Research And Application Of A New Method For Determination Of Algal Toxins In Seawater By Solid-phase Extraction-LC-MS

With the increasing impact of human activities and global climate change on marine ecosystems,the occurrence of harmful algal blooms(HABs)has been increasing in frequency and scope,and the pollution risk of algal toxins produced by some harmful algae to the nearshore aquatic environment has also been increasing.Domoic acid(DA)is a neurotoxin produced by the toxic marine diatom genus Pseudo-nitzschia.In recent years,the toxigenic algae of DA has been found in the sea area of China.In addition,lipophilic marine algal toxins(LMATs)were found widely in the inshore and offshore sea area of China.The coastal aquatic environment is the habitat of marine fishery organisms,and the concentration of DA and LMATs in seawater will directly affect the food safety of seafood.Therefore,it is very important to monitor the DA and LMATs in the coastal seawater environment.At present,there is still a lack of relevant technical standards for the detection of DA and LMATS in seawater in the world,and the efficient detection technology needs to be further developed.Aiming at the above problems,efficient methods for the detection of DA and LMATs in seawater by using offline/online solid phase extraction combined with liquid chromatography-tandem mass spectrometry were developed,and the proposed methods were applied to study the distribution of DA and LMATs in the Bohai Sea and the Yellow Sea in China.The details are as follows:In order to realize the accurate determination of DA in seawater,a series of methods for the determination of DA in seawater have been established using offline/online solid phase extraction(SPE),liquid chromatography-diode array detector(LC-DAD)and liquid chromatography-tandem mass spectrometry(LC-MS/MS),through the optimization of experimental conditions.Under the optimum conditions,2.0 L seawater samples were enriched by offline SPE disk and detected by LC-DAD.The linear relationship was good in the range of 5.0-500.0 ng/L concentration(R~2=0.9998).The limit of detection(LOD)of DA was 2.5 ng/L,with good recovery(?89.3%)and precision(relative standard deviation,RSD?4.0%).After simple treatment of seawater samples by filter and acidification,0.6 m L of seawater sample was injected directly for online SPE-LC-MS/MS detection.The linear relationship was good in the range of 12.0-500.0 ng/L concentration(R~2=0.9992).LOD of DA was 4.0 ng/L,with good recovery(?81.0%)and precision(RSD?4.2%).After the DA in 80.0 m L seawater sample were enriched by offline SPE,the injection of 0.6 m L sample was used for online SPE-LC-MS/MS detection.DA in spiked blank seawater sample showed a good linear relationship in the concentration range of 0.3-50.0 ng/L(R~2=0.9990),and the LOD was 0.1 ng/L.The above results show that all the three developed methods can meet the requirements of determination of DA in seawater,and the suitable method can be selected according to the sample concentration and laboratory instrument conditions.DA in the surface seawater of the Bohai Sea and the Northern Yellow Sea was measured.The detection rate of DA was high,and the concentration range was 1.65-34.90 ng/L,with an average concentration of 10.34 ng/L.In general,DA concentration is higher in nearshore than in offshore.The higher concentration of DA appear in the Northern Yellow Sea.It is necessary