毛细管电泳在几种药物分析中的应用
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摘要
毛细管电泳(Capillary Electrophoresis，CE)以其分离效率高，分析时间短、样品需要量少、操作费用低等优点，日益成为一种高效分离分析方法。但由于其进样体积小和检测光程短，对常用的紫外检测器而言，CE的一个主要缺陷是灵敏度较低，解决此问题的一个有效途径是采用离线或在线样品富集的方法。在线富集技术具有简单经济的特点，无需对商品仪器进行改造。样品在线富集方法如推扫、阴阳离子耗尽进样-推扫、场放大样品堆积、pH调制酸(碱)堆积、pH梯度-推扫等已经成功应用于各种样品的分析中。在系统查阅有关文献资料的基础上，进行了以下研究工作：
(1)建立了毛细管区带电泳法测定阿魏酸、绿原酸、咖啡酸等有机酸的方法。以pH=8．7的150mmol／L硼酸为缓冲溶液，在0．5psi进样8s，分离电压23kV，检测波长320nm，温度25℃的条件下进行测定。阿魏酸、绿原酸、咖啡酸的浓度在0．01～0．2mg／mL范围内与峰面积呈良好的线性关系，线性相关系数分别为0．999，0．997和0．999，检出限分别为0．2，0．4，0．2μg／mL。该方法已用于金银花、蒲公英、蜂胶、感冒止咳颗粒等实际样品中阿魏酸、绿原酸、咖啡酸含量的测定，其样品平均回收率在96．0％～100．4％之间，取得了满意的结果。
(2)建立了在线推扫富集毛细管电泳紫外检测测定药物和人血清中尼莫地平浓度的方法。以pH=1．45的50mmol／LH_3PO_4，20％乙腈和100mmol／LSDS为分离缓冲溶液，在0．5psi下进样270s，检测波长235nm，分离电压为-20kV的条件下进行测定。尼莫地平的浓度在0．2～15μg／mL范围内与其峰面积呈良好的线性关系，线性相关系数为0．998，检出限为0．1μg／mL(信噪比S／N=3：1)。
(3)建立了场放大富集毛细管电泳测定中草药草乌、川乌样品中的乌头生物碱的方法。以pH=8．0的60mmol／L硼砂，20％乙腈为分离缓冲溶液，在10kV下进样20s，分离温度20℃，检测波长200nm，分离电压10kV的条件下进行测定。乌头碱、次乌头碱和新乌头碱浓度在0．5～10μg／mL范围内与峰面积呈良好线性关系，线性相关系数分别为0．992，0．985和0．993，检出限分别为0．10，0．13和0．20μg／mL(信噪比S／N=3：1)。
Capillary electrophoresis (CE) is increasingly recognized as a highly attractive separation technique because of its high separation efficiencies, short analysis time, small sample requirements and low operation cost. However, the main drawback of CE is the poor concentration sensitivity due to the small injection volumes and a short optical path length in the most commonly used UV detection. One solution to the problems is to apply off-line or on-line sample concentration methods. The on-line concentration techniques have advantages of simplicity and economy because of no requirements of modification in CE instrumentation. Some on-line concentration techniques such as sweeping, anion or cation selective exhaustive injection-sweeping, large volume sample stacking, field-enhanced sample stacking, acid or base pH-mediated stacking and dynamic pH junction-sweeping have enjoyed much success to the analysis of many types of samples. This thesis is mainly concerned with the following aspects:
(1) A simple capillary zone electrophoresis method was developed for the determination of chlorogenic acid, caffeic acid and ferulic acid in some Chinese herbal medicines. The influence of the pH and concentration of the running buffer, separation voltage and injection time on separation was investigated. The best separation was achieved on a fused-silica capillary column (50 cm×75μm I. D.) in a running buffer of 150 mmol/L boric acid (pH = 8.7) with injection time of 8s at 0.5 psi and an applied voltage of 23 kV at 25℃. The detection was performed with diode array detection at 320 nm. The calibration curve was linear over a range of 0.01～0.2 mg/mL for ferulic acid, chlorogenic acid and caffeic acid, with correlation coefficients of 0.999, 0.997 and 0.999, respectively. The detection limits(S/N = 3:1)for ferulic acid, chlorogenic acid and caffeic acid were 0.2, 0.4 and 0.2μg/ml, respectively. The average recoveries for ferulic acid, chlorogenic acid and caffeic acid were 96.0～100.4 %. The CZE method has been successfully applied to the analysis of some organic acids in honeysuckle, taraxacum, propolis and gan mao zhi ke ke li.
