微囊藻毒素总量的快速检测方法研究

发布时间：2018-05-20 09:50

本文选题：2-甲基-3-甲氧基-4-苯基丁酸(MMPB) + 微囊藻毒素　；参考：《江南大学》2016年硕士论文

【摘要】：水体富营养化(Eutrophication)致使藻类大量繁殖,引发蓝藻水华在不同水域频繁发生,导致自然生态系统失衡。微囊藻毒素(Microcystins,MCs)是由蓝藻产生且具有强烈肝毒性、促癌性的代谢产物。MCs可通过多种途径进入生物体内,与生物体的肝脏、肾脏等器官作用导致生理病变,极大地危害了人类的健康和生态环境安全。因此,迫切需要一种先进、可靠、快速、高效的方法测定不同环境中MCs的存在。事实上,研究报道MCs的种类已超过九十种以上。目前,国内外利用色谱质谱联用技术检测单种MC的研究很多,但由于受到标准品稀缺的制约,无法全面真实地反映藻毒素污染的水平。因此,建立一种灵敏度高、准确性好的测定不同基质中MCs总量的方法具有重要意义。Adda侧链((2s,3s,8s,9s)-3-氨基-9-甲氧基-10-苯基-2,6,8-三甲基-10-苯基-4,6-二烯酸)是MCs的特征结构。特定条件下,通过氧化MCs分子中Adda侧链的共轭双键,可生成名为2-甲基-3-甲氧基-4-苯基丁酸(MMPB)的化合物。因而,通过测定MMPB含量可以间接获知不同环境基质中MCs总量。文献报道,多采用Lemieux oxidation法将MCs转化为MMPB。但是,Lemieux oxidation法存在步骤复杂、耗时长等缺点。相反,臭氧具有极强的氧化性能,被誉为清洁、高效的氧化剂。此外,为了未来能够从环境藻类样品中进行大规模地纯化、制备MCs作为试验材料以进行深入研究,还对从蓝藻中提取、分离、净化MCs的条件进行探究和优化。具体研究内容包括:(1)详细探究利用臭氧氧化MCs生成MMPB的方法条件。同时,优化MMPB与氯甲酸甲酯(MCF)进行甲酯化反应的条件。结果表明,常温酸性条件下(p H 3.0),臭氧可高效地定量氧化MCs,氧化产物MMPB与MCF在水相中快速反应,经氯仿萃取后进行气相色谱串联质谱(GC-MS)分析。该方法在MCs浓度10~300μg L-1的范围内,MMPB-OCH3和内标物4-PB-OCH3峰面积比值与MCs浓度之间具有良好的线性关系(R20.996),方法检出限为0.34μg L-1(S/N=3)。样品加标回收率为77.7%~80.2%,相对标准偏差为4.1~6.5%。结果说明,该方法具有良好的回收率与重现性并可应用于水体中MCs总量的分析。(2)对从蓝藻中提取、分离、净化MCs的条件进行探究和优化。同时,还定性分析了蓝藻中MCs的种类并利用MMPB法对其总量进行测定。研究证明,室温条件下,采用搅拌和超声粉碎联用方法,利用70%甲醇水溶液对蓝藻样品进行提取可获得良好效果。同时,为了能够去除藻胆蛋白的影响,利用等电点沉淀法将溶液p H值调节至4.0左右,使蛋白质变性析出。随后采用SPE柱对其进一步富集纯化,SPE的实验条件为:20%甲醇为淋洗剂,90%甲醇(含0.10%三氟乙酸)为洗脱剂。采用上述已优化的实验条件,对不同区域的蓝藻样品中MCs种类进行定性分析。结果显示,MCs种类与数量随时间、地点不同进行变更。MMPB法测定MCs总量的结果高于单种MC含量之和,说明蓝藻样品中可能存在未知MC种类,但由于缺乏相应的标准品,无法全面准确定性分析未知MCs的种类。然而,针对能够准确定性的MCs如MC-RR、MC-LR等,本研究的优化方法可为其未来从环境藻类样品中进行大量制备奠定良好的基础。(3)采用加速溶剂萃取(ASE)和超高效液相色谱-串联质谱(UPLC-MS)联用技术,建立了快速提取和测定水产品中毒性最强的两种游离态藻毒素(MC-RR、MC-LR)含量的方法。通过对实验条件的筛选和优化,确定ASE的优化条件:85%甲醇水溶液为萃取剂,弗罗里硅土为吸附剂,萃取温度85oC,静态循环2次,最终萃取体积20 m L。实验结果表明,微囊藻毒素的质量浓度在0.025~8μg/g范围内,峰面积与其质量浓度线性关系良好(R20.997)。样品加标回收率为68.5~86.3%,相对标准偏差为7.0%~9.8%。该研究适用于淡水水产品中游离态单种MC分析测定。因为水产品体内部分MCs与蛋白磷酸酶通过共价键结合后,采用常规的提取手段无法将其分离出来。另外,受到商品化MC标准品稀缺的限制,无法全面地检测单种藻毒素的含量。为准确、真实反映水产品中MCs的污染水平,研究通过MMPB法检测水产品中MCs总量,采用固相萃取手段对MMPB进行富集和净化,甲酯化处理后进行GC-MS分析。结果表明,MCs浓度在0.02~10μg/g范围内具有良好的线性关系(R20.995)。加标回收率范围为72~85%,相对标准偏差为8.2~10.5%。实验证明,这种方法较好的满足了水产品中MCs总量的分析要求。
[Abstract]:Water eutrophication (Eutrophication) causes a large number of algae to reproduce, causing cyanobacteria blooms frequently occurring in different waters, leading to the imbalance of natural ecosystems. Microcystins (MCs) is produced by cyanobacteria and has strong hepatotoxicity, and the carcinogenic metabolite.MCs can enter organisms through a variety of ways, and the liver of organisms. Organs such as dirty, kidney and other organs lead to physiological diseases, which greatly harm human health and ecological environment safety. Therefore, an advanced, reliable, rapid and efficient method for the determination of MCs in different environments is urgently needed. In fact, more than ninety kinds of MCs have been reported. There are many studies on the detection of single MC, but due to the shortage of standard products, it is impossible to fully reflect the level of algae toxin pollution. Therefore, it is of great significance to establish a high sensitivity and accurate method for the determination of the total amount of MCs in different substrates (2S, 3S, 8s, 9S) -3- amino -9- methoxy -10- phenyl -2,6,8- three methyl - 10- phenyl -4,6- two enoic acid) is a characteristic structure of MCs. Under specific conditions, a compound named 2- methyl -3- methoxy -4- phenyl butyric acid (MMPB) can be produced by oxidizing the conjugated double bonds of the Adda side chain of the MCs molecule. Thus, the MCs amount in different environmental bases can be indirectly known by the determination of MMPB content. The method transforms MCs into MMPB., but the Lemieux oxidation method has the disadvantages of complex steps and long time consuming. On the contrary, the ozone has very strong oxidation performance and is known as clean and efficient oxidizing agent. In addition, in order to be able to purify from the environmental algae samples in a large scale in the future, the MCs is prepared as a test material for in-depth study, and also from blue. The conditions of extraction, separation and purification of MCs are explored and optimized. The specific research contents include: (1) the conditions of using ozone to oxidize MCs to produce MMPB are discussed in detail. At the same time, the conditions for the methylene esterification