Study On Liquid Chromatographic-Mass Spectrometric Analysis Of Lipophilic Shellfish Toxins

In recent years,red tides occurred frequently in domestic and overseas,a large area of water and Marine organisms were polluted,causing enormous harm to the Marine ecological environment,fishery resources and human physical and mental health.When red tides occur,toxic algae proliferate and produce shellfish toxins,which mainly accumulate and metabolize in bivalve molluscs,and are transmitted to consumers through the food chain to produce toxic effects.The fat-soluble characteristics of lipophilic shellfish toxins(LSTs)lead to the elimination of the half-life of LSTs in Shellfish and other organisms as long as tens of days or even months,bringing potential harm to consumers.Over the past four decades,there have been numerous reports of shellfish toxins causing mass poisoning and even death around the world.Therefore,it is of great significance to carry out research on shellfish toxin analysis and establish a unified,efficient and wide range of shellfish toxin detection method for the control and monitoring of shellfish toxin in aquatic products and sea areas,to understand the occurrence and distribution characteristics of shellfish toxin in coastal areas of China,and to ensure the safety of aquatic products and public health.In this study,common lipophilic shellfish toxins were selected as the research object.In view of the characteristics of lipophilic shellfish toxins detected in different substrates,a liquid chromatography-tandem mass spectrometry method was established for the analysis of lipophilic shellfish toxins in shellfish and seawater samples by studying different pretreatment methods.The thesis mainly includes the following three aspects:1.Established an HPLC-MS/MS method for the analysis and detection of lipophilic shellfish toxins.By optimizing the conditions of high performance liquid chromatography and mass spectrometry,focusing on the optimization of mass spectrometry parameters such as precursor ion,product ion,fragment voltage and collision energy,chromatography conditions such as gradient elution procedure and flow phase,the qualitative and quantitative detection of 10 kinds of LSTs(including OAs,AZAs,YTXs,GYM and PTX2)were realized,and the HPLC-MS/MS detection of 10 kinds of LSTs was established.Methodological verification showed that the method has good stability.Each LSTs had a good linearity within the quantitative linear range(R~2>0.997),and the LOD for 10 LSTs were 0.01～3.27μg/L(S/N=3),and the accuracy and precision of intra-day(N=3)and inter-day(N=6)were good.2.Established an HPLC-MS/MS method for the simultaneous analysis and detection of lipophilic shellfish toxins in shellfish.By optimizing the pretreatment process of shellfish samples,including the liquid-liquid extraction and solid-phase extraction conditions,which effectively solved the problems of SPE column blockage and large-volume solvent concentration,and took into account the continuity and timeliness of operation.The effective enrichment of LSTs(including OAs,AZAs,YTXs,GYM,PTX2)in shellfish samples was realized,and the method of HPLC-MS/MS analysis and detection of lipophilic shellfish toxins in shellfish samples was established.The specific method was as follows:methanol was used as the extraction solvent,n-hexane was used for liquid-liquid extraction twice,the OASIS HLB solid-phase extraction column was used for enrichment and purification,the eluent was concentrated by rotary evaporation,and the HPLC-MS/MS was used for detection after redissolution and centrifugation.Methodological verification showed that the method had good stability.Each LSTs had a good linearity within the quantitative linear range(R~2>0.997),and the LOD for 10 LSTs are 0.04～3.50μg/kg(S/N=3),the average recoveries of 10 lipophilic shellfish toxins ranged from 77.54%to 116.9%,and the accuracy and precision of intra-day(N=3)and inter-day(N=6)were good.Six kinds of actual shellfish samples(including mussels,clams,green clams,constricted clams,clams and scallops)were tested,and some samples were detected LSTs,and the content of LSTs was lower than the international safety limits of LSTs.3.Established an HPLC-MS/MS method for the analysis and detection of trace lipophilic shellfish toxins in seawater.It was the first attempt to apply macroporous resin to the pretreatment process of seawater samples.The enrichment of trace LSTs(including OAs,AZAs and PTX2)in seawater samples was effectively solved by screening macroporous resin models and optimizing solid phase extraction conditions,and a HPLC-MS/MS method for the analysis and detection of trace LSTs in seawater was established.The specific method was as follows:NKA-9(analysis of OAs)and S-8(analysis of AZAs and PTX2)were used to fill the self-made SPE column,and the seawater samples were filtered and directly pre-treated with the self-made SPE column.After elution by methanol,the eluent was concentrated by rotary evaporation,and the HPLC-MS/MS was used for detection after redissolution and centrifugation.Methodological verification showed that the method had good stability.Each LSTs had a good linearity within the quantitative linear range(R~2>0.999),and the LOD for 7LSTs are 0.10～2.45 ng/L(S/N=3),and the accuracy and precision of intra-day(N=3)and inter-day(N=6)were good.The average recoveries of LSTs ranged from 81.10%to92.67%.Compared with the solid phase adsorption and toxin tracking(SPATT)technique,the LSTs cannot be quantitatively analyzed by the SPATT technique,and this method effectively solved the problem that the long enrichment time of seawater sample.This method was successfully applied to the concentration analysis of group OA toxins in the culture medium of P.lima,which provided a technical method for the follow-up research.In summary,this thesis with the ocean lipophilic shellfish toxins as the research object,made atmost use of the SPE technology advantage,through different sample pretreatment technology,established methods for the analysis and detection of LSTs in shellfish and seawater by HPLC-MS/MS,which provided a reference for the safety detection of seafood and a new idea for real-time monitoring of LSTs in the sea.