Separation And Detection Of Lipophilic Shellfish Toxins In Marine Shellfish And Typical Toxigenic Marine Microalgae

During the past decades,harmful algae blooms?HAB?occurrence is becoming more frequent.The lipophilic shellfish toxins?LSTs?,secondary metabolites of algae,are likely to be accumulated in marine shellfishes and can cause human poisoning incidents.Therefore,it is necessary to carry out toxin screening and detection of the LSTs in algaes and marine shellfishes.It is important to assess the safety risk of edible marine shellfishes.In this study,High performance liquid chromatography-multi-stage mass spectrometry?HPLC-MS/MS?was applied to simultaneously determine several common LSTs in marine shellfishes,and a new method based on the combined contamination of LSTs was established to evaluate the safety risk of commercial marine shellfishes,and the new method was used to assess the food safety risk of seafood shellfishes from Qingdao market.Besides that,high performance liquid chromatography-high-resolution mass spectrometry?HPLC-TOF/MS?method and HPLC-MS/MS were applied to screen and indentify diarrhetic shellfish poisoning?DSPs?and their derivatives of the Prorocentrum lima?P.lima?.The details are as follows:1.In view of the present situation that edible marine shellfishes are combinedly contaminated by different kinds of lipophilic toxins,common lipophilic shellfish toxins in marine shellfishes were simultaneously detected by liquid chromatography-tandem mass spectrometry,and the safety risk of commercial marine shellfish was evaluated using the risk assessment method based on combined contamination of various toxins.Under the optimum conditions,satisfactory recoveries?63.22%⁸8.77%?,precision?relative standard deviations?RSD??14.5%?and sensitivity?limit of detection in the range of 0.5².7 ng/g?of the method were achieved for all the analytes.Among the 129commercially available shellfish samples,41.86%of the samples were detected for at least a kind of toxin.The highest average content was 43.75?g/kg of DTX1,which was the most serious contaminant for marine shellfishes.The total Expose Risk Index??ERI?was calculated based on Tolerable Daily Intake?TDI?and Acute Reference Dose?ARfD?of each toxin to evaluate the safety risk of commercial marine shellfish.The results showed that the ratio of commercially available marine shellfish with safety risk was 15.51%and the food safety risk of scallop was the highest.In summary,a new method based on the combined contamination of lipophilic shellfish toxins was successfully developed for risk assessment of the commercial marine shellfish.The proposed method is harsher compared with the European Food Safety Authority?EFSA?regulation and can make shellfish consumers better to avoid the risk of poisoning.2.In this study,the HPLC-TOF/MS and HPLC-MS/MS combined with PP2A inhibition assay were applied to screen and identify diarrheic shellfish poisoning?DSPs?and their derivatives of the Prorocentrum lima.The experimental results showed that eight kinds of known OAs and a kind of new compound"816"were found in P.lima.The new compound did not report and may be a toxin.Then,the compound was purified by microporous resin,high-speed countercurrent chromatography and semi-preparative HPLC,sequentially.The evaluation of PP2A inhibition by this compound showed that the IC₅₀ value was 1.6412?g/L,which was between OA and DTX1,indicating that the compound has diarrhea-inducing toxicity.The mass spectrometry identification of the compound showed that the molecular weight was 816.4 and the molecular formula was C₄₅H₆₈O₁₃.The structure of "816" based on DTX1 may form a keto group of the C₂₇ or form C₂₈=C₂₉.This study systematically identified DSP toxins and their derivatives in P.lima and provided scientific evidence for the improvement of DSP toxin detection standards in the marine environment and marine shellfishes.