(2) A sensitive and simple on-line sweeping concentration method with photodiode array detection was developed for the determination of anti hypertensive drug nimodipine. The effect of the pH and concentration of the running buffer solution, organic modifier, voltage and injection time on the concentration efficiency was investigated. An untreated fused-silica capillary was used (50 cm, effective length, 40 cm, 75μm i.d.) for the analysis. The background solution (BGS) was 50 mmol/L H_3PO_4 (pH 1.45) and 100 mmol/L SDS containing 20 % acetonitrile with an applied voltage of -20 kV at 25℃. Sample introduction was performed at 0.5 psi for 270 s with diode array detection at 235 nm. In the method validation, the calibration curve was linear over a range of 0.2～15μg/mL for nimodipine with a correlation coefficient of 0.998. The detection limit (S/N = 3:1) of nimodipine was 0.10μg/mL. About 100-fold improvement in concentration sensitivity was achieved in terms of peak height by the sweeping method compared to conventional injection method. The Sweeping-MEKC method has been successfully applied to the analysis of nimodipine in tablet and human serum.
(3) A novel method for the determination of aconitum alkaloid in Chinese herbs by capillary electrophoresis field-enhanced sample stacking technology was developed. The effect of the pH and concentration of the running buffer solution, organic modifier, voltage and injection time on the concentration efficiency were investigated. After experimental optimizations, the best separation was achieved in 60 mmol/L borax (pH 8.0) containing 20 % (v/v) acetonitrile, with an applied voltage of 10 kV at 20℃. The detection was performed with diode array detection at 200 nm. The calibration curve was linear over a range of 0.5～10μg/mL for aconitine, hypaconitine, and mesaconitine, with correlation coefficients of 0.992, 0.985 and 0.993, respectively. The detection limits (S/N = 3:1) for aconitine, mesaconitine, and hypaconitine were 0.10, 0.13 and 0.20μg/ml, respectively.
(1)建立了毛细管区带电泳法测定阿魏酸、绿原酸、咖啡酸等有机酸的方法。以pH=8．7的150mmol／L硼酸为缓冲溶液，在0．5psi进样8s，分离电压23kV，检测波长320nm，温度25℃的条件下进行测定。阿魏酸、绿原酸、咖啡酸的浓度在0．01～0．2mg／mL范围内与峰面积呈良好的线性关系，线性相关系数分别为0．999，0．997和0．999，检出限分别为0．2，0．4，0．2μg／mL。该方法已用于金银花、蒲公英、蜂胶、感冒止咳颗粒等实际样品中阿魏酸、绿原酸、咖啡酸含量的测定，其样品平均回收率在96．0％～100．4％之间，取得了满意的结果。
(2)建立了在线推扫富集毛细管电泳紫外检测测定药物和人血清中尼莫地平浓度的方法。以pH=1．45的50mmol／LH_3PO_4，20％乙腈和100mmol／LSDS为分离缓冲溶液，在0．5psi下进样270s，检测波长235nm，分离电压为-20kV的条件下进行测定。尼莫地平的浓度在0．2～15μg／mL范围内与其峰面积呈良好的线性关系，线性相关系数为0．998，检出限为0．1μg／mL(信噪比S／N=3：1)。
(3)建立了场放大富集毛细管电泳测定中草药草乌、川乌样品中的乌头生物碱的方法。以pH=8．0的60mmol／L硼砂，20％乙腈为分离缓冲溶液，在10kV下进样20s，分离温度20℃，检测波长200nm，分离电压10kV的条件下进行测定。乌头碱、次乌头碱和新乌头碱浓度在0．5～10μg／mL范围内与峰面积呈良好线性关系，线性相关系数分别为0．992，0．985和0．993，检出限分别为0．10，0．13和0．20μg／mL(信噪比S／N=3：1)。
Capillary electrophoresis (CE) is increasingly recognized as a highly attractive separation technique because of its high separation efficiencies, short analysis time, small sample requirements and low operation cost. However, the main drawback of CE is the poor concentration sensitivity due to the small injection volumes and a short optical path length in the most commonly used UV detection. One solution to the problems is to apply off-line or on-line sample concentration methods. The on-line concentration techniques have advantages of simplicity and economy because of no requirements of modification in CE instrumentation. Some on-line concentration techniques such as sweeping, anion or cation selective exhaustive injection-sweeping, large volume sample stacking, field-enhanced sample stacking, acid or base pH-mediated stacking and dynamic pH junction-sweeping have enjoyed much success to the analysis of many types of samples. This thesis is mainly concerned with the following aspects:
