复杂体系中多目标化合物定性和定量分析技术和策略研究

发布时间：2018-06-14 08:23

  本文选题：复杂体系 + 代谢组学　； 参考：《北京协和医学院》2016年博士论文

【摘要】：复杂体系中多目标化合物的定性和定量分析一直是分析化学领域研究的难点。在药物分析领域,复杂体系分析主要包括了中草药及中药组方中有效成分或违禁物质的分析,生物样本中药物及其代谢产物以及内源性代谢物的分析等。一方面这类样本的成分和基质都很复杂,一种中草药可能就含有成千上百种化学成分,而在生物样本中更含有种类繁多数量巨大的内源性物质；另一方面很多目标化合物是微量甚至痕量的,并且结构是未知的。因此,如何从复杂体系中快速灵敏地发现并确证已知目标化合物,并且有效排除干扰发现并鉴定未知的目标化合物,以及实现对微量目标化合物的准确定量一直是近年来研究的热点。本论文选择了几种常见的复杂体系样本,针对不同的分析目标进行分析技术和分析策略的探索性研究。论文第一部分选择微量生物样本的代谢组学分析技术为研究目标,针对目前代谢组学研究面临的生物样本微量,但是需要高覆盖不同丰度和理化性质的内源性代谢物的需求,建立了一种新的生物样品前处理方法,该方法首先向100μl血浆或组织匀浆液中加入内标溶液和3ml甲醇/甲基叔丁基醚(1：1,v/v)并涡旋沉淀蛋白,经离心后弃去蛋白沉淀,溶液部分加入3.5ml甲基叔丁基醚和1.2ml去离子水后涡旋萃取,脂质代谢物被萃取到有机相,极性小分子代谢物被萃取到水相,离心后分别将有机相和水相用氮气吹干后复溶。该方法可以实现从同一份微量生物样本(100μl)中同时提取并分离脂质和极性小分子内源性代谢物。在此基础上,采用对强极性化合物具有良好保留作用的亲水相互作用色谱和灵敏度高、定量准确的三重四极杆质谱联用技术新建了靶向极性小分子代谢组学的分析方法,结合实验室已有的靶向脂质组学分析方法和脂肪酸分析方法,使得生物样本经前处理后可以按照代谢物极性和丰度不同采用上述系列液质联用分析方法进行分析,形成一个新的基于微量生物样本的高覆盖靶向代谢组学分析技术平台。该平台实现了同时覆盖包括脂肪酸代谢、鞘脂代谢、磷脂和甘油酯代谢、氨基酸代谢、能量代谢和核昔酸代谢等主要代谢通路中808种代谢物的靶向分析。采用该平台基于代谢组学研究了银杏类提取物(银杏叶提取物和内酯提取物)对心肌缺血损伤大鼠的心脏保护作用。结果发现银杏叶提取物和内酯提取物能够显著调节由于心肌缺血损伤造成的脂质代谢、氨基酸代谢和能量代谢紊乱,发现了血浆中5种脂肪酸、1种鞘脂、4种磷脂、9种甘油酯、8种氨基酸以及心肌中2种鞘脂、12种磷脂、1种甘油酯、7种极性小分子代谢物可以作为生物标志物,指征银杏叶提取物的心脏保护作用,发现了血浆中5种脂肪酸、10种鞘脂、2种磷脂、9种甘油酯、7种极性小分子化合物以及心肌中3种鞘脂、6种磷脂、1种甘油酯、11种极性小分子代谢物可以作为生物标志物,指征内酯提取物的心脏保护作用。这种调节作用与它们的抗氧化、抗血小板凝集和降血脂的作用有关。为了进一步研究银杏叶提取物和内酯提取物发挥心脏保护作用的物质基础,进行了体外成分的分析和体内代谢产物的研究。首先建立了提取物成分和体内代谢产物的液相色谱-高分辨质谱联用技术的分析方法,该方法可以同时采集提取物和生物样本中目标化合物的高分辨质谱数据和多级质谱数据。然后经过对两种提取物和大鼠给药后尿液、粪便、血浆和心肌组织的分析,并采用质谱树状图相似度过滤技术寻找代谢物,结果在银杏叶提取物中鉴定了六大类48种成分,在银杏叶提取物给药大鼠体内发现18种原型成分和21种甲基化、葡萄糖醛酸化和硫酸化代谢物；在内酯提取物给药大鼠体内发现3种原型成分和1种甲基化代谢物。论文第二部分针对目前保健食品中违禁物质种类繁多、不断出现新的结构类似物,一种方法很难同时实现发现、确证和定量多种违禁物质的问题,选择保健食品作为研究对象,建立了一种新的基于高效液相色谱-高分辨质谱联用技术和质谱树状图相似度过滤技术(MTSF)的保健食品中违禁物质的分析策略。第一步,运用67种已知违禁物质对照品建立分析方法,该方法前处理采用甲醇超声提取,色谱柱为资生堂Capcell Core C18 (50×2.1mm,2.7μm),流动相为缓冲溶液(含10mM甲酸铵和0.1%乙酸)和乙腈,梯度洗脱,流速：0.3ml/min,柱温：30℃,进样量：2μl；质谱采用线性离子阱／傅立叶变换离子回旋共振质谱仪,ESI离子源,正离子检测模式,扫描方式为全扫描以及通过数据依赖扫描模式对最强和次强的离子进行碰撞诱导解离(CID)获得三级碎片离子。然后采集每种违禁物质对照品的高分辨质谱数据、多级质谱数据和保留时间。第二步,将采集到的已知违禁物质的质谱数据生成质谱树状图,并建立违禁物质质谱树状图数据库。第三步,待测样品经甲醇超声提取后采用上述分析方法分析,分析结果首先通过保留时间和高分辨质量数进行初筛。如果样品中含有已知违禁物质,则采用MTSF技术将样品中可疑物质的质谱树状图和数据库中违禁物质的质谱树状图进行相似度比对,如果相似度得分大于950,则样品中的违禁物质得到确证。并且该违禁物质可进一步被定量。第四步,为发现样品中含有的未知违禁物质,采用MTSF技术将样品中所有物质的质谱树状图和数据库中违禁物质的质谱树状图进行相似度比对。相似度得分大于200的化合物认为是潜在的违禁物质。根据这些潜在的违禁物质的质谱树状图和精确质量数推导出其结构,通过购买或者合成对照品后,将其建立到数据库中,从而对新的违禁物质进行确证和定量。目前该策略包含抗疲劳、降糖、镇咳、镇静催眠、激素和减肥六大类67种违禁物质和结构类似物。通过测定市售的50种保健食品和中药组方制剂,证明该分析策略快速而且可靠。论文第三部分针对目前蛋白同化激素类兴奋剂代谢个体差异大而且复杂,特征代谢物类型、体内停留时间和体内浓度水平具有个体差异性,阳性结果不易判断的瓶颈问题,选择服药后人体尿样作为研究对象,建立了基于高效液相色谱-多反应监测技术的兴奋剂代谢物发现新策略。首先建立了司坦唑醇及其代谢物在尿样中的分析方法,该分析方法采用甲基叔丁基醚提取Ⅰ相代谢物,固相萃取法分离Ⅱ相代谢物,色谱柱为Waters Symmetry C18(2.1×100mm,3.5μm),流动相为0.1%甲酸水溶液和乙腈,梯度洗脱,流速：0.3ml/min,柱温：35℃,进样量：5μl。质谱采用三重四极杆质谱, ESI离子源,正离子检测模式,扫描模式为多反应检测(MRM)。其次,以司坦唑醇其主要代谢物的特征碎片离子作为子离子,根据可能的Ⅰ相和Ⅱ相代谢反应类型推测母离子,通过MRM扫描在尿样中全面寻找司坦唑醇的代谢物,特别是未知的和低浓度的代谢物。结果共发现司坦唑醇Ⅰ相代谢物27种,Ⅱ相代谢物21种,其中14种Ⅰ相代谢物和14种Ⅱ相代谢物未见文献报道,9种代谢物在停药后15天仍可在尿液中检测到。因此该策略可有效用于表征蛋白同化激素类兴奋剂的体内代谢轮廓并发现其未知和长时间停留的代谢物,延长其检测窗口期,增强对这类兴奋剂的检测能力。
