本文選題：親水色譜 + 內(nèi)源性碳水化合物　； 參考：《上海交通大學(xué)》2014年碩士論文

【摘要】：隨著代謝組學(xué)的迅速發(fā)展,發(fā)現(xiàn)了越來越多的疾病標(biāo)志物,對這些標(biāo)志物以及代謝途徑上下游的代謝物進(jìn)行深入研究有助于人們揭示疾病的發(fā)生及發(fā)展的機理、臨床診斷、藥理學(xué)研究等。然而,這些物質(zhì)大多數(shù)為極性小分子,在實際分析研究中存在同分異構(gòu)多、待測物含量低、雜質(zhì)干擾多、無與之相對應(yīng)的空白基質(zhì)等一系列亟待解決的問題。目前親水色譜(hydrophilic interaction chromatography,HILIC)及其與液質(zhì)(liquid chromatography-tandem mass spectrometry,LC-MS/MS)聯(lián)用技術(shù)的發(fā)展,為這些極性化合物的分離、分析提供了新手段。在對生物樣品如血清、血漿和尿液的代謝輪廓研究時發(fā)現(xiàn),內(nèi)源性糖類物質(zhì)通常作為差異物出現(xiàn)在不同疾病中;在神經(jīng)傳遞過程中,5-羥色胺起著重要的作用,常有報道稱,5-羥色胺失調(diào)現(xiàn)象出現(xiàn)在各種單胺類神經(jīng)遞質(zhì)紊亂和精神疾病包括抑郁、焦慮、精神分裂癥的研究中。本論文基于親水色譜的LC-MS/MS體系對生物基質(zhì)中的兩類極性小分子物質(zhì),內(nèi)源性糖類和內(nèi)源性5-羥色胺,進(jìn)行研究,其內(nèi)容包括:1)系統(tǒng)考察色譜柱、流動相組成、柱溫對糖類物質(zhì)的保留與分離行為的影響,簡單研究糖類物質(zhì)在HILIC amide色譜柱上的保留機制;尋找合適的內(nèi)源性替代基質(zhì);對蛋白沉淀溶劑體系進(jìn)行優(yōu)化;按要求進(jìn)行方法學(xué)驗證;最后對大鼠血漿樣品進(jìn)行分析。結(jié)果表明:采用冷的甲醇/乙腈(v/v,50/50)沉淀,渦旋1 min,并于-20℃放置20 min后再離心沉淀的前處理方式最佳;方法驗證中的標(biāo)準(zhǔn)曲線、精密度、基質(zhì)效應(yīng)、回收率和穩(wěn)定性的考察結(jié)果表明該方法符合內(nèi)源性物質(zhì)血漿樣品的分析要求。2)系統(tǒng)考察色譜柱、流動相組成、柱溫對5-羥色胺的保留與分離行為的影響,簡單研究該物質(zhì)在HILIC amide色譜柱上保留機制;尋找合適的內(nèi)源性替代基質(zhì);對蛋白沉淀溶劑體系進(jìn)行優(yōu)化;按要求進(jìn)行方法學(xué)驗證;最后對大鼠血漿樣品進(jìn)行分析。結(jié)果表明:采用冷的甲醇/乙腈(v/v,50/50)沉淀,渦旋1 min,并于-20℃放置20 min后再離心沉淀的前處理方式最佳;方法驗證中的標(biāo)準(zhǔn)曲線、精密度、基質(zhì)效應(yīng)、回收率和穩(wěn)定性的考察結(jié)果表明該方法符合內(nèi)源性物質(zhì)血漿樣品的分析要求。本研究針對16個碳水化合物及5-羥色胺分別建立的兩個親水色譜的UHPLC-MS/MS方法,具有簡單、快速、靈敏、準(zhǔn)確等特點。特別是糖類物質(zhì)的分析方法的建立彌補了目前對糖類物質(zhì)分析研究的不足。另外,建立方法過程中對化合物在HILIC amide色譜柱上的分離機理進(jìn)行的研究,為科研或者工業(yè)生產(chǎn)中類似極性化合物分析方法的建立提供了一種思路與解決辦法。
[Abstract]:With the rapid development of metabonomics, more and more disease markers have been found. The further study of these markers and metabolites in the upstream and downstream of metabolic pathways is helpful to reveal the mechanism of disease occurrence and development and clinical diagnosis. Pharmacological studies, etc. However, most of these substances are polar small molecules, and there are a series of problems to be solved in practical analysis, such as more isomerism, lower content of substance to be tested, more interference from impurities, and no corresponding blank matrix. At present, the development of hydrophilic interaction chromatographyHILICand its combination with liquidid chromatography-tandem mass spectrometric LC-MS / MSprovides a new method for the separation and analysis of these polar compounds. In the study of metabolic profiles of biological samples such as serum, plasma and urine, it was found that endogenous carbohydrates are commonly found as differentials in different diseases; serotonin plays an important role in neurotransmission. Serotonin disorders are often reported in various monoamine neurotransmitter disorders and psychiatric disorders including depression, anxiety, and schizophrenia. In this paper, based on the LC-MS / MS system of hydrophilic chromatography, two kinds of polar small molecular substances, endogenous carbohydrates and endogenous 5-hydroxytryptamine, were studied in biological matrix. The effect of column temperature on the retention and separation behavior of carbohydrates was studied. The retention mechanism of carbohydrates on HILIC amide column was studied, the suitable endogenous substitute matrix was found, and the solvent system of protein precipitation was optimized. The methodology was validated according to the requirements. Finally, the plasma samples of rats were analyzed. The results show that the best pretreatment method is the cold methanol / acetonitrile / v / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / The results of recovery and stability showed that the method met the analytical requirements of endogenous substance plasma samples. 2) the effects of chromatographic column, mobile phase composition and column temperature on the retention and separation behavior of 5-hydroxytryptamine were investigated systematically. The retention mechanism of the substance on HILIC amide column was studied briefly; the suitable endogenous substitute matrix was found; the solvent system of protein precipitation was optimized; the methodology was verified according to the requirements; and the plasma samples of rats were analyzed. The results show that the best pretreatment method is the cold methanol / acetonitrile / v / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / V / The results of recovery and stability showed that the method was suitable for the analysis of endogenous substance plasma samples. In this study, two UHPLC-MS / MS methods were developed for 16 carbohydrates and 5-hydroxytryptamine, respectively, which have the characteristics of simplicity, rapidity, sensitivity and accuracy. In particular, the establishment of analysis methods for carbohydrates makes up for the deficiency in the analysis of carbohydrates. In addition, the study on the separation mechanism of compounds on HILIC amide column during the establishment of the method provides a way of thinking and solution for the establishment of analytical methods for similar polar compounds in scientific research or industrial production.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R917;O657.63

