毛细管电泳—电化学发光用于含胺类药物的检测
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摘要
电致化学发光(Electrochemiluminescence,简称ECL),是在化学发光基础上发展起来的一种新的分析方法,它将电化学与化学发光结合起来,使该方法具有灵敏、原位、线性范围宽、稳定性好和选择性强等特点。毛细管电泳(Capillary electrophoresis,简称CE)是二十世纪八十年代兴起的液相分离分析技术,具有分离效率高、分析速度快、样品用量少、装置简单等优点。将毛细管电泳(CE)分离-联吡啶钌[Ru(bpy)32+]-电致化学发光(ECL)检测联用(CE-ECL)是最近几年兴起的分离分析技术,它兼有CE的微量、高效、快速及ECL的灵敏、原位、高选择性和高重现性等优点。该方法在药物特别是含胺类药物的分析方面已经显示出了强大的优势,已成功应用于药物、氨基酸、毒品和环境等方面的检测。CE-ECL联吡啶钌联用技术作为一种分析测试手段在医学、环境科学及生命科学等方面有着广阔的应用前景。
本论文中,主要应用毛细管电泳-电化学发光对含胺类药物进行检测。各项研究工作简述如下:
1.基于甲磺酸培氟沙星(Pefloxacin Mesylate,简称PM)对联吡啶钌在铂电极上的电致化学发光信号有增敏作用,与毛细管电泳结合,建立了一种研究PM药代动力学的新方法。在最佳条件下,该药物在0.05～10 mg/L范围内峰高与PM浓度呈良好的线性关系,该方法检出限为0.006 mg/L,峰高相对标准偏差为2.4%(n=6),尿样回收率在92.9～112%之间。该方法用于人尿中PM的排泄速率测定,1.5～2 h内平均排泄速率最大,48 h内原型药物排泄率为13.5%。
2.基于盐酸丁咯地尔(Buflomedil Hydrochloride,简称BUF)和盐酸维拉帕米(Verapamil hydrochloride,简称,VER)在铂电极上对联吡啶钌电化学发光强烈的增敏作用,与毛细管电泳联用,同时测定人血浆中的BUF和VER。为了提高灵敏度,采用0.1 mmol/L醋酸作为溶剂。在最佳条件下,BUF的线性范围为0.001～3 mg/L,检出限为0.0007 mg/L,VER的线性范围为0.01～12 mg/L,检出限为0.0047 mg/L。BUF和VER的回收率分别为85～92%和85～93%。该方法应用于人血浆中BUF和VER的测定,血浆中杂质与样品能够完全分离。
3.首次应用CE-ECL对盐酸麻黄碱(Ephedrine Hydrochloride,简称EP)进行检测,为提高灵敏度,用乙醛对其进行衍生,在最佳条件下,衍生后光强增强了近50倍。该药物的线性范围为0.078～11.7 mg/L,检出限为0.002 mg/L,回收率在93～108%之间,RSD低于5.9%。将此法应用于被盐酸麻黄碱稀释过的人血浆中测定,血浆中杂质与样品能够完全分离。
Electrochemiluminescence (ECL), which combines the characteristics of electrochemistry and chemiluminescence, is proved to be a powerful analytical tool with advantages of sensitivity, in situ, wide linear range, and good selectivity etc. Capillary electrophoresis (CE) has been greatly evolved since 1980s, now it has been proved to be an effective technique for separation with short analysis time, high resolution and small sample volume. The combination of capillary electrophoresis with tris (2, 2’-bipyridyl) ruthenium (II)-based electrochemiluminescence detection (CE-ECL) shows many advantages, for instance, high selectivity, good efficiency, fast analysis speed, and easy operation, etc. Now, the method has been widely used for the detection of nitrogen-containing compounds, and it has been successfully applyzed to pharmaceuticals, amino acids, narcotics, and environmental samples etc. As a method of analysis, CE-ECL will be more widely use in medical science, environmental science, and life science.
This thesis is concentrated on the application of CE-ECL for the analyses of amine-containing pharmaceuticals. Its contents were intruduced as follows:
1. A novel and simple pharmacokinetics of Pefloxacin mesylate (PM) in the urine of a healthy adult was developed. The proposed methodology was based on the ECL intensity of ruthenium (II) tris (bipyridine) at a platinum electrode was greatly enhanced by PM. Under the optimal conditions, the calibration curve was linear over the range from 0.05 to 10 mg/L with a detection limit of 0.006 mg/L (σ=3). The relative standard deviation of the peak height was 2.4% (n=6). The recovery in human urine was between 92.9% and 112%. The highest excretion rate was observed during 1.5 h to 2 h after oral administration. The urinary excretion ratio of PM was 13.5% within 48 h.
2. Based on electrochemiluminescence (ECL) intensity of ruthenium(II) tris (bipyridine) at a platinum electrode was greatly enhanced by Buflomedil hydrochloride (BUF) and Verapamil hydrochloride (VER), the simultaneous determination of BUF and VER in human plasma has been developed using capillary electrophoresis with ECL. In order to increase the sensitivity, 0.1 mmol/L acetic acid was selected as sample solvent. Under the optimal conditions,the linear ranges were 0.001～3 mg/L for BUF and 0.01～12 mg/L for VER. The detection limits (3σ) for BUF and VER were 0.0007 and 0.0047 mg/L, respectively. The recoveries of the two drugs at different concentrations were between 85% and 92% for BUF and 85% to 93% for VER. Plasma constituents did not interfer with the determination of BUF and VER.