[Abstract]:Qualitative and quantitative analysis of Multiobjective compounds in complex systems has been a difficult problem in the field of analytical chemistry. In the field of drug analysis, the analysis of complex systems mainly includes the analysis of effective components or prohibited substances in Chinese herbal medicine and Chinese herbal medicines, and the analysis of drugs and their metabolites in biological samples and the analysis of endogenous metabolites. The composition and matrix of such samples are very complex, and a Chinese herbal medicine may contain hundreds of chemicals, and a great variety of endogenous substances in the biological samples; on the other hand, many target compounds are trace and trace, and the structure is unknown. Therefore, how to quickly get from the complex system. The rapid and sensitive detection and identification of known target compounds, and the effective elimination of interference detection and identification of unknown target compounds, and the accurate quantification of trace target compounds have always been the focus of research in recent years. The first part of this paper is to select the metabonomics analysis technique of microbiological samples as the research goal, aiming at the current biological samples in the metabonomics, but the need to cover the endogenous metabolites with high abundance and physicochemical properties is needed, and a new pretreatment side for biological samples is established. The method first added internal standard solution and 3ml methanol / methyl tert Ding Ji ether (1:1, v/v) and precipitate protein into the 100 u plasma or tissue homogenate. After centrifugation, the protein was abandoned and precipitated. The solution was partially added to the 3.5ml methyl tert Ding Ji ether and 1.2ml deionized water after the vortex extraction. The lipid metabolite was extracted into the organic phase, and the polar small molecule was extracted. The metabolites are extracted into the aqueous phase, and after the centrifugation, the organic phase and the water phase are dried and redissolved with nitrogen, respectively. This method can be used to extract and separate the lipid and polar small endogenous metabolites from the same trace biological sample (100 L). On this basis, the hydrophilic interaction with good retention of strong polar compounds is adopted. The analytical method of target polar small molecular metabolomics is built by the method of interaction chromatography, high sensitivity and quantitative and accurate three heavy quadrupole mass spectrometry. Combined with the existing target liposome analysis and fatty acid analysis methods in the laboratory, the biological samples can be used in accordance with the polarity and abundance of metabolites. A new high coverage targeting metabonomics analysis platform based on microbiological samples was developed. The platform was used to cover 80 major metabolic pathways, including fatty acid metabolism, sphingolipid metabolism, phospholipid and glyceride metabolism, amino acid metabolism, energy metabolism, and the metabolism of celecoxib metabolism. The target analysis of 8 metabolites. The protective effect of Ginkgo biloba extract (Ginkgo leaf extract and lactone extract) on cardiac ischemic injury in rats was studied based on this platform. The results showed that Ginkgo biloba extract and lactone extract could significantly regulate the lipid metabolism caused by myocardial ischemia injury. Metabolic and energy metabolic disorders, 5 kinds of fatty acids, 1 sphingolipids, 4 kinds of phospholipids, 9 glycerides, 8 kinds of amino acids, 2 kinds of sphingolipids in the heart, 12 kinds of phospholipids, 1 glycerides, 7 polar small molecule metabolites can be used as biomarkers, indicating the protective effect of the extract of Ginkgo biloba, 5 fatty acids in plasma, 10 in plasma, and 