【共引文獻(xiàn)】

相關(guān)期刊論文 前8條

1 戴玉成;崔寶凱;;藥用真菌桑黃種類研究[J];北京林業(yè)大學(xué)學(xué)報;2014年05期

2 李媛媛;張海霞;;親水作用色譜填料的研究進(jìn)展[J];分析測試技術(shù)與儀器;2015年02期

3 李瑞萍;袁琴;黃應(yīng)平;;硅膠色譜柱的親水作用保留機理及其影響因素[J];色譜;2014年07期

4 張英;晉艷;李海芳;林金明;;植物中單糖的樣品前處理及色譜分析方法研究進(jìn)展[J];食品安全質(zhì)量檢測學(xué)報;2014年05期

5 許麗珠;李秀英;黃金鳳;陳曉丹;鄧偉恒;黃偉乾;冼燕萍;曾慶祝;郭新東;;超濾離心凈化-高效液相色譜法同時測定配方乳粉中的單糖、雙糖和低聚果糖[J];食品安全質(zhì)量檢測學(xué)報;2015年02期

6 胡貝貞;董文洪;夏碧琪;宋偉華;韓超;;高效陰離子交換色譜-積分脈沖安培檢測法同時測定黃酒中的單糖和低聚糖及其指紋圖譜的構(gòu)建[J];色譜;2015年06期

7 張仟春;羅夏琳;李攻科;肖小華;;復(fù)雜樣品痕量極性小分子化合物的樣品前處理及分析方法研究進(jìn)展[J];色譜;2015年09期

8 閆競宇;郭志謀;丁俊杰;沈愛金;王紀(jì)霞;金高娃;梁鑫淼;;萬古霉素及其雜質(zhì)的親水作用色譜分析[J];色譜;2015年09期

相關(guān)博士學(xué)位論文 前1條

1 高方圓;基于新型樣品預(yù)處理技術(shù)結(jié)合多維色譜—組合質(zhì)譜法的白芷化合物組與代謝物組研究[D];第二軍醫(yī)大學(xué);2014年

相關(guān)碩士學(xué)位論文 前5條

1 楊總;同位素稀釋質(zhì)譜法測定奶粉基體中藥物殘留的基質(zhì)效應(yīng)研究[D];中國計量科學(xué)研究院;2013年

2 羅浩師;動物性食品中甲氧芐啶和二甲氧芐啶殘留量的測定[D];中國農(nóng)業(yè)科學(xué)院;2013年

3 張聰;貫流硅膠微球在固定化脂質(zhì)體色譜和半制備親水作用色譜中的應(yīng)用[D];華中科技大學(xué);2012年

4 王亞麗;中藥材水煎液鉻形態(tài)分析方法研究及異丹葉大黃素小鼠血藥濃度研究[D];北京協(xié)和醫(yī)學(xué)院;2014年

5 張英;單糖和糖肽的樣品前處理及其分析方法研究[D];北京化工大學(xué);2014年

，

本文編號：2021231