| The rapid development of the mariculture industry and the frequently occurring harmful algae blooms along Chinese coast have led more and more attention to the seafood safety issue, which was directly related to the health of the consumers. Toxins produced during the toxic algae blooms could be retained by the wild or cultured shellfishes. However, the capability in detection and analysis of marine biotoxins is still not very strong in China. New techniques, such as liquid chromatography-mass spectrometry (LC-MS) haven't been used in this field in China before. The advantages of the LC-MS technique itself have made it especially useful in analyzing marine biotoxins. Therefore, there is an urgent need to develop analytical methods for marine biotoxins using LC-MS. In this thesis, the LC-MS analytical methods for toxins such as TTX, DA, OA, DTX1, GYM, SPX and PTX2 have been developed and used to analyze the toxin components in the shellfish and algae samples. During the last three years, large scale poisoning incidents caused by consumption of snails Nassarius spp. have been frequently reported in China. However, the toxins responsible for the poisoning incidents were still not confirmed. To elucidate the enigma, the LC-MS method for TTX analysis has been developed to analyze the role of TTX in the poisoning incidents. According to the analytical results, TTX and its seven derivatives, including 4-epiTTX, 6-epiTTX, 4,9-anhydroTTX, 6-epi-4,9-anhydroTTX, 5-deoxyTTX, 5,6,11-trideoxyTTX and 11-oxoTTX, were detected in the toxic Nassarius gastropods using the LC-MS method. The toxins were confirmed to be responsible for the poisoning incidents through the comparison with mouse bioassay results. It was found out that the content of 5,6,11-trideoxyTTX in the toxic snails was the highest through the select ion monitoring (SIM) analysis, followed by TTX (the responses of different toxins in SIM mode were assumed to be the same). There was no dramatic difference in toxin profile among the toxic species. Toxin distribution among different tissues in the toxic snails collected in Xianyou district of Fujian Province in June, 2004, was also studied. For the distribution of different derivatives, 5,6,11-trideoxyTTX and 5-deoxyTTX had the highest proportion in the muscle, while 11-oxoTTX had the higher proportion in the gullet, TTX and 4,9-anhydroTTX had the highest proportion in the digestive tissues. Total fifty-one Nassarius spp. samples collected from Xiamen, Putian and Ningde of Fujian Province from March, 2005 to July, 2005 were also screened with mouse bioassay method and LC-MS method for TTX, the results showed that one sample is toxic and the toxicity was 4,138MU/100g (about 0.7mg TTX/100g). The toxicity was higher than the safety criteria for consumption of puffer fishes in Japan (10MU/g). Besides, Mysis shrimp Neomysis awatschensis was tested for its potential in monitoring the toxicity of the gastropod Nassarius. It was found that the median lethal time (LT50) of the shrimps correlated well with the toxicity of the snail samples, and the detection limit was about 1,044MU/100g, which is comparable to the safety criteria for consumption of puffer fishes in Japan (10MU/g). The LC-MS methods for the most popular phycotoxins were developed in the lab and the methods were used to analyze a part of shellfish and algae samples collected along the Chinese coast. The detailed works include: (1) LC-MS method for analysis of OA and DTX1 using negative mode LC-MS has been developed, and the detection limit (S/N=3) was 110pg and 220pg for OA and DTX1, respectively. The standard addition method was applied to the analysis of an oyster sample, which was suspected to be toxic after mouse bioassay. The results showed that there were no OA and DTX1 in the sample, but trace amount of OA and DTX1 ester, which could be hydrolyzed to produce OA and DTX1, were considered to be existed in the sample. The result was also testified by the data from protein phosphates inhibition assay of the same sample. (2) The method for the simultaneous analysis of OA, DTX1, GYM, SPX and PTX2 using LC-MS-SIM in positive mode were developed, the detection limit (S/N=3) is 49.6pg,84.6pg,2.1pg,2.8pg and 2.9pg, respectively. The method was also used to screen the toxic oyster sample, however, all the toxins listed above were below the detection limit. (3) Analytical method using LC-MSn detection of DA was developed. The detection limit (S/N=3) of LC-MS1, LC-MS2,and LC-MS3 were 0.15ng,0.05ng and 0.42ng, respectively. The method was used to analyze the toxicity of a strain Pseudonitzschia pungens (PP0201-01) from the East China Sea. However, it was found that the strain could not produce DA under the culture condition of laboratory. All the experimental results suggested that the LC-MS technique has great potential in the analysis of marine biotoxins which might lead to the contamination of shellfish products. This was further confirmed by the bioassay results from mouse bioassay, protein phosphates inhibition assay and Mysis shrimp bioassay. The great advantages of the LC-MS technique, such as the capability for simultaneous analysis of multiple toxins, high analytical efficiency, and the qualitative analytical capability, make the technique a preferential choice in the analysis of aquatic products. |
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