基于亲水液相色谱—串联质谱联用技术的大鼠血浆中内源性糖类和5-羟色胺的定量方法学研究

发布时间：2018-06-15 07:32

本文选题：亲水色谱 + 内源性碳水化合物　；参考：《上海交通大学》2014年硕士论文

【摘要】：随着代谢组学的迅速发展,发现了越来越多的疾病标志物,对这些标志物以及代谢途径上下游的代谢物进行深入研究有助于人们揭示疾病的发生及发展的机理、临床诊断、药理学研究等。然而,这些物质大多数为极性小分子,在实际分析研究中存在同分异构多、待测物含量低、杂质干扰多、无与之相对应的空白基质等一系列亟待解决的问题。目前亲水色谱(hydrophilic interaction chromatography,HILIC)及其与液质(liquid chromatography-tandem mass spectrometry,LC-MS/MS)联用技术的发展,为这些极性化合物的分离、分析提供了新手段。在对生物样品如血清、血浆和尿液的代谢轮廓研究时发现,内源性糖类物质通常作为差异物出现在不同疾病中;在神经传递过程中,5-羟色胺起着重要的作用,常有报道称,5-羟色胺失调现象出现在各种单胺类神经递质紊乱和精神疾病包括抑郁、焦虑、精神分裂症的研究中。本论文基于亲水色谱的LC-MS/MS体系对生物基质中的两类极性小分子物质,内源性糖类和内源性5-羟色胺,进行研究,其内容包括:1)系统考察色谱柱、流动相组成、柱温对糖类物质的保留与分离行为的影响,简单研究糖类物质在HILIC amide色谱柱上的保留机制;寻找合适的内源性替代基质;对蛋白沉淀溶剂体系进行优化;按要求进行方法学验证;最后对大鼠血浆样品进行分析。结果表明:采用冷的甲醇/乙腈(v/v,50/50)沉淀,涡旋1 min,并于-20℃放置20 min后再离心沉淀的前处理方式最佳;方法验证中的标准曲线、精密度、基质效应、回收率和稳定性的考察结果表明该方法符合内源性物质血浆样品的分析要求。2)系统考察色谱柱、流动相组成、柱温对5-羟色胺的保留与分离行为的影响,简单研究该物质在HILIC amide色谱柱上保留机制;寻找合适的内源性替代基质;对蛋白沉淀溶剂体系进行优化;按要求进行方法学验证;最后对大鼠血浆样品进行分析。结果表明:采用冷的甲醇/乙腈(v/v,50/50)沉淀,涡旋1 min,并于-20℃放置20 min后再离心沉淀的前处理方式最佳;方法验证中的标准曲线、精密度、基质效应、回收率和稳定性的考察结果表明该方法符合内源性物质血浆样品的分析要求。本研究针对16个碳水化合物及5-羟色胺分别建立的两个亲水色谱的UHPLC-MS/MS方法,具有简单、快速、灵敏、准确等特点。特别是糖类物质的分析方法的建立弥补了目前对糖类物质分析研究的不足。另外,建立方法过程中对化合物在HILIC amide色谱柱上的分离机理进行的研究,为科研或者工业生产中类似极性化合物分析方法的建立提供了一种思路与解决办法。
[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.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R917;O657.63

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