Preliminary Study On Preparation Of Diarrheic Shellfish Poisoning (DSP) Toxins Okadaic Acid (OA) And Dinophysistoxin-1 (DTX1)

Toxins corresponding for the diarrhetic shellfish poisoning (DSP) incidents are a group of phycotoxins produced by toxic dinoflagellates, some of which can form harmful algal blooms (HAB). Seafood contamination by DSP toxins has been reported from time to time along the coast of China, posing potent threats on the health of seafood consumers. However, the lack of DSP toxin standards hampers the effective management and sanitation control of shellfish products.In the present study, the protocol for preparation of okadaic acid (OA) and dinophysistoxin-1 (DTX1),two representative DSP toxins, was studied. Toxins were extracted from the cells of a toxic strain of Prorocentrum lima (Ehrenberg) Dodge, and columns of silica gel, Sephadex LH20 and C18 were used in turn to separate and purify OA and DTX1. The preparative high performance liquid chromatography with a C18 column was employed at the final step to further purify the toxins. Samples collected during the purification process were derivatized with 4-bromomethyl-7-methoxycoumarin (BrMmc) and then analyzed with a high perfomance liquid chromatography-fluoresence detection (HPLC-FLD) method, based on which the toxin preparation protocol was optimized.The prepared products were analyzed by HPLC-FLD, high performance liquid chromatography-ultraviolet detection (HPLC-UV), flow injection-mass spectrometry detection and high performance liquid chromatography-mass spectrometry detection (HPLC-MS), to determine their identity and purity. The presence of OA and DTX1 in the prepared products has been confirmed and and the purity of the products are high. According to the results of HPLC-FLD and HPLC-UV, the content of purified OA and DTX1 is about 2 mg. OA and DTX1 products are suitable to be useed as a standard in routine analysis of shellfish samples and toxicological studies as well.In summary, based on the current study, the toxin preparation protocol was primarily established, and high-purity OA and DTX1 products were prepared. Studies on optimization of the preparation and quality control protocols of OA and DTX1 are still needed to realized the domestically production of DSP toxin standards, which will significantly promote the management of DSP contamination in seafoods in China.