微乳毛细管电动色谱同时分离水溶性和脂溶性维生素
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摘要
维生素是维持机体正常生长的必不可少的化合物,对人体和动物的生理活动有非常重要的作用,可以分为水溶性维生素和脂溶性维生素。微乳毛细管电动色谱(MEEKC)可以同时分离分析水溶性和脂溶性的物质。本论文的研究主要基于MEEKC的分析测定技术,建立快速分析脂溶性物质的新型微乳体系,并建立快速分析水溶性和脂溶性维生素的MEEKC的方法。本文分为以下四部分。
第一部分:对各种维生素进行了简介,对电化学分析法、分光光度法、荧光光度法、高效液相色谱法、毛细管电泳法等各种分析方法在维生素测定中的应用进行了综述,并介绍了MEEKC法的基本原理、影响其分离的因素以及应用。
第二部分:在MEEKC经典微乳体系的基础上进行了改进,建立了新型微乳体系,旨在解决MEEKC分析脂溶性物质时间太长的问题,而解决问题的关键是减少离子表面活性剂十二烷基硫酸钠(SDS)的用量。为了减少SDS的用量,又能保证微乳体系的稳定性,同时具备足够的分离效率,本文采用了两种方式改进微乳体系:一、采用非离子表面活性剂与SDS复配;二、大幅度增加助表面活性剂。研究表明:两种体系均能使脂溶性物质分析速度加快,但前者分离效率不如后者。大量的助表面活性剂使微乳粒子变得松散,有利于脂溶性维生素在油相和水相之间的进出,缩短了脂溶性物质的分离时间,提高了分离效率。第三部分:维生素A、D3、E是三种最常见的脂溶性维生素,利用新型微乳体系,建
立了MEEKC快速分析脂溶性维生素A、D3、E的方法。该微乳液的组成为:1.2%(W/W)SDS-21%(V/V)正丁醇-18%(V/V)乙腈-0.8%(W/W)正己烷-20 mmol/LpH8.4 H3BO3-Na2B4O7缓冲液。该微乳体系中,助表面活性剂正丁醇和有机溶剂乙腈对脂溶性维生素的分离起到了重要的作用。当分离电压为25 kV,柱温为25℃时,维生素A、D3、E在13 min内达到基线分离。三种脂溶性维生素的迁移时间和峰面积的RSD(n= 5)小于2.5%和4.5%;维生素A、D3、E分别在20~1000μg/mL,5~1000μg/mL,5~1000μg/mL范围内与峰面积呈线性关系;检出限(S/N=3)分别为12、0.72、0.29μg/mL。该体系直接应用于市售维生素E胶囊的测定,测定结果与标示值相符。
第四部分:利用该微乳体系对13种脂溶性维生素和水溶性维生素进行了快速分析。该微乳体系中,微乳体系中缓冲溶液的pH和浓度以及正丁醇和乙腈的用量分别对水溶性维生素和脂溶性维生素的分离起到了重要的作用。当分离电压为25 kV,柱温为25℃时,13种维生素在30 min内达到基线分离。表明该MEEKC是一个快速、简单的分离测定脂溶性维生素和水溶性维生素的有效方法。该体系适用于市售多维维生素片的测定,准确性高,灵敏度好。各种维生素的回收率在97%~105%之间。除了维生素C,样品测定值与药品标示值相符。该MEEKC方法适用于维生素相关药物制品的质量控制,具有应用前景。
Vitamins are a broad group of organic compounds that are minor, but essential, constituents of food required for the normal growth, self-maintenance and functioning of human and animal bodies. These compounds can be classified in two main groups: water- and fat-soluble vitamins. Microemulsion electrokinetic chromatography (MEEKC) is an electrodriven separation technique. The target of the present work was to separate water- and fat-soluble vitamins simultaneously by MEEKC within a short time. The thesis mainly includes the following four parts.
1. The analytical methods for determination of vitamins were reviewed in this part, and the advantage and disadvantage of these diferent analytical methods was discussed. The principle, the important factors and the applications of MEEKC in various fields were also summarized, especially, the applications in analysis of vitamins, pharmaecutical, natural products and environmental analysis.
2. Based on the common SDS microemulsion system used in MEEKC, the novel microemulsion systems were studied to shorten analysis time for fat-soluble analytes. Since Low SDS concentration could speed migration of the analytes due to decrease of backward electrophoretic mobility of microemulsion droplet and increase of electroosmotic flow (EOF), it is the key to lower SDS concentration, as well as to ensure the stability of microemulsion systems. In this part, two schemes were applied to solve the difficulty. Non-ionic surfactant, together with SDS constituted mixed surfactants used in microemulsion to low SDS content. On the other hand, a lot of 1-butanol was added to help reduce the tension between two layer of oil and water, stabilize the microemulsion system. Experimental results showed these two methods could shorten the analysis time, and the latter has more separation efficiency. Therefore, the novel microemulsion has potential to solve two difficulties of long migration time and poor resolution.
3. The novel microelusion system was applied to rapid determine fat-soluble vitamins A, D3, and E with MEEKC. The microemulsion consisted of 1.2% (W/W) SDS, 21% (V/V) 1-butanol, 18%(V/V) acetonitrile, 0.8% (W/W) n-hexane and 20 mM borax buffer ( pH 8.4). The three analytes were baseline separated within 13 min at 25 kV and 25℃. The RSD values (n= 5) of migration time and peak area of the analytes were less than 2.5% and 4.5%, showing high reproducibility. Good linear relations between peak areas and concentrations of analytes were established in the range of 20-1000μg /mL for vitamin A, 5-1000μg /mL for vitamin D3, and 5-1000μg /mL for vitamin E. The detection limits (S/N=3) were 12μg/mL, 0.72μg/mL, 0.29μg/mL respectively. This method has been successfully applied for analysis commercial vitamin capsules, the determination results were accorded with the declared in the label.
4. A rapid, reliable and reproducible method based on MEEKC for simultaneous determination of thirteen kinds of water- and fat-soluble vitamins has been developed in this work. The novel microemulsion system consisting of 1.2% (W/W) SDS, 21% (V/V) 1-butanol, 18% (V/V) acetonitrile, 0.8% (W/W) n-hexane, 20 mM borax buffer (pH 8.7) was applied to improve selectivity and efficiency, as well as shorten analysis time. Acetonitrile as the organic solvent modifier, pH of the running buffer and 1-butanol as the cosurfactant played the most important roles for separation of the fat-soluble vitamins, water-soluble vitamins and stabilization of system, respectively. The 13 water- and fat-soluble vitamins were baseline separated within 30 min. The system was applied to determine of water- and fat-soluble vitamins in commercial multivitamin pharmaceutical formulation, good accuracy and precision were obtained with recoveries between 97% and 105%, acceptable quantitative results corresponding to label claim except vitamin C.
第一部分:对各种维生素进行了简介,对电化学分析法、分光光度法、荧光光度法、高效液相色谱法、毛细管电泳法等各种分析方法在维生素测定中的应用进行了综述,并介绍了MEEKC法的基本原理、影响其分离的因素以及应用。
第二部分:在MEEKC经典微乳体系的基础上进行了改进,建立了新型微乳体系,旨在解决MEEKC分析脂溶性物质时间太长的问题,而解决问题的关键是减少离子表面活性剂十二烷基硫酸钠(SDS)的用量。为了减少SDS的用量,又能保证微乳体系的稳定性,同时具备足够的分离效率,本文采用了两种方式改进微乳体系:一、采用非离子表面活性剂与SDS复配;二、大幅度增加助表面活性剂。研究表明:两种体系均能使脂溶性物质分析速度加快,但前者分离效率不如后者。大量的助表面活性剂使微乳粒子变得松散,有利于脂溶性维生素在油相和水相之间的进出,缩短了脂溶性物质的分离时间,提高了分离效率。第三部分:维生素A、D3、E是三种最常见的脂溶性维生素,利用新型微乳体系,建
立了MEEKC快速分析脂溶性维生素A、D3、E的方法。该微乳液的组成为:1.2%(W/W)SDS-21%(V/V)正丁醇-18%(V/V)乙腈-0.8%(W/W)正己烷-20 mmol/LpH8.4 H3BO3-Na2B4O7缓冲液。该微乳体系中,助表面活性剂正丁醇和有机溶剂乙腈对脂溶性维生素的分离起到了重要的作用。当分离电压为25 kV,柱温为25℃时,维生素A、D3、E在13 min内达到基线分离。三种脂溶性维生素的迁移时间和峰面积的RSD(n= 5)小于2.5%和4.5%;维生素A、D3、E分别在20~1000μg/mL,5~1000μg/mL,5~1000μg/mL范围内与峰面积呈线性关系;检出限(S/N=3)分别为12、0.72、0.29μg/mL。该体系直接应用于市售维生素E胶囊的测定,测定结果与标示值相符。
第四部分:利用该微乳体系对13种脂溶性维生素和水溶性维生素进行了快速分析。该微乳体系中,微乳体系中缓冲溶液的pH和浓度以及正丁醇和乙腈的用量分别对水溶性维生素和脂溶性维生素的分离起到了重要的作用。当分离电压为25 kV,柱温为25℃时,13种维生素在30 min内达到基线分离。表明该MEEKC是一个快速、简单的分离测定脂溶性维生素和水溶性维生素的有效方法。该体系适用于市售多维维生素片的测定,准确性高,灵敏度好。各种维生素的回收率在97%~105%之间。除了维生素C,样品测定值与药品标示值相符。该MEEKC方法适用于维生素相关药物制品的质量控制,具有应用前景。
Vitamins are a broad group of organic compounds that are minor, but essential, constituents of food required for the normal growth, self-maintenance and functioning of human and animal bodies. These compounds can be classified in two main groups: water- and fat-soluble vitamins. Microemulsion electrokinetic chromatography (MEEKC) is an electrodriven separation technique. The target of the present work was to separate water- and fat-soluble vitamins simultaneously by MEEKC within a short time. The thesis mainly includes the following four parts.
1. The analytical methods for determination of vitamins were reviewed in this part, and the advantage and disadvantage of these diferent analytical methods was discussed. The principle, the important factors and the applications of MEEKC in various fields were also summarized, especially, the applications in analysis of vitamins, pharmaecutical, natural products and environmental analysis.
2. Based on the common SDS microemulsion system used in MEEKC, the novel microemulsion systems were studied to shorten analysis time for fat-soluble analytes. Since Low SDS concentration could speed migration of the analytes due to decrease of backward electrophoretic mobility of microemulsion droplet and increase of electroosmotic flow (EOF), it is the key to lower SDS concentration, as well as to ensure the stability of microemulsion systems. In this part, two schemes were applied to solve the difficulty. Non-ionic surfactant, together with SDS constituted mixed surfactants used in microemulsion to low SDS content. On the other hand, a lot of 1-butanol was added to help reduce the tension between two layer of oil and water, stabilize the microemulsion system. Experimental results showed these two methods could shorten the analysis time, and the latter has more separation efficiency. Therefore, the novel microemulsion has potential to solve two difficulties of long migration time and poor resolution.
3. The novel microelusion system was applied to rapid determine fat-soluble vitamins A, D3, and E with MEEKC. The microemulsion consisted of 1.2% (W/W) SDS, 21% (V/V) 1-butanol, 18%(V/V) acetonitrile, 0.8% (W/W) n-hexane and 20 mM borax buffer ( pH 8.4). The three analytes were baseline separated within 13 min at 25 kV and 25℃. The RSD values (n= 5) of migration time and peak area of the analytes were less than 2.5% and 4.5%, showing high reproducibility. Good linear relations between peak areas and concentrations of analytes were established in the range of 20-1000μg /mL for vitamin A, 5-1000μg /mL for vitamin D3, and 5-1000μg /mL for vitamin E. The detection limits (S/N=3) were 12μg/mL, 0.72μg/mL, 0.29μg/mL respectively. This method has been successfully applied for analysis commercial vitamin capsules, the determination results were accorded with the declared in the label.
4. A rapid, reliable and reproducible method based on MEEKC for simultaneous determination of thirteen kinds of water- and fat-soluble vitamins has been developed in this work. The novel microemulsion system consisting of 1.2% (W/W) SDS, 21% (V/V) 1-butanol, 18% (V/V) acetonitrile, 0.8% (W/W) n-hexane, 20 mM borax buffer (pH 8.7) was applied to improve selectivity and efficiency, as well as shorten analysis time. Acetonitrile as the organic solvent modifier, pH of the running buffer and 1-butanol as the cosurfactant played the most important roles for separation of the fat-soluble vitamins, water-soluble vitamins and stabilization of system, respectively. The 13 water- and fat-soluble vitamins were baseline separated within 30 min. The system was applied to determine of water- and fat-soluble vitamins in commercial multivitamin pharmaceutical formulation, good accuracy and precision were obtained with recoveries between 97% and 105%, acceptable quantitative results corresponding to label claim except vitamin C.
引文
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