Study On Detection Of Brevetoxin And Its Distribution,Accumulation And Elimination In Shellfish

In recent years,harmful algae bloom frequently occur in china and pose a serious threat to food hygiene and human health.Brevetoxin is a kind of red tide toxic with extremely harmful.Brevetoxins can be accumulated in marine shellfishes and atomized in marine aerosol,causing serious harm to the human body.In this study,the method of high performance liquid chromatography combine with tandem mass spectrometry?HPLC-MS/MS?was developed to detect brevetoxins in seawater and shellfish,and the distribution,accumulation and elimination of brevetoxins in Mytilus coruscus were explored.The main contents and results of this study are as follows:1.The method of high performance liquid chromatography combine with tandem mass spectrometry?HPLC-MS/MS?was developed to detect3 brevetoxins?BTX-1?BTX-2?BTX-3?.BTXs were detected in the multi-reaction monitoring mode,using electrospray positive ion mode,selecting[M+H]⁺as the characteristic ion and acetonitrile as the solvent-soluble solvent.The optimum conditions of liquid chromatography and mass spectrometry were determined.The correlation coefficients?R²?of the three BTXs in the range of 1.0-200.0 ng/mL were all above 0.999.The relative standard deviations of retention time,ion abundance ratio and peak area were less than 5%.The retention time and ion abundance ratio of BTXs were consistent at different concentrations.2.The HPLC-MS/MS method was developed to detect 3 brevetoxins in shellfish by using Liquid-liquid extraction.The analysis volume of seawater was reduced to 20 mL by using HPLC-MS/MS.The extraction conditions of brevetoxins in seawater were optimized by response surface:6.00 g of sodium chloride,the vortex time was 60 s,and extracted twice with 5 mL of ethyl acetate.The limit of detection for 3 brevetoxins were0.02?g/mL,and the limit of quantification were 0.05?g/mL.The average recoveries of the method were 95.4-104.3%,the intra-day precisions were2.17-5.26%,and the inter-day precisions were 2.95-4.54%.This method reduces the analysis volume,improves the pretreatment efficiency,and realizes high-efficiency trace analysis of brevetoxins in seawater.3.The HPLC-MS/MS method was developed to detect 3 brevetoxins in shellfish by using solid phase extraction.The samples were extracted by acetone and cleaned up by C₁₈ solid phase extraction column.The optimum conditions of liquid chromatography and mass spectrometry were determined.This method reduces influence of matrix effect,improves the experimental efficiency,and realizes high-efficiency enrichment and purification of brevetoxins in shellfish.The limit of detection for 3brevetoxins were 1.0?g/kg,and the limit of quantification were 3.0?g/kg.The average recoveries of the method were 81.2-88.4%,the intra-day precisions were 1.80-6.47%,and the inter-day precisions were 3.18-4.96%.4.The distribution,accumulation and elimination of brevetoxin in Mytilus coruscus were explored.In this study,BTX-3 was selected as brevetoxin maker,and Mytilus coruscus was used as the analyses object.The exposure of Mytilus coruscus behavior in a simulated red tide by brevetoxin extract was studied.The results showed that when Mytilus coruscus was exposed to brevetoxin,the BTX-3 was easily accumulated in Mytilus coruscus.Brevetoxin was mainly distributed in the visceral mass and gonad of Mytilus coruscus.The accumulation content of tissue from high to low was visceral mass,mantle,gill,gonad,and adductor muscle.Tissue concentration in Mytilus coruscus was 817?g/kg?above the 800?g/kg limit?after 4 days of exposure to brevetoxin extract.During the course of exposure,tissue concentrations steadily increased until day 7 was1207?g/kg and the average accumulation rate was 143?g/kg/d.On the first day of depuration,BTX-3 fell back to the safe level.Tissue concentration was 70?g/kg?below 800?g/kg limit?after 13 days of depuration.The half-life period of BTX-3 in Mytilus coruscus was 3.23 d.