of MMPB and methyl chloroformate (MCF) are optimized. The results show that ozone can efficiently oxidize MCs and oxygen at the ambient temperature acidity (P H 3). The product MMPB and MCF reacted rapidly in the water phase and analyzed by gas chromatography tandem mass spectrometry (GC-MS) after chloroform extraction. The method has a good linear relationship between the 4-PB-OCH3 peak area ratio of MMPB-OCH3 and the internal standard and the MCs concentration in the range of MCs concentration 10~300 uh g L-1 (R20.996). The detection limit is 0.34 mu g. The standard recovery rate is 77.7%~80.2%, and the relative standard deviation is 4.1~6.5%.. The method has good recovery and reproducibility and can be applied to the analysis of the total amount of MCs in water. (2) the conditions of extracting, separating and purifying MCs from cyanobacteria are explored and optimized. At the same time, the species of MCs in cyanobacteria are analyzed and MMPB method is used. The total amount is measured. It has been proved that, under the condition of room temperature, the extraction of cyanobacteria with 70% methanol solution can be obtained by mixing and ultrasonic comminution. At the same time, in order to remove the effect of alginin, the P H value of the solution is adjusted to about 4 by isoelectric point precipitation method, and then the protein denaturation can be precipitated. The SPE column was used to further enrich and purify it. The experimental conditions of SPE were 20% methanol as the eluent and 90% methanol (containing 0.10% three FLUOROACETIC acid) as eluant. The MCs species in the samples of cyanobacteria in different regions were qualitatively analyzed with the above optimized experimental conditions. The results showed that the variety and quantity of MCs changed with the time and place to change the.MMPB method. The results of the total MCs determination are higher than the sum of the single MC content, indicating that there may be unknown MC species in the cyanobacteria samples. However, due to the lack of corresponding standard products, the species of unknown MCs can not be comprehensively and accurately analyzed. However, the optimization method for the accurate qualitative MCs, such as MC-RR, MC-LR, etc., can be used for the future from the environmental algae samples. A good foundation is laid for a large number of preparation. (3) the rapid extraction and determination of the two most toxic free algae toxins (MC-RR, MC-LR) in aquatic products by accelerated solvent extraction (ASE) and super high performance liquid chromatography tandem mass spectrometry (UPLC-MS) are established. Through screening and optimization of the experimental conditions, the advantages of ASE are determined. Conditions: 85% methanol water solution is an extractant and Florida siliceous soil is an adsorbent. The extraction temperature is 85oC, the static cycle is 2 times, the final extraction volume 20 m L. shows that the mass concentration of microcystin is in the range of 0.025~8 g/g, and the peak area has a good linear relationship with the mass concentration (R20.997). The recovery rate of the sample is 68.5~86.3%, and the ratio of the sample addition is 68.5~86.3%. The standard deviation is 7.0%~9.8%., which is suitable for the determination of isolated MC in the middle reaches of freshwater aquatic products. Because some of the MCs and protein phosphatase in the aquatic products are combined with the covalent bonds, they can not be separated by conventional methods. In addition, the limitation of the scarcity of commercialized MC standard products can not fully detect the single species of algae. In order to accurately reflect the pollution level of MCs in aquatic products, the total amount of MCs in aquatic products was detected by MMPB method. Solid phase extraction was used to enrich and purify the MMPB and GC-MS analysis after methyl esterification. The results showed that the MCs concentration had a good linear relationship (R20.995) in the range of 0.02~10 mu g/g. The rate range is 72~85% and the relative standard deviation is 8.2~10.5%.. Experiments show that this method can better meet the analysis requirements of MCs in aquatic products.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.63;TS254.7

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