Research On Standardization Method Of Dete-Ction And Preparation The Pectenotoxin

Nowadays the shellfish poisoning events become more frequent worldwide and put the public health into great risk when consuming the contaminated seafood.Therefore,the approaches to qualify and quantify the poisonings are in great importance to ensure the food safety.In the present study,one of the lipophilic shellfish toxins(LSTs),pectenotoxin-2(PTX2),was studied.A whole system,including collecting,extracting,purification and detection of standard PTX2 was established.PTX2 toxin here was purified for standard use for the first time by extracting from large biomass of Dinophysis culture in the laboratory and HP20 resin samples collected in the field.On the basis of the established system,the lipophilic shellfish toxins in the water and oysters of Guangxi Beibu Gulf were investigated.First,a detection method of liquid chromatography-quadrupole/linear ion trap tandem mass spectrometry(LC-Q/Trap-MS)in multiple reaction monitoring(MRM)was optimized to analyse 17 lipophilic poisoning toxins,including Okadaic acid(OA)?Dinophysistoxin-1(DTX1)?DTX2?Pectenotoxin-1(PTX1),PTX2?PTX2sa?PTX11?PTX12?Gymnodimine(GYM)?Spirolides-1(SPX1)?Azaspiracids-1(AZA1)?AZA2?AZA3?Yessotoxin(YTX)and its derivatives,45 OH-YTX? homoYTX and 45-OH-homoYTX.Within these toxins,PTX1,PTX2sa,PTX11,PTX12,45 OH-YTX,homo YTX and 45-OH-homoYTX were only for qualification.The optimized detection method can be used for the selecting the most efficient extraction and purification methods for PTX2 toxin in the following studies,as well as for the lipophilic shellfish toxins monitoring.Second,the protocol for preparation the PTX2 from large biomass of Dinophsis acuminata was conducted by high performance liquid chromatography(HPLC).Methods of cell disruption,toxin extraction and HPLC purification were selected and optimized during the preparation process.The results indicated that the most efficient methods of cell disruption and toxin extraction were achieved with the combination of freezing plus thawing and ultrasonic methods,resulting to 99.2%and 81.5%respectively.PTX2 were extracted using liquid-liquid extraction(methanol,actone,diethyl ether and dichloromethane)and Solid Phase Extraction columns(Oasis HLB SPE,Phenomenex Strata-X SPE,Sep-Pak C18 SPE).PTX2 extraction efficiency were maximum(78.19%)when extracted by diethyl ether and dichloromethane.Studies on optimization conditions of HPLC showed that the pH value of the mobile phase had an impact on the chromatographic reaction.When mobile phase was in alkaline conditions(both containing 6.7 mM ammonium hydroxide in acetonitrile/water,pH=10.94),PTX2 could be better chromatographic separated by Phenomenex Kinetex C18(4.8 mm×250 mm,5?m).The final purified PTX2 reached 93.2%.Solid Phase Adsorption Toxin Tracking(SPATT)is a new in situ monitoring tool that is distinguished from traditional monitoring methods.In this study,SPATT filling in DIAION(?)HP20 resin was applied to the Beibu Gulf of Guangxi and PTX2 toxin was extracted and purified from these SPATT samples.Here the PTX2 toxin was separated using Oasis HLB SPE,and the HPLC conditions for the isolation and purification of the PTX2 toxin were optimized,resulting to 88.9%of PTX2 from the SPATT sample.On the basis of the established detection system,the analysis of the toxins in the Beibu Gulf seawater proved that the high-purity PTX2 prepared can be used for routine detection and screening of shellfish toxins.The monitoring of LSTs in Beibu Bay showed that oysters were most contaminated in Summer but safe to eat in Winter.Finally,the preparation of PTX2 in accordance with national standards of technical requirements for test of homogeneity,stability,uncertainty and ture value were conducted to ensure the validity of the sample preparation process.The current result discovered in present study will be benefit to the control and monitoting the PTX2 contamination in China.