本文選題：復(fù)雜體系 + 代謝組學(xué)�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：復(fù)雜體系中多目標化合物的定性和定量分析一直是分析化學(xué)領(lǐng)域研究的難點。在藥物分析領(lǐng)域,復(fù)雜體系分析主要包括了中草藥及中藥組方中有效成分或違禁物質(zhì)的分析,生物樣本中藥物及其代謝產(chǎn)物以及內(nèi)源性代謝物的分析等。一方面這類樣本的成分和基質(zhì)都很復(fù)雜,一種中草藥可能就含有成千上百種化學(xué)成分,而在生物樣本中更含有種類繁多數(shù)量巨大的內(nèi)源性物質(zhì)；另一方面很多目標化合物是微量甚至痕量的,并且結(jié)構(gòu)是未知的。因此,如何從復(fù)雜體系中快速靈敏地發(fā)現(xiàn)并確證已知目標化合物,并且有效排除干擾發(fā)現(xiàn)并鑒定未知的目標化合物,以及實現(xiàn)對微量目標化合物的準確定量一直是近年來研究的熱點。本論文選擇了幾種常見的復(fù)雜體系樣本,針對不同的分析目標進行分析技術(shù)和分析策略的探索性研究。論文第一部分選擇微量生物樣本的代謝組學(xué)分析技術(shù)為研究目標,針對目前代謝組學(xué)研究面臨的生物樣本微量,但是需要高覆蓋不同豐度和理化性質(zhì)的內(nèi)源性代謝物的需求,建立了一種新的生物樣品前處理方法,該方法首先向100μl血漿或組織勻漿液中加入內(nèi)標溶液和3ml甲醇/甲基叔丁基醚(1：1,v/v)并渦旋沉淀蛋白,經(jīng)離心后棄去蛋白沉淀,溶液部分加入3.5ml甲基叔丁基醚和1.2ml去離子水后渦旋萃取,脂質(zhì)代謝物被萃取到有機相,極性小分子代謝物被萃取到水相,離心后分別將有機相和水相用氮氣吹干后復(fù)溶。該方法可以實現(xiàn)從同一份微量生物樣本(100μl)中同時提取并分離脂質(zhì)和極性小分子內(nèi)源性代謝物。在此基礎(chǔ)上,采用對強極性化合物具有良好保留作用的親水相互作用色譜和靈敏度高、定量準確的三重四極桿質(zhì)譜聯(lián)用技術(shù)新建了靶向極性小分子代謝組學(xué)的分析方法,結(jié)合實驗室已有的靶向脂質(zhì)組學(xué)分析方法和脂肪酸分析方法,使得生物樣本經(jīng)前處理后可以按照代謝物極性和豐度不同采用上述系列液質(zhì)聯(lián)用分析方法進行分析,形成一個新的基于微量生物樣本的高覆蓋靶向代謝組學(xué)分析技術(shù)平臺。該平臺實現(xiàn)了同時覆蓋包括脂肪酸代謝、鞘脂代謝、磷脂和甘油酯代謝、氨基酸代謝、能量代謝和核昔酸代謝等主要代謝通路中808種代謝物的靶向分析。采用該平臺基于代謝組學(xué)研究了銀杏類提取物(銀杏葉提取物和內(nèi)酯提取物)對心肌缺血損傷大鼠的心臟保護作用。結(jié)果發(fā)現(xiàn)銀杏葉提取物和內(nèi)酯提取物能夠顯著調(diào)節(jié)由于心肌缺血損傷造成的脂質(zhì)代謝、氨基酸代謝和能量代謝紊亂,發(fā)現(xiàn)了血漿中5種脂肪酸、1種鞘脂、4種磷脂、9種甘油酯、8種氨基酸以及心肌中2種鞘脂、12種磷脂、1種甘油酯、7種極性小分子代謝物可以作為生物標志物,指征銀杏葉提取物的心臟保護作用,發(fā)現(xiàn)了血漿中5種脂肪酸、10種鞘脂、2種磷脂、9種甘油酯、7種極性小分子化合物以及心肌中3種鞘脂、6種磷脂、1種甘油酯、11種極性小分子代謝物可以作為生物標志物,指征內(nèi)酯提取物的心臟保護作用。這種調(diào)節(jié)作用與它們的抗氧化、抗血小板凝集和降血脂的作用有關(guān)。為了進一步研究銀杏葉提取物和內(nèi)酯提取物發(fā)揮心臟保護作用的物質(zhì)基礎(chǔ),進行了體外成分的分析和體內(nèi)代謝產(chǎn)物的研究。首先建立了提取物成分和體內(nèi)代謝產(chǎn)物的液相色譜-高分辨質(zhì)譜聯(lián)用技術(shù)的分析方法,該方法可以同時采集提取物和生物樣本中目標化合物的高分辨質(zhì)譜數(shù)據(jù)和多級質(zhì)譜數(shù)據(jù)。然后經(jīng)過對兩種提取物和大鼠給藥后尿液、糞便、血漿和心肌組織的分析,并采用質(zhì)譜樹狀圖相似度過濾技術(shù)尋找代謝物,結(jié)果在銀杏葉提取物中鑒定了六大類48種成分,在銀杏葉提取物給藥大鼠體內(nèi)發(fā)現(xiàn)18種原型成分和21種甲基化、葡萄糖醛酸化和硫酸化代謝物；在內(nèi)酯提取物給藥大鼠體內(nèi)發(fā)現(xiàn)3種原型成分和1種甲基化代謝物。