5 fatty acids in plasma, 10. Sphingolipids, 2 kinds of phospholipids, 9 glycerides, 7 polar small molecular compounds, 3 sphingolipids, 6 kinds of phospholipids, 1 glycerides, 11 polar small molecule metabolites can be used as biomarkers, indicating the protective effect of lactone extract. This regulation is related to their antioxidation, antiplatelet agglutination and hyperlipidemia. In order to further study the material basis of the extract of Ginkgo biloba leaves and lactone extract to play the protective effect of the heart, the analysis of components in vitro and the metabolites in the body were carried out. First, the analysis method of the extract composition and the liquid chromatography high resolution mass spectrometry of the metabolites in the body was established. The method can be collected and extracted at the same time. High resolution mass spectrometry data and multilevel mass spectrometry data of the target compounds in the biological samples and the analysis of urine, feces, plasma and myocardial tissue after the two extracts and rats were given, and the mass spectrum dendrogram similarity filtration technique was used to find the metabolites, and the results were identified in the extract of Ginkgo biloba leaves with six categories and 48 components. 18 prototypes and 21 kinds of methylation, glucuronidization and sulfated metabolites were found in the rats of Ginkgo biloba extract, and 3 prototypes and 1 methylation metabolites were found in the rat body of lactone extract. The second part of the paper was aimed at a wide variety of prohibited substances in the present health food, and a new knot appeared. A method is difficult to realize the discovery, confirm and quantify a variety of prohibited substances and choose health food as the research object. A new analysis strategy based on high performance liquid chromatography high resolution mass spectrometry (high resolution mass spectrometry) and mass spectrum dendrogram similarity filtration technology (MTSF) is established. One step, 67 kinds of known contraband substances were used to establish an analytical method. The method was processed by ultrasonic extraction of methanol. The chromatographic column was Shiseido Capcell Core C18 (50 x 2.1mm, 2.7 mu m). The mobile phase was buffered solution (including 10mM ammonium formate and 0.1% acetic acid) and acetonitrile, gradient elution, flow velocity: 0.3ml/min, column temperature: 30 C, 2 mu L; The spectrum uses linear ion trap / Fu Liye transform ion cyclotron resonance mass spectrometer, ESI ion source, positive ion detection mode, scanning mode for full scanning and collision induced dissociation (CID) for the strongest and second strong ions through data dependent scanning mode to obtain three fragment ions. Then, the high resolution of each contraband substance is collected. Mass spectrometric data, multilevel mass spectrometry data and retention time. The second step is to generate mass spectrogram of mass spectra of known prohibited substances and establish a database of mass spectrum dendrims of prohibited substances. The third step is to analyze the samples after methanol ultrasonic extraction, and the analysis results first pass the retention time and high resolution. The quality number is screened. If the sample contains known prohibited substances, the MTSF technique is used to compare the mass spectrum of the