新型β-环糊精液相色谱键合相的制备及性能评价

发布时间：2018-10-21 16:03

【摘要】：手性是生命体进化和发展的自然属性之一,蛋白质等许多生命物质都是手性的。手性药物和农药进入人体后,对映体在药效、毒理和代谢途径的不同,这与人们的身体健康密切相关,正日益引起国际社会的高度重视,如何高效分离和准确测定对映体的含量将成为药物安全和食品安全分析的新课题。不断发展新型分离材料与色谱技术是解决上述手性问题的关键。β-环糊精类手性固定相具有优良的手性拆分性能、色谱重现性和广泛的包结客体,且制备方法简便、成本较低、实用性强。特别是基于β-环糊精端口的功能化,引入多种作用位点,与腔体包结作用形成合力,进一步地提高其手性识别能力,更好地满足对映体快速拆分的需要,为保障食品药品安全,乃至生态环境的可持续性发展服务。本论文主要包含五个部分的研究工作:论文第一部分回顾了各类手性拆分手段和基本原理,系统地归纳了几类应用较为广泛的高效液相色谱手性固定相的发展历程和优缺点,着重介绍了环糊精类固定相的相关性质和研究进展。同时对有序介孔材料SBA-15作为色谱键合相基质的发展也进行了论述,以此作为研究工作的理论支撑。论文第二至第五部分依次将芳基脲氢键型配体、乙二胺配位剂和弱离子化苯硼酸引入到β-环糊精端口,以有序介孔SBA-15硅胶为基质、制备和表征了3个新品种的手性固定相,分别考察了它们的基本手性分离能力,并初步用于手性农药和药物对映体分离分析,为将来的实际应用提供实验数据。论文第二部分制备了对甲基苯脲修饰β-环糊精手性固定相(UCDP),采用质谱、透射电镜、固体核磁等表征新固定相的结构与形貌。首先测定柱效,然后评价新固定相分离硝基苯胺位置异构体等的能力,实验表明固定相有较高的异构体分离选择性,对位异构体由于进入环糊精腔体更深,在最后被洗脱。接着以戊唑醇等10种三唑类杀菌剂为探针,研究了UCDP的手性色谱性能。结果表明,使用水和甲醇或乙腈简单的流动相,常温下新制备的环糊精类固定相拆分三唑类杀菌剂对映体的效果好,其中己唑醇和粉唑醇的分离度分别达到2.50和1.81,且分析时间较短(30 min)。研究了流动相的组成及柱温对分离度的影响,并测定了相关热力学参数,计算结果表明其手性分离是焓驱动的结果。结合三唑类杀菌剂和固定相的化学结构,探讨UCDP对该类农药的手性识别机理。β-环糊精配体的空腔对溶质的包结作用、端口的氢键作用和空间位阻等协同作用增强了对三唑类杀菌剂的对映体识别能力。论文第三部分采用对甲苯脲修饰β-环糊精手性固定相(UCDP),通过进一步优化流动相组成和比例、柱温等色谱操作参数,在反相色谱条件下对粉唑醇和己唑醇对映体的分离度达到2.02和2.42,可同时检测双组分的对映体,分析时间不到20 min。在此基础上建立了HPLC-MS法通过选择离子监测四种果蔬基质中的粉唑醇和己唑醇对映体,采用简易的磁回收技术进行果蔬样品前处理,结合QuEChERS法的改进,增强方法的实用性。粉唑醇和己唑醇的每个对映体均在0.05~12.5 mg/L浓度范围内线性相关度良好(r≥0.999),最低检出限均为0.01 mg/L,准确度、精密度和稳定性较高(平均回收率为81.93%~101.10%,RSD为1.05%~7.11%,n=5)。此方法简便快速,结果准确,为果蔬中农药对映体残留量分析提供了一种新方法。论文第四部分制备了乙二胺修饰β-环糊精手性固定相(EASP),表征了其结构。阿替洛尔是一种治疗心血管疾病的常用手性药物,洛尔类药物手性分离通常很困难,β-环糊精端口引入乙二胺配位剂有利于手性拆分。本研究采用极性有机模式,通过优化淋洗剂中甲醇含量、冰醋酸、三乙胺的含量和柱温等操作条件,有效地拆分了阿替洛尔药物对映体,分离度达到1.72,分析时间在25 min以内。建立了高效液相色谱荧光(HPLC-FLD)检测了阿替洛尔药品的对映体含量的新方法,两对映体在0.05~5.0 mg/L浓度范围内具有良好的线性关系,最低检出限均为0.02 mg/L,准确度高,灵敏度高,分析快速,为阿替洛尔对映体含量药物质量监测和临床相关药理、代谢研究提供新方法。论文第五部分以EDC为脱水剂,4-羧基苯硼酸与乙二胺-β-环糊精进行脱水反应,并键合到SBA-15硅胶上,制备一种弱离子化的苯硼酸单取代β-环糊精手性固定相(PHBSP),采用红外光谱、元素分析和热重分析等表征新制备的固定相的结构。以8种黄酮类化合物、12种β-受体阻滞剂和6种三唑类杀菌剂为溶质,反相模式下成功地拆分了黄酮类化合物,分析时间均在30 min以内,其中2'-羟基黄烷酮分离度为3.15;成功地拆分了部分β-受体阻滞剂,其中阿替洛尔分离度为1.59,分析时间在15 min以内;成功地拆分了大部分三唑类杀菌剂,其中己唑醇们分离度为1.96。分别探讨了PHBSP对此三类手性物质的拆分机理,弱离子化的苯硼酸基环糊精对可电离的极性溶质有较好的分离选择性,衍生弱离子化的苯硼酸基团后不会带来离子交换作用,仍适用于常见的反相色谱流动相。
[Abstract]:Hand is one of the natural attributes of evolution and development of living organisms, and many vital substances, such as proteins, are chiral. When chiral drugs and pesticides enter the human body, the enantiomers are different in drug effect, toxicology and metabolic pathway, which is closely related to the health of people, and is becoming more and more important to the international community. How to efficiently separate and accurately determine the content of enantiomers will become a new subject of drug safety and food safety analysis. The development of new separation materials and chromatographic techniques is the key to solve the above-mentioned chiral problems. the cyclodextrin-cyclodextrin chiral stationary phase has excellent chiral separation performance, chromatographic reproducibility and wide packet junction object, and the preparation method is simple and convenient, the cost is lower, and the practicability is strong. In particular, based on the functionalization of the poly-cyclodextrin port, a plurality of action sites are introduced, a resultant force is formed with the action of the cavity bag junction, the chiral recognition capability is further improved, the need of rapid separation of enantiomers is better met, and the food and drug safety is guaranteed, and even the sustainable development service of the ecological environment. This paper mainly includes five parts of research work: the first part of this paper reviews the methods and basic principles of hand-splitting, and systematically summarizes the development course and advantages and disadvantages of several kinds of high performance liquid chromatography chiral stationary phases. In this paper, the related properties and research progress of cyclodextrin-based stationary phase are introduced. At the same time, the development of the ordered mesoporous material SBA-15 as chromatographic bond matrix is also discussed, as a theoretical support for the research work. in that second to fifth part of the thesis, the aryl group hydrogen bond type ligand, the ethylenediamine coordination agent and the weakly ionized phenylboronic acid are sequentially introduced into the poly-cyclodextrin port, and the chiral stationary phase of the three new varieties is prepared and characterized by taking the ordered mesoporous SBA-15 silica gel as the substrate, Their basic chiral separation ability was investigated, and the analysis of chiral pesticides and drug enantiomers was used to provide experimental data for practical application in the future. In the second part, the structure and morphology of the new stationary phase were characterized by mass spectrometry, transmission electron microscope, solid nuclear magnetic and the like. Firstly, the column effect is determined, then the ability of the new fixed phase to separate the position isomer of nitroaniline is evaluated. The experimental results show that the fixed phase has higher isomer separation selectivity, and the para-isomer is finally eluted by entering the cyclodextrin cavity. The chiral chromatographic performance of UCDP was studied by using 10 kinds of carbamazepine as probe. The results showed that the separation degree of hexyl alcohol and alcohol was 2.50 and 1.81 respectively, and the analysis time was shorter (30 min). The influence of the composition of mobile phase and column temperature on the degree of separation is studied, and the relevant thermodynamic parameters are measured. The results show that their chiral separation is the result of hydrostatic drive. In this paper, the chiral recognition mechanism of UCDP on the pesticide is discussed in this paper. The synergistic effects of the cavity of the poly-cyclodextrin ligand on solute inclusion, hydrogen bonding of the port and steric hindrance have enhanced the ability to identify the enantiomers of the carbamazepine bactericide. In the third part of the thesis, the chiral stationary phase (UCDP) was modified with toluene diethylamine. By further optimizing the flow phase composition and ratio, column temperature and other chromatographic operation parameters, the separation degree of the enantiomers of the alcohol and hexyl alcohol was 2.02 and 2.42 under the condition of reversed phase chromatography. the enantiomers of the two components can be simultaneously detected, and the analysis time is less than 20 minutes. On the basis of this, a HPLC-MS method was established to monitor the enantiomers of powder and hexyl alcohol in four kinds of fruit and vegetable substrates by selecting ions. The pretreatment of fruit and vegetable samples was carried out by simple magnetic recovery technology, and the practicability of the method was improved by combining with the improvement of the QuEChERS method. The average recoveries were 0. 01 mg/ L, accuracy, precision and stability (average recovery was 81. 93% ~ 101. 10%, RSD was 1. 05% ~ 7.11%, n = 5). The method is simple and rapid, the result is accurate, and a new method is provided for the analysis of pesticide residues in fruits and vegetables. In the fourth part of the thesis, ethylenediamine modified poly-cyclodextrin chiral stationary phase (EASP) was prepared and its structure was characterized. Atiolol is a commonly used chiral drug for treating cardiovascular diseases. The chiral separation of metoprolol is usually very difficult. In this study, the polar organic model was used to effectively split the enantiomers of Atiolol by optimizing the conditions of methanol content, glacial acetic acid, triethylamine content and column temperature, and the analysis time was within 25 minutes. High performance liquid chromatography (HPLC-FLD) has been established to detect the enantiomer content of Atiolol. The two enantiomers have a good linear relationship in the range of 0.05 to 5.0 mg/ L. The lowest detection limit is 0.02mg/ L, the accuracy is high, the sensitivity is high, and the analysis is rapid. To provide a new method for the quality monitoring and clinical related pharmacological and metabolic studies of Atiolol enantiomers. The fifth part of the thesis is prepared by dehydration reaction of EDC, 4-dimethylphenylboronic acid and ethylenediamine-NHS-cyclodextrin, and bonding on SBA-15 silica gel to prepare a weakly ionized phenylboronic acid mono-substituted cis-cyclodextrin chiral stationary phase (PHBSP), which adopts the infrared spectrum. The structure of the newly prepared fixed phase was characterized by elemental analysis and thermal analysis. The flavonoid compounds were successfully resolved in the anti-phase mode with 8 flavonoids, 12 proton pump-receptor blockers and 6 anti-inflammatory agents, and the analysis time was within 30 minutes, among which 2 The separation degree of "-hydroxyflavonane" was 3. 15. Partial proton pump-receptor blocker was successfully resolved, in which the separation degree of Atiolol was 1. 59, the analysis time was within 15 min, and most of the anti-inflammatory agents were successfully resolved, among which the separation degree of hexyl alcohol was 1. 96. The separation mechanism of the three chiral substances of PHBSP is discussed. The weakly ionized phenylboronic acid-based cyclodextrins have better separation selectivity for the ionizable polar solute, and the weakly ionized phenylboronic acid group does not lead to ion exchange. It is still suitable for the common reversed phase chromatography mobile phase.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.72

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