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题名: 液相色谱-质谱联用测定生物样品中肝毒性微囊藻毒素及其代谢物
作者: 戴明
答辩日期: 2008-06-16
导师: 谢平
专业: 水生生物学
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 液相色谱-质谱联用 ; 微囊藻毒素 ; 代谢物 ; 定量测定 ; 方法验证 ; 提取制备 ; ; 大鼠
其他题名: Determination of hepatotoxic microcystin and its metabolites in biological samples by liquid chromatography-electrospray tandem mass spectrometry
摘要: 随着人类社会生产力的大幅提高及人口压力的不断增大,全球范围的淡水水体富营养化现象也日趋严重,由此引起的蓝藻水华在世界范围内频繁暴发,并释放出大量有害次生代谢产物。微囊藻毒素是一种在蓝藻水华污染中出现频率最高、产毒量最大、危害最严重的一类藻毒素。微囊藻毒素进入人体可以通过很多途径,包括饮用和接触受污染的水体或口服受到污染的蓝藻类保健品,或特殊情况下通过静脉输入影响一些脆弱的透析患者。同时MC不仅可以在鱼体内蓄积,还会在虾、贝、蛤等软体动物体内蓄积,通过水产品的形式进入人体,从而对人类健康造成潜在的威胁。 目前对于微囊藻毒素代谢物及其解毒过程的研究,大多集中于体外实验,而且也局限于定性研究,对于定量研究报道较少。同时由于微囊藻毒素及其代谢产物在生物样品中的浓度通常很低,干扰物质较多,因而特别需要建立可靠、稳定的分析方法,并通过有效的分离、纯化、富集过程实现高灵敏度、高专属性分析。本论文实验的目的主要是对微囊藻毒素在动物体内吸收、分布、代谢和排泄等过程进行探讨并建立高效的分析检测方法,分别研究了微囊藻毒素在鱼和大鼠体内组织和生物流体(血浆和尿等)中的分布和代谢情况。建立了定量测定鱼组织、大鼠血浆、尿样和组织中微囊藻毒素及其代谢产物的LC/MS/MS方法。同时利用液相色谱-离子阱质谱定性研究了大鼠尿样中微囊藻毒素及其代谢产物。并根据实验需要系统研究了微囊藻毒素的提取和纯化,实现毫克级制备,为开展微囊藻毒素代谢及毒理学研究奠定了基础。 1、本研究利用凝胶色谱的独特的色谱保留行为,发展了基于柱色谱技术的微囊藻毒素的分离提纯方法。该方法简便、高效、使用溶剂简单,便于溶剂的回收利用,减少环境的污染。相对现有方法更为经济、环保。制备了两种微囊藻毒素的纯品,其纯度经液相色谱检测,纯度均大于95%,方便了后续代谢、毒理研究的展开。通过麦克尔加成反应合成了一系列微囊藻毒素的代谢产物,为定性、定量研究MC-LR及其代谢物提供了纯品。首次利用离子阱质谱强大的定性功能,对以上一系列化合物进行了多级扫描,对相关碎片离子峰进行了归属。既确定了以上化合物的结构,又比较了它们在(+)-ESI 条件下的裂解机理,为分析和鉴定生物样品中微囊藻毒素及其代谢产物提供了基础。 2、利用固相萃取(SPE)、液相色谱-离子阱串接质谱等手段首次建立了同时测定鱼组织中MC-LR及其谷胱甘肽结合物的分析方法。比较了不同提取溶剂对分析物样品回收率的影响,优化了色谱和质谱条件,从而可在较低浓度下检测微囊藻毒素及其谷胱甘肽结合物产物。利用上述方法测定了腹腔注射的鲫鱼体内肝脏和肾脏等主要代谢器官中MC-LR及其谷胱甘肽结合产物的含量,确定了其浓度-时间分布曲线。 3、通过蛋白质沉淀与固相萃取(SPE)组合净化,液相色谱-离子阱串接质谱检测,建立了在大鼠血浆中同时测定MC-RR、-LR、MC-[Dha7]-LR的分析方法,利用该方法研究了大鼠血浆、肝、肾等组织中MC-RR、-LR、MC-[Dha7]-LR的含量和分布。 4、利用液相色谱-离子阱串接质谱定性检测了大鼠尿样中微囊藻毒素及其代谢产物。确证了大鼠尿样中MC-LR-GSH和MC-LR-Cys结合产物的存在。改进了样品制备、纯化过程,优化了实验步骤,对分析方法的选择性、回收率、检测限、精密度和基质效应等指标进行评价;建立了大鼠血浆和尿中MC-LR及其谷胱甘肽结合产物的LC/MS/MS方法,并利用该方法定量研究了腹腔注射的Wistar大鼠血浆和尿样中MC-LR及其谷胱甘肽结合产物的含量与分布。
英文摘要: Dense blooms of cyanobacterial (blue–green) algae are one of the consequences of the increasing eutrophication in many waters worldwide. The frequency of cyanobacterial blooms in freshwater has increased dramatically in recent years. It is estimated that 50% of cyanobacterial blooms are toxic, producing hepatotoxins, neurotoxins and lipopolysaccharide endotoxins. Microcystins (MCs), produced as the secondary metabolites by cyanobacteria, appear to be the most common cyanotoxins. Microcystins, especially MC-LR, cause adverse effects on mammals, birds and fish, and pose a public threat to human health through contaminated drinking water and fisheries products such as fish, shrimp, shellfish and clam. So far, studies on the metabolites of microcystin and its detoxication mechanism have been mainly focused on in vitro experiments, and few in vivo studies were reported. Due to trace amount of microcystin and its metabolites, and complex matrix in biological samples, it is needed to establish a selective and sensitive analytical technology for identification and/or quantification of these compounds in metabolic organs (e.g., liver and kidney) and biological fluid (e.g., plasma and urine). This dissertation aims to determine the hepatotoxic microcystin and its metabolites in biological samples by using liquid chromatography-electrospray tandem mass spectrometry. The LC/MS/MS methods were developed and validated for the simultaneous determination of MC-LR and its glutathione conjugate in fish tissues, and plasma and urine of rat. Microcystins were prepared from cyanobacteria cell which were collected from laboratory cultures