Study On The Method Of Rapid Analysis Of Total Microcystins

The water eutrophication caused outbreak of cyanobacterial blooms in different waters, leading to natural ecosystems imbalance. Microcystins(MCs) are metabolites of cyanobacteria and have strong hepatotoxicities, cancer-promoting properties. MCs can enter the living body through a variety of pathways that pose a serious threat to ecological environment security and cause a major challenge for public health due to their capability of accumulation in animal tissues. In recent years, more than 90 kinds of MCs had been found. At present, there are many researches on detecting a single MC by using chromatography-mass spectrometry in the domestic and abroad. However, due to the constraints of standards, it can’t fully reflect true pollution level of microcystins. Therefore, it is of great significance to establish a high sensitivity and good accuracy measurement method for the determination of the total amount of MCs in different sample matrices. Under certain conditions, by the cleavage of the chemical structure Adda to form 2-methyl-3-methoxy-4-phenylbutyric acid(MMPB) for indirect quantification of the total MCs. At present, all MC variants containing Adda give rise to one MMPB molecule. Therefore, we can use this MMPB method for total MCs determination in a range of sample matrices. In literatures, the Lemieux oxidation is used to transform MCs into MMPB. However, The Lemieux oxidation involves complex procedures, which is not only time consuming, but also uses a variety of organic solvents. In contrast, ozone has a strong oxidation property, known as a clean and efficient oxidant. In addition, for the future to be able to carry out large-scale preparation of MCs, which use environmental cyanobacterial samples as a raw material. The conditions of extraction, separation and purification of MCs from cyanobacteria were investigated and optimized.(1) To determine the total microcystins, a novel analytical method, including ozonolysis, methylation of MMPB with methylchloroformate(MCF) and gas chromatography mass spectrometry(GC-MS) detection was developed. The results show that MCs can be oxidized by ozone to produce MMPB at ambient temperature. The oxidation conditions as well as the esterification process were optimized and, subsequently applied to analysis of environmental samples. The method showed the ratio of the peak area of MMPB-OCH3 and 4PB-OCH3, and the amount of MCs(10 to 300 μg L-1) had good linear relationship and high sensitivity with a detection limit of 0.34 μg L-1. The recoveries for spiked samples were in the range from 77.7% to 80.2% with RSD between 4.1%-6.5%. The established method was successfully applied to the analysis of microcystins in water samples.(2) The conditions of extraction, separation and purification of MCs from cyanobacteria were investigated and optimized. Qualitative analysis of the MCs species in cyanobacteria and detect the total amount of MCs by MMPB method. Studies show that at room temperature, combined with the use of agitation and sonication, the cyanobacteria samples were extracted to obtain good results by using 70% Me OH. At the same time, in order to remove the influence of the phycobiliprotein, adjusting the p H to about 4 to denature and precipitate proteins. The optimum conditions for SPE extraction were as followed: wash with 20%Me OH; eluted with 90%Me OH /0.10% TFA. Qualitative analysis of MC species in cyanobacteria samples from different regions. Results showed that the type and quantity of MC changed with time and place. Total MCs analysis by MMPB method were higher than the total sum of single MC content, which indicated that there may be unknown MC species in cyanobacteria, but due to the lack of appropriate standards, some MC congeners that would otherwise go undetected. However, in view of the MCs, which can be accurate qualitative such as MC-LR, MC-RR, etc. The optimization method can lay good foundation for the future preparation of MCs from the environmental cyanobacteria samples.(3) A method for rapid extraction and determination of free microcystin(MC-RR、MC-LR) in aquatic products by accelerated solvent extraction(ASE) coupled with Ultra performance liquid chromatography-mass spectrometry(UPLC-MS) was proposed. Through the screening and optimization of experimental conditions, the results showed that the optimum conditions for extraction were as followed: 85% Me OH as solvent, extraction temperature of 85 oC, static loop of two times and final extraction volume 20 m L. Experimental results proved that peak area and concentration of the microcystins had good linear relationship with a linear range between 0.025 and 8 μg/g(R2 >0.997). The recoveries for spiked samples were in the range from 68.5% to 86.3%, the relative standard deviations were from7.0% to 9.8%. The established method was suitable for determination of free microcystins in aquatic products.On one hand, due to MCs can covalently bound to protein phosphatases in the aquatic product body, using conventional extraction methods can’t sufficiently separated MCs from sample matrices. On the other hand, limited by the market commercialization of MCs standards, the content of the single species of microcytin can’t be fully tested. In order to accurately and truly reflect the pollution level of MCs in aquatic products, MMPB method was used to detect total MCs in aquatic products, and MMPB was enriched and purified by solid phase extraction. Results showed that the MMPB method had good linear relationship with a linear range between 0.02 and 10 μg /g(R2 >0.995). The recoveries were in the range from 72% to 85%, the relative standard deviations were from8.2% to 10.5%. The results show that this method can meet the requirements of the total amount of MCs in aquatic products.