新型β-環(huán)糊精液相色譜鍵合相的制備及性能評價
發(fā)布時間：2018-10-21 16:03
【摘要】：手性是生命體進化和發(fā)展的自然屬性之一,蛋白質(zhì)等許多生命物質(zhì)都是手性的。手性藥物和農(nóng)藥進入人體后,對映體在藥效、毒理和代謝途徑的不同,這與人們的身體健康密切相關(guān),正日益引起國際社會的高度重視,如何高效分離和準確測定對映體的含量將成為藥物安全和食品安全分析的新課題。不斷發(fā)展新型分離材料與色譜技術(shù)是解決上述手性問題的關(guān)鍵。β-環(huán)糊精類手性固定相具有優(yōu)良的手性拆分性能、色譜重現(xiàn)性和廣泛的包結(jié)客體,且制備方法簡便、成本較低、實用性強。特別是基于β-環(huán)糊精端口的功能化,引入多種作用位點,與腔體包結(jié)作用形成合力,進一步地提高其手性識別能力,更好地滿足對映體快速拆分的需要,為保障食品藥品安全,乃至生態(tài)環(huán)境的可持續(xù)性發(fā)展服務。本論文主要包含五個部分的研究工作:論文第一部分回顧了各類手性拆分手段和基本原理,系統(tǒng)地歸納了幾類應用較為廣泛的高效液相色譜手性固定相的發(fā)展歷程和優(yōu)缺點,著重介紹了環(huán)糊精類固定相的相關(guān)性質(zhì)和研究進展。同時對有序介孔材料SBA-15作為色譜鍵合相基質(zhì)的發(fā)展也進行了論述,以此作為研究工作的理論支撐。論文第二至第五部分依次將芳基脲氫鍵型配體、乙二胺配位劑和弱離子化苯硼酸引入到β-環(huán)糊精端口,以有序介孔SBA-15硅膠為基質(zhì)、制備和表征了3個新品種的手性固定相,分別考察了它們的基本手性分離能力,并初步用于手性農(nóng)藥和藥物對映體分離分析,為將來的實際應用提供實驗數(shù)據(jù)。論文第二部分制備了對甲基苯脲修飾β-環(huán)糊精手性固定相(UCDP),采用質(zhì)譜、透射電鏡、固體核磁等表征新固定相的結(jié)構(gòu)與形貌。首先測定柱效,然后評價新固定相分離硝基苯胺位置異構(gòu)體等的能力,實驗表明固定相有較高的異構(gòu)體分離選擇性,對位異構(gòu)體由于進入環(huán)糊精腔體更深,在最后被洗脫。接著以戊唑醇等10種三唑類殺菌劑為探針,研究了UCDP的手性色譜性能。結(jié)果表明,使用水和甲醇或乙腈簡單的流動相,常溫下新制備的環(huán)糊精類固定相拆分三唑類殺菌劑對映體的效果好,其中己唑醇和粉唑醇的分離度分別達到2.50和1.81,且分析時間較短(30 min)。研究了流動相的組成及柱溫對分離度的影響,并測定了相關(guān)熱力學參數(shù),計算結(jié)果表明其手性分離是焓驅(qū)動的結(jié)果。結(jié)合三唑類殺菌劑和固定相的化學結(jié)構(gòu),探討UCDP對該類農(nóng)藥的手性識別機理。β-環(huán)糊精配體的空腔對溶質(zhì)的包結(jié)作用、端口的氫鍵作用和空間位阻等協(xié)同作用增強了對三唑類殺菌劑的對映體識別能力。論文第三部分采用對甲苯脲修飾β-環(huán)糊精手性固定相(UCDP),通過進一步優(yōu)化流動相組成和比例、柱溫等色譜操作參數(shù),在反相色譜條件下對粉唑醇和己唑醇對映體的分離度達到2.02和2.42,可同時檢測雙組分的對映體,分析時間不到20 min。在此基礎(chǔ)上建立了HPLC-MS法通過選擇離子監(jiān)測四種果蔬基質(zhì)中的粉唑醇和己唑醇對映體,采用簡易的磁回收技術(shù)進行果蔬樣品前處理,結(jié)合QuEChERS法的改進,增強方法的實用性。粉唑醇和己唑醇的每個對映體均在0.05~12.5 mg/L濃度范圍內(nèi)線性相關(guān)度良好(r≥0.999),最低檢出限均為0.01 mg/L,準確度、精密度和穩(wěn)定性較高(平均回收率為81.93%~101.10%,RSD為1.05%~7.11%,n=5)。此方法簡便快速,結(jié)果準確,為果蔬中農(nóng)藥對映體殘留量分析提供了一種新方法。論文第四部分制備了乙二胺修飾β-環(huán)糊精手性固定相(EASP),表征了其結(jié)構(gòu)。阿替洛爾是一種治療心血管疾病的常用手性藥物,洛爾類藥物手性分離通常很困難,β-環(huán)糊精端口引入乙二胺配位劑有利于手性拆分。本研究采用極性有機模式,通過優(yōu)化淋洗劑中甲醇含量、冰醋酸、三乙胺的含量和柱溫等操作條件,有效地拆分了阿替洛爾藥物對映體,分離度達到1.72,分析時間在25 min以內(nèi)。建立了高效液相色譜熒光(HPLC-FLD)檢測了阿替洛爾藥品的對映體含量的新方法,兩對映體在0.05~5.0 mg/L濃度范圍內(nèi)具有良好的線性關(guān)系,最低檢出限均為0.02 mg/L,準確度高,靈敏度高,分析快速,為阿替洛爾對映體含量藥物質(zhì)量監(jiān)測和臨床相關(guān)藥理、代謝研究提供新方法。論文第五部分以EDC為脫水劑,4-羧基苯硼酸與乙二胺-β-環(huán)糊精進行脫水反應,并鍵合到SBA-15硅膠上,制備一種弱離子化的苯硼酸單取代β-環(huán)糊精手性固定相(PHBSP),采用紅外光譜、元素分析和熱重分析等表征新制備的固定相的結(jié)構(gòu)。以8種黃酮類化合物、12種β-受體阻滯劑和6種三唑類殺菌劑為溶質(zhì),反相模式下成功地拆分了黃酮類化合物,分析時間均在30 min以內(nèi),其中2'-羥基黃烷酮分離度為3.15;成功地拆分了部分β-受體阻滯劑,其中阿替洛爾分離度為1.59,分析時間在15 min以內(nèi);成功地拆分了大部分三唑類殺菌劑,其中己唑醇們分離度為1.96。分別探討了PHBSP對此三類手性物質(zhì)的拆分機理,弱離子化的苯硼酸基環(huán)糊精對可電離的極性溶質(zhì)有較好的分離選擇性,衍生弱離子化的苯硼酸基團后不會帶來離子交換作用,仍適用于常見的反相色譜流動相。
[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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.72
本文編號：2285624
[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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.72
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