Studies On Extraction,Purification And Preparation Technology Of Tetrodotoxin In Nassarius

Tetrodotoxin（TTX）was a higly toxic non-protein neurotoxin,it was widely used in medicine and could be used as analgesic drug and anesthetic.TTX standards were also needed in aquatic product testing.Due to the difficulties in purification and preparation of TTX,the high purity of TTX standards were in short supply.In this paper,TTX content analysis was performed on samples of Nassarius collected from Zhanjiang,Guangdong Province.Purification of extracts by cation exchange resin and Sephadex Gel,sample solution was concentrated and purified by high performance liquid chromatography（HPLC）.In this paper,Nassarius as raw material for innovative preparation of TTX product at an efficient,simple and cost-effective production method.The research results were as follows:1.TTX analysis of samples of Nassarius collected from Zhanjiang,Guangdong Province in April,July,September and October.Analyzed by liquid chromatography tandem mass spectrometry（LC-MS/MS）by immunoaffinity column pretreatment.All samples were positive with an average concentration of 1.8³.1 mg/kg.The lowest concentration came from July and the highest concentration came from October.The difference between the highest concentration and the lowest concentration were relatively large,concentration of TTX in Nassarius changes with the month and was related to the environment.2.The TTX extraction method was optimized for Nassarius,and the extraction rate of the water bath combined with ultrasound-assisted extraction and the traditional water bath method was compared.The yield of the former was 21.8%higher than that of the latter.Compared the factors such as the solvent type,water bath temperature,ratio of liquid to material in the extraction process and determine the optimum extraction conditions after optimization:water bath combined with ultrasound extraction,1%acetic acid methanol as the extraction solvent,60°C water bath temperature,liquid to material ratio 5:1,repeated extraction 1 time.3.Several common solid phase extraction（SPE）columns used for the purification of TTX were subjected to experimental analysis.A comparative analysis of the MCX,MAX,C₁₈,WCX columns found that the WCX column performed well.Optimized the use conditions of the WCX column,determined the loading pH 6.4,washed with 5 mL of0.01%aqueous ammonia and 5 mL of methanol,and eluted with 5 m L of 2%acetic acid in4.Three kinds of common cation resins D113,D152,724 were tested and analyzed.Through comparison of adsorption rate and desorption rate,it was determined that D152cation resin was used as a purification material.Different conditions were optimized to determine the column pH 5.0⁵.5 of the extract,2%acetic acid-methanol（40:60）as eluent,and the elution dose was 10 BV（bed volume,column volume）.The sample solution that has been purified by the D152 resin was again purified with G-10 Sephadex Gel.The Sephadex Gel purification conditions were:The sample solution was adjusted to pH 6.5 to7.5.The sample was washed with pure water and eluted with 1%acetic acid solution.The elution dose was 6 BV.The obtained purified liquid was concentrated to 10 mL,adjusted to pH 8.5-9.0 with ammonia water,and after vacuum freeze-drying,15.58 mg of white crystals were precipitated,and further pure product was prepared by HPLC.5.The white crystals were dissolved with 10 mL of a 0.1%formic acid-acetonitrile（1:1,v/v）solution.The TTX standard solution was prepared to obtain the standard chromatogram.According to the established standard conditions,the peak time of the toxic peak was 3.752 min and the acquisition time was determined to be 3.40-4.00 min.After HPLC preparation,a total of 54 mL of purified liquid was obtained.The purified solution was concentrated by using rotary evaporator and concentrated to 10 mL.The pH was adjusted to 8.5-9.0 with ammonia water and freeze-dried to obtain 8.58 mg of pure product.6.1.00 mg pure product was weighed and dissolved in 10 mL of a 0.1%formic acid-acetonitrile（1:1,v/v）solution.The product was analyzed by LC-MS/MS.The concentration of the product was 32.65μg/mL,and the purity of the product was 32.65%.Pure product was white crystal,odorless and stable.