or a water bloom in Lake Dianchi (China). The main results are as follows: 1. Microcystins (MCs) are potent monocyclic heptapeptides produced by many members of cyanobacteria. They are receiving increasing attention around the world as a public health concern. Microcystins with high purity are very important for research and application including toxicological, biochemical studies and develop- ment of detection method. Base on the different retention behavior of microcystins on the gel permeation chromatography, a simplified and efficient procedure for purification of microcystins was developed. Purified microcysins were obtained with simple operation, low cost, light pollution, recovered organic solvent. To investigate the possible metabolites of MC-LR and MC-RR, four compounds including MC-LR-GSH, MC-LR-Cys, MC-LR-NacCys and MC-RR-GSH were synthesized according to the method by Kondo (1992). The fragment ions in the (+)-ESI-MSn of the above compounds were characterized and the amino acid sequences for these fragments were given. 2. A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01 M EDTA-Na2-5% acetic acid, followed by solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5 µg g-1 dry weight [DW]) and ranged from 91% to 103% for MC-LR and from 65.0% to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0 µg g-1 DW, with a limit of quantification (LOQ) of 0.02 µg g-1 DW. The limit of detection (LOD) of the method was 0.007 µg g-1 DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR. 3. A LC/MS/MS bioanalytical method was developed and validated for the simultaneous determination of MCYST-RR, -LR, and MC-[Dha7]-LR in rat plasma, Protein precipitation and solid-phase extraction (SPE) were applied to clean the biological samples. Acceptable recoveries of analytes were obtained at three concentrations (low, medium and high), which were in the range 70.8–88.7% for the MCs in rat plasma. Validation results demonstrated that this method can be used for determining low concentrations of MCs in complex biological matrices such as rat plasma. It was successfully applied to determine the concentration-time profiles in pharmacokinetic studies. By slightly modifying the conditions in the Chapter 3, the concentration-time profiles in rat liver and kidney were determined and analyzed. 4. Liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MSn) was employed to investigate the in vivo metabolites of micro- cystin-LR. Urine and plasma samples were collected after intraperitoneal injection of 50 µg kg-1 microcystin-LR to healthy rats. Urine samples were cleaned up by solid-phase extraction procedures (HLB cartridges). LC-ESI-MSn was used for the separation and identification of the metabolites using C18 column with mobile phase of CH3CN (0.05% formic acid) and H2O (0.05% formic acid). By comparing with the characteristic fragment ions of MC-LR-GSH, MC-LR-Cys and MC-LR-NacCys, the results revealed that MC-LR-GSH and MC-LR-Cys were the two metabolites in rat urine. And some dubious fragment ions were detected in urine and maybe represent three possible metabolites, which should be confirmed by further experiments. MC-LR and MC-LR-GSH in rat urine were quantitatively determined. The results showed that concentration of MC-LR-GSH was very low and was be detectable in urine from 12 to 18h.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12290
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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