3. A novel method for the determination of ephedrine hydrochloride (EP) was proposed. In order to improve the sensitivity, the precolumn derivatization of EP with acetaldehyde was proposed. Under the optimal conditions, the ECL intensity obtained about 50-fold improvement, compared to that wasn’t derived. The calibration curve was linear over the range from 0.078 to 11.7 mg/L with a detection limit of 0.002 mg/L (σ=3). All of the values of RSD were lower than 5.9% (n=6), and the recovery was between 93% and 108%. The mothod was applied to the detection of EP extracted from human plasma. The impurities in the plasma didn't interfer with the determination of EP.
本论文中,主要应用毛细管电泳-电化学发光对含胺类药物进行检测。各项研究工作简述如下:
1.基于甲磺酸培氟沙星(Pefloxacin Mesylate,简称PM)对联吡啶钌在铂电极上的电致化学发光信号有增敏作用,与毛细管电泳结合,建立了一种研究PM药代动力学的新方法。在最佳条件下,该药物在0.05～10 mg/L范围内峰高与PM浓度呈良好的线性关系,该方法检出限为0.006 mg/L,峰高相对标准偏差为2.4%(n=6),尿样回收率在92.9～112%之间。该方法用于人尿中PM的排泄速率测定,1.5～2 h内平均排泄速率最大,48 h内原型药物排泄率为13.5%。
2.基于盐酸丁咯地尔(Buflomedil Hydrochloride,简称BUF)和盐酸维拉帕米(Verapamil hydrochloride,简称,VER)在铂电极上对联吡啶钌电化学发光强烈的增敏作用,与毛细管电泳联用,同时测定人血浆中的BUF和VER。为了提高灵敏度,采用0.1 mmol/L醋酸作为溶剂。在最佳条件下,BUF的线性范围为0.001～3 mg/L,检出限为0.0007 mg/L,VER的线性范围为0.01～12 mg/L,检出限为0.0047 mg/L。BUF和VER的回收率分别为85～92%和85～93%。该方法应用于人血浆中BUF和VER的测定,血浆中杂质与样品能够完全分离。
3.首次应用CE-ECL对盐酸麻黄碱(Ephedrine Hydrochloride,简称EP)进行检测,为提高灵敏度,用乙醛对其进行衍生,在最佳条件下,衍生后光强增强了近50倍。该药物的线性范围为0.078～11.7 mg/L,检出限为0.002 mg/L,回收率在93～108%之间,RSD低于5.9%。将此法应用于被盐酸麻黄碱稀释过的人血浆中测定,血浆中杂质与样品能够完全分离。
Electrochemiluminescence (ECL), which combines the characteristics of electrochemistry and chemiluminescence, is proved to be a powerful analytical tool with advantages of sensitivity, in situ, wide linear range, and good selectivity etc. Capillary electrophoresis (CE) has been greatly evolved since 1980s, now it has been proved to be an effective technique for separation with short analysis time, high resolution and small sample volume. The combination of capillary electrophoresis with tris (2, 2’-bipyridyl) ruthenium (II)-based electrochemiluminescence detection (CE-ECL) shows many advantages, for instance, high selectivity, good efficiency, fast analysis speed, and easy operation, etc. Now, the method has been widely used for the detection of nitrogen-containing compounds, and it has been successfully applyzed to pharmaceuticals, amino acids, narcotics, and environmental samples etc. As a method of analysis, CE-ECL will be more widely use in medical science, environmental science, and life science.
This thesis is concentrated on the application of CE-ECL for the analyses of amine-containing pharmaceuticals. Its contents were intruduced as follows:
1. A novel and simple pharmacokinetics of Pefloxacin mesylate (PM) in the urine of a healthy adult was developed. The proposed methodology was based on the ECL intensity of ruthenium (II) tris (bipyridine) at a platinum electrode was greatly enhanced by PM. Under the optimal conditions, the calibration curve was linear over the range from 0.05 to 10 mg/L with a detection limit of 0.006 mg/L (σ=3). The relative standard deviation of the peak height was 2.4% (n=6). The recovery in human urine was between 92.9% and 112%. The highest excretion rate was observed during 1.5 h to 2 h after oral administration. The urinary excretion ratio of PM was 13.5% within 48 h.
2. Based on electrochemiluminescence (ECL) intensity of ruthenium(II) tris (bipyridine) at a platinum electrode was greatly enhanced by Buflomedil hydrochloride (BUF) and Verapamil hydrochloride (VER), the simultaneous determination of BUF and VER in human plasma has been developed using capillary electrophoresis with ECL. In order to increase the sensitivity, 0.1 mmol/L acetic acid was selected as sample solvent. Under the optimal conditions,the linear ranges were 0.001～3 mg/L for BUF and 0.01～12 mg/L for VER. The detection limits (3σ) for BUF and VER were 0.0007 and 0.0047 mg/L, respectively. The recoveries of the two drugs at different concentrations were between 85% and 92% for BUF and 85% to 93% for VER. Plasma constituents did not interfer with the determination of BUF and VER.
3. A novel method for the determination of ephedrine hydrochloride (EP) was proposed. In order to improve the sensitivity, the precolumn derivatization of EP with acetaldehyde was proposed. Under the optimal conditions, the ECL intensity obtained about 50-fold improvement, compared to that wasn’t derived. The calibration curve was linear over the range from 0.078 to 11.7 mg/L with a detection limit of 0.002 mg/L (σ=3). All of the values of RSD were lower than 5.9% (n=6), and the recovery was between 93% and 108%. The mothod was applied to the detection of EP extracted from human plasma. The impurities in the plasma didn't interfer with the determination of EP.
引文
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