Studies On Detection Of Multiple Marine Biotoxins In Nassarius By Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

In recent years,red tides occured frequently, the Marine environment pollution increased rapidly along with constant consumers poisoning events that eating various kinds of seafood. Poisoning caused by eating Nassarius increasing seriously in Fujian Province.Aquatic products safety is directly related to life safety and health of consumers. At present, the importance of Marine biotoxins is not enough in our country, the corresponding group event monitoring system is not established, liquid-mass spectrometry as a new kind of detection technology has broad prospect of application in Marine biotoxins. Thus it is very important to develop a rapid and accurate Marine Biotoxins detection method and use this method to determine the Nassarius toxins pollution condition in our province, and according to the test results we put forward the corresponding prevention countermeasures is particularly important.Therefore,in this paper,we develop a analytical methods to detect Tetrodotoxin(TTX)?Diarrhetic shellfish poisoning(DSP) ? Paralytic shellfish poisoning(PSP)by ultra performance liquid chromatography-mass/mass spectrometry(UPLC-MS/MS)and use the method to determine Marine biotoxins residues in practical Nassarius samples that collected from Putian and Ningde in Fujian Province for two years(2014 and 2015).It was found that the three kinds of toxins were all detected in Nassarius samples in two cities,the pollution is serious.1?Analyze tetrodotoxin residual in Nassarius by UPLC-MS/MS.Objective: Confirmed the Chromatography mass spectrometry analysis condition of tetrodotoxin, developed a method for the determination of TTX by ultra performance liquid chromatography-mass/mass spectrometry(UPLC-MS/MS).Methods:The Nassariidae samples were extracted by 3% acetic acid/methanol solution,Nassariidae extract solution was purifies by MCX solid-phase extraction cartridges. The TTX were separated on Atlantis HILIC Silica column, using acetonitrile and 10 mmo L/L formic acid-5mmo L/L ammonium formate solution as mobile phase and then detected by ultra performance liquid chromatography-mass/mass spectrometry under multiple reaction monitoring(MRM) mode.Results: Tthe method showed a good linearity over the rang of 10~100ng/m L. The quantification limit(LOD) of the method were3.3?g/kg.The recoveries ranged from 80.5% to 92.4%and the relative standard deviation less than 5%Conclusion: This method is suitable for TTX determination in Nassarius,less matrix interference, can satisfy the detection requirements.2. Analyze Diarrhetic shellfish poisoning residual in Nassarius by UPLC-MS/MS.Objective:Confirmed the chromatography mass spectrometry analysis condition of DSP, developed a method for the determination of DSP by ultra performance liquid chromatography-mass/mass spectrometry(UPLC-MS/MS).Methods:The Nassariidae samples were extracted by methanol and water(80:20,V/V). Nassariidae extract solution was purifies by C18 solid-phase extraction cartridges. The DSP were separated on a reversed phase ACQUITY UPLC BEH C18 column, using acetonitrile-10 mmo L/L formic acid-5mmo L/L ammonium formate solution as mobile phase and then detected by ultra performance liquid chromatography-mass/mass spectrometry under multiple reaction monitoring(MRM) mode.Results:The method showed a good linearity over the rang of 10~100ng/mL. The OA?DTX-1?DTX-2 quantification limit(LOD) of the method were 6?g/kg?3?g/kg?4.5?g/kg.The recoveries ranged from 80.3 % to 93.6 % and the relative standard deviation less than 5%.Conclusion: This method is suitable for DSP determination in Nassarius,fast, simple and with good reproducibility, can satisfy Nassarius actual testing requirements.3. Analyze Paralytic shellfish poisoning residual in Nassarius by UPLC-MS/MS.Objective:Confirmed the chromatography mass spectrometry analysis condition of PSP, developed a method for the determination of PSP by ultra performance liquid chromatography-mass/mass spectrometry(UPLC-MS/MS).Methods: The Nassariidae samples were extracted by 0.2 mol/L acetic acid solution. Nassariidae extract solution was purifies by C18 solid-phase extraction cartridges. The PSP were separated on a Atlantis HILIC Silica column, using acetonitrile-2mmo L/L ammonium formate solution as mobile phase and then detected by ultra performance liquid chromatography-mass/mass spectrometry under multiple reaction monitoring(MRM) mode.Results:The method showed a good linearity over the rang of10~100 ng/m L. The STX?dc STX?GTX-1.4?GTX-2.3 quantification limit(LOD)of the method were 1.1?g/kg?1.8?g/kg?2.5?g/kg?2.3?g/kg.The recoveries ranged from 85.6% to 95.4%and the relative standard deviation less than 5%.Conclusion: This method is suitable for PSP determination in Nassarius,the method is fast and efficient and has high sensitivity, can satisfy the requirements of the Nassarius daily inspection.4.Detect toxins residual of TTX?DSP?PSP in Nassarius practical samplesThis established method was applied to marine biotoxins detection work in Nassarius of Putian and Ningde in fujian province in 2014 and 2015.Based on the high sensitivity, low detection limit of the method,we have toxins residual analysis for TTX?DSP?PSP in Nassarius samples. We detected three batch, 96 sanmples, have different degrees of toxins residual, it is recommended that the relevant regulatory authorities should strengthen the regulation of Nassarius market, reduce or eliminate Nassarius poisoning threat to people. The research provides technique support for the detection of marine biotoxins in Nassarius and other marine products, it has great theoretical value and good application prospect.