suspected substance in the sample with the mass spectrum dendrims of the prohibited substances in the database. If the similarity score is greater than 950, the prohibited substance in the sample is confirmed. And the prohibited substance can be further carried out. Fourth step, in order to discover the unknown prohibited substances in the sample, the MTSF technique is used to compare the mass spectra of all the substances in the sample with the mass spectra of the mass spectra of the prohibited substances in the database. The compounds with the similarity score greater than 200 are considered as potential contraband. According to the mass spectra of these potential prohibited substances The structure is derived from the dendrims and the number of exact mass. By buying or synthesizing control products, they are established in a database to confirm and quantify the new prohibited substances. The strategy currently includes anti fatigue, hypoglycemic, antitussive, sedative and hypnotic, 67 kinds of prohibited substances and structural analogues of the six major categories of hormones and weight loss. 50 kinds of health food and traditional Chinese medicine prescription preparation, proved that the analysis strategy is fast and reliable. The third part of the paper is aimed at the large and complex metabolism of the protein anabolic stimulants, the characteristic metabolite type, the body residence time and the body concentration level have individual difference, the positive result is not easy to judge the bottleneck problem. A new strategy was established for the discovery of stimulant metabolites based on high performance liquid chromatography and multi reaction monitoring technology. Firstly, the analytical method of Coran and its metabolites in urine was established. The analytical method used methyl tert butyl ether to extract I phase metabolite and solid phase extraction to separate the phase of phase metabolism. The chromatographic column is Waters Symmetry C18 (2.1 x 100mm, 3.5 mu m), the flow phase is 0.1% formic acid water solution and acetonitrile, gradient elution, flow velocity: 0.3ml/min, column temperature: 35 C, sample quantity: 5 mu L. mass spectrometry adopts three heavy quadrupole mass spectrometry, ESI ion source, positive ion detection mode, and scanning mode for multi reaction detection (MRM). Secondly, mainly the scans of the main The characteristic fragment ions of metabolites as subions, according to the possible type of phase I and phase II metabolic reactions to speculate the mother ion, through MRM scanning in the urine sample to find the metabolites of the polyosinol, especially the unknown and low concentrations of metabolites. The results were found to be 27 kinds of metabolites, 21 kinds of phase metabolites, 14 kinds of metabolites. The phase I metabolites and 14 types of second phase metabolites have not been reported, and the 9 metabolites can still be detected in the urine at 15 days after stopping the drug. Therefore, this strategy can be used to characterize the metabolic profiles of the protein assimilation hormone stimulants in vivo and to discover their unknown and long stay metabolites, prolong the detection window period, and enhance this kind of excitement. The testing ability of the agent.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R917
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本文编号：2016733