(1) A simple capillary zone electrophoresis method was developed for the determination of chlorogenic acid, caffeic acid and ferulic acid in some Chinese herbal medicines. The influence of the pH and concentration of the running buffer, separation voltage and injection time on separation was investigated. The best separation was achieved on a fused-silica capillary column (50 cm×75μm I. D.) in a running buffer of 150 mmol/L boric acid (pH = 8.7) with injection time of 8s at 0.5 psi and an applied voltage of 23 kV at 25℃. The detection was performed with diode array detection at 320 nm. The calibration curve was linear over a range of 0.01～0.2 mg/mL for ferulic acid, chlorogenic acid and caffeic acid, with correlation coefficients of 0.999, 0.997 and 0.999, respectively. The detection limits(S/N = 3:1)for ferulic acid, chlorogenic acid and caffeic acid were 0.2, 0.4 and 0.2μg/ml, respectively. The average recoveries for ferulic acid, chlorogenic acid and caffeic acid were 96.0～100.4 %. The CZE method has been successfully applied to the analysis of some organic acids in honeysuckle, taraxacum, propolis and gan mao zhi ke ke li.
(2) A sensitive and simple on-line sweeping concentration method with photodiode array detection was developed for the determination of anti hypertensive drug nimodipine. The effect of the pH and concentration of the running buffer solution, organic modifier, voltage and injection time on the concentration efficiency was investigated. An untreated fused-silica capillary was used (50 cm, effective length, 40 cm, 75μm i.d.) for the analysis. The background solution (BGS) was 50 mmol/L H_3PO_4 (pH 1.45) and 100 mmol/L SDS containing 20 % acetonitrile with an applied voltage of -20 kV at 25℃. Sample introduction was performed at 0.5 psi for 270 s with diode array detection at 235 nm. In the method validation, the calibration curve was linear over a range of 0.2～15μg/mL for nimodipine with a correlation coefficient of 0.998. The detection limit (S/N = 3:1) of nimodipine was 0.10μg/mL. About 100-fold improvement in concentration sensitivity was achieved in terms of peak height by the sweeping method compared to conventional injection method. The Sweeping-MEKC method has been successfully applied to the analysis of nimodipine in tablet and human serum.
(3) A novel method for the determination of aconitum alkaloid in Chinese herbs by capillary electrophoresis field-enhanced sample stacking technology was developed. The effect of the pH and concentration of the running buffer solution, organic modifier, voltage and injection time on the concentration efficiency were investigated. After experimental optimizations, the best separation was achieved in 60 mmol/L borax (pH 8.0) containing 20 % (v/v) acetonitrile, with an applied voltage of 10 kV at 20℃. The detection was performed with diode array detection at 200 nm. The calibration curve was linear over a range of 0.5～10μg/mL for aconitine, hypaconitine, and mesaconitine, with correlation coefficients of 0.992, 0.985 and 0.993, respectively. The detection limits (S/N = 3:1) for aconitine, mesaconitine, and hypaconitine were 0.10, 0.13 and 0.20μg/ml, respectively.
引文
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