論文第二部分針對目前保健食品中違禁物質(zhì)種類繁多、不斷出現(xiàn)新的結(jié)構(gòu)類似物,一種方法很難同時實現(xiàn)發(fā)現(xiàn)、確證和定量多種違禁物質(zhì)的問題,選擇保健食品作為研究對象,建立了一種新的基于高效液相色譜-高分辨質(zhì)譜聯(lián)用技術(shù)和質(zhì)譜樹狀圖相似度過濾技術(shù)(MTSF)的保健食品中違禁物質(zhì)的分析策略。第一步,運用67種已知違禁物質(zhì)對照品建立分析方法,該方法前處理采用甲醇超聲提取,色譜柱為資生堂Capcell Core C18 (50×2.1mm,2.7μm),流動相為緩沖溶液(含10mM甲酸銨和0.1%乙酸)和乙腈,梯度洗脫,流速：0.3ml/min,柱溫：30℃,進樣量：2μl；質(zhì)譜采用線性離子阱／傅立葉變換離子回旋共振質(zhì)譜儀,ESI離子源,正離子檢測模式,掃描方式為全掃描以及通過數(shù)據(jù)依賴掃描模式對最強和次強的離子進行碰撞誘導(dǎo)解離(CID)獲得三級碎片離子。然后采集每種違禁物質(zhì)對照品的高分辨質(zhì)譜數(shù)據(jù)、多級質(zhì)譜數(shù)據(jù)和保留時間。第二步,將采集到的已知違禁物質(zhì)的質(zhì)譜數(shù)據(jù)生成質(zhì)譜樹狀圖,并建立違禁物質(zhì)質(zhì)譜樹狀圖數(shù)據(jù)庫。第三步,待測樣品經(jīng)甲醇超聲提取后采用上述分析方法分析,分析結(jié)果首先通過保留時間和高分辨質(zhì)量數(shù)進行初篩。如果樣品中含有已知違禁物質(zhì),則采用MTSF技術(shù)將樣品中可疑物質(zhì)的質(zhì)譜樹狀圖和數(shù)據(jù)庫中違禁物質(zhì)的質(zhì)譜樹狀圖進行相似度比對,如果相似度得分大于950,則樣品中的違禁物質(zhì)得到確證。并且該違禁物質(zhì)可進一步被定量。第四步,為發(fā)現(xiàn)樣品中含有的未知違禁物質(zhì),采用MTSF技術(shù)將樣品中所有物質(zhì)的質(zhì)譜樹狀圖和數(shù)據(jù)庫中違禁物質(zhì)的質(zhì)譜樹狀圖進行相似度比對。相似度得分大于200的化合物認為是潛在的違禁物質(zhì)。根據(jù)這些潛在的違禁物質(zhì)的質(zhì)譜樹狀圖和精確質(zhì)量數(shù)推導(dǎo)出其結(jié)構(gòu),通過購買或者合成對照品后,將其建立到數(shù)據(jù)庫中,從而對新的違禁物質(zhì)進行確證和定量。目前該策略包含抗疲勞、降糖、鎮(zhèn)咳、鎮(zhèn)靜催眠、激素和減肥六大類67種違禁物質(zhì)和結(jié)構(gòu)類似物。通過測定市售的50種保健食品和中藥組方制劑,證明該分析策略快速而且可靠。論文第三部分針對目前蛋白同化激素類興奮劑代謝個體差異大而且復(fù)雜,特征代謝物類型、體內(nèi)停留時間和體內(nèi)濃度水平具有個體差異性,陽性結(jié)果不易判斷的瓶頸問題,選擇服藥后人體尿樣作為研究對象,建立了基于高效液相色譜-多反應(yīng)監(jiān)測技術(shù)的興奮劑代謝物發(fā)現(xiàn)新策略。首先建立了司坦唑醇及其代謝物在尿樣中的分析方法,該分析方法采用甲基叔丁基醚提取Ⅰ相代謝物,固相萃取法分離Ⅱ相代謝物,色譜柱為Waters Symmetry C18(2.1×100mm,3.5μm),流動相為0.1%甲酸水溶液和乙腈,梯度洗脫,流速：0.3ml/min,柱溫：35℃,進樣量：5μl。質(zhì)譜采用三重四極桿質(zhì)譜, ESI離子源,正離子檢測模式,掃描模式為多反應(yīng)檢測(MRM)。其次,以司坦唑醇其主要代謝物的特征碎片離子作為子離子,根據(jù)可能的Ⅰ相和Ⅱ相代謝反應(yīng)類型推測母離子,通過MRM掃描在尿樣中全面尋找司坦唑醇的代謝物,特別是未知的和低濃度的代謝物。結(jié)果共發(fā)現(xiàn)司坦唑醇Ⅰ相代謝物27種,Ⅱ相代謝物21種,其中14種Ⅰ相代謝物和14種Ⅱ相代謝物未見文獻報道,9種代謝物在停藥后15天仍可在尿液中檢測到。因此該策略可有效用于表征蛋白同化激素類興奮劑的體內(nèi)代謝輪廓并發(fā)現(xiàn)其未知和長時間停留的代謝物,延長其檢測窗口期,增強對這類興奮劑的檢測能力。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R917
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本文編號：2016733