导出新的组分保留因子表达式k_(CEC)=k′-μ_(ep)/(μ_(eo)+μ_(ep))(Ⅰ)及k_(CEC)=k′-(μ_(ep)-μ_(ep)~0)/(μ_(eo)+μ_(ep))(Ⅱ)，两者互为补充。同时对毛细管电色谱文献中组分保留因子的两种表达式k_(CEC)=k′+k′μ_(ep)/μ_(eo)+μ_(ep)/μ_(eo)(Ⅲ)及k_(CEC)=(k′-μ_(ep)/μ_(eo))/(1+μ_(ep)/μ_(eo))(Ⅳ)进行了讨论，指出了表达式(Ⅲ)推导中引用组分电泳迁移距离的错误。当μ_(eo)=0时，(Ⅱ)式能够弥补表达式(Ⅰ)和(Ⅳ)式的不足，有效反映组分的μ_(ep)和k′对k_(CEC)的影响。(Ⅰ)和(Ⅱ)式能够使组分保留因子始终反映出组分色谱与电泳行为的综合作用。
3．2制备了四种毛细管电色谱整体柱：分别以BMA和LMA为单体，均含电渗流引发剂AMPS的两种整体柱与分别以BMA和LMA为单体均不含AMPS的两种整体柱。结果表明，采用阴离子表面活性剂对不含电渗流引发剂的两种电色谱整体柱进行表面修饰，电渗流产生状况一致，分离性能相似。而含电渗流引发剂的LMA柱电渗流小且不稳定，组分峰形差。电渗流引发剂似乎只适用于短碳链固定相的情况。
3．3采用含电渗流引发剂的BMA柱对联磺甲氧苄啶片中的三组分进行了分离，对曲克芦丁片中曲克芦丁的含量、冬虫夏草中胞苷和腺苷的含量进行了测定。结果表明整体毛细管电色谱法对于中药和西药的分析都具有很大的应用潜力。虽柱效较低，但方法的重现性和耐用性等优于相应的毛细管区带电泳。整体毛细管电色谱在药物分析中的应用前景广阔。
Theories of capillary zone electrophoresis, microfluidic chip analysis and monolithic capillary electrochromatography and their applications in pharmaceutical analysis were investigated and demonstrated in the paper.1 Theoretical studies on CE and the applications of CE in pharmaceutical analysis1.1 An expression that permits the determination of the resolution froman electropherogram was firstly formulated. The least length of a capillary column was t_sv_1. The expression should be useful in the optimization of common chromatographicanalysis and CEC.1.2 The conflicts between the motivity of EOF and the experimental phenomena in CE were described, and a hypothesis on the mechanism of EOF development was presented for the first time: under the applied voltage, the diffuse layer of the EDL acted as a stationary phase and had a chromatographic function to the ions in buffer, the co-ions in buffer which had the same charge of the ions in the diffuse layer were "lubricated", while the counter-ions in buffer were "retarded". So, the balance of the push forces to the bulk buffer in two directions was broken and a net push force produced, and thus formed the motivity of EOF in the direction of the electrophoresis of the ions in the diffuse layer. The inhibition of the capillary inner wall to the bulk solution was also played an important role in the process of EOF. The flat profile mechanism and how the capillary inside diameter, buffer concentration, temperature and added modifiers, etc. influenced EOF were re-explained according to the assumption, and this will be helpful for the design of different kinds of monolithic capillary electrochromatographic bed and the optimization of its operational condition.1.3 Factors that influenced the reproducibility in CE and its controls were summarized and discussed. The reproducibility of the results could be improved when the analytical process was synthetically controlled.1.4 The content of troxerutin in troxerutin preparations and the contents of Cytidine, Adenosine, Guanosine and Undine in Banlangen injection were determined by CE. Troxerutin and its impurities were also identified by CE-MS/MS.2 The establishment of microfluidic chip analytical systems and their applications in pharmaceutical analysis
A small-scale LIF detector with simple orthogonal structure for the chip analysis was developed. It was seasoned with chips of different size and channel network. The position of the detection could be adjusted conveniently. The replacement of the laser and the filter with different wavelength could adapt to the corresponding condition. The turn-induced broadening named turn effect was observed through a set of homemade micro=imagining system. The expression t_(inj)=(l_(inj)·E_(sep))/(l_(sep)·E_(inj))·t_m, which described the relationship between the injection time and the migration time of a component in chip analysis, was given. Vitamin B_2 was analyzed with excited wavelength 473nm and emission wavelength 520nm. The design of the channel structure, the treatment of the channel, the operational condition optimizing and the reproduction of the results were investigated. The primary separation of amphetamine kind drugs on microchip was also carried out. A set of UV detection system for chip analysis was also established since many medicines had UV absorbanee. Three components in sulfamethoxazole, sulfadiazine and trimethoprim tablets and three nueleotides in aweto were analyzed by the designed UV system, using the hybridized chip with a length of fused silica capillary was conglutinated to the channel end. The results demonstrated the feasibility of chip analysis for pharmaceutical analysis. Chip analysis is of value for further investigation.
3 Theoretical study on CEC and the applications of CEC in pharmaceutical analysis
3.1 On the assumption that component electrophoresis factor basically did not have parametric interaction with its chromatographic behavior in CEC, two new retention factor expressions k_(CEC)=k′-μ_(ep)/(μ_(eo)+μ_(ep))(Ⅰ)and k_(CEC)=k′-(μ_(ep)-μ_(ep)~0)/(μ_(eo)+μ_(ep))(Ⅱ) was deduced, and they can complement each other. Two expressions of component retention factor in literatures k_(CEC)=k′+k′μ_(ep)/μ_(eo)+μ_(ep)/μ_(eo)(Ⅲ) and k_(CEC)=(k′-μ_(ep)/μ_(eo))/(1+μ_(ep)/μ_(eo))(Ⅳ) were diseussed. Wrong citation of the component electrophoresis distance expression in the deduction of expression (Ⅲ) was pointed out. The expression (Ⅱ) could make up the limitation of above expressions, especially whenμ_(eo)=0. Expressions (Ⅰ)and (Ⅱ)could reflect the integrated effect of component chromatographic and electrophoresis behaviors.
3.2 Four kinds of MLCEC column with or without AMPS as an EOF initiator using BMA and LMA, respectively, were prepared. The performance of the EOF on the four columns was investigated. The results indicated that the performances of the two columns without AMPS were consistent when modified using anionic surfactant. The EOF on the LMA column with AMPS was smaller and not stable causing poor peak shape. EOF initiator seems only applicable to the short chain stationary phase.
3.3 The separation of three components in sulfamethoxazole, sulfadiazine and trimethoprim tablets and the determination of two nucleotides in aweto, and troxerutin in troxerutin tablets were performed by the BMA column with AMPS. The reproducibility and the ruggedness of MLCEC were better than those of CE though with lower column efficiency. The results indicated that M.LCEC had the broad prospects in pharmaceutical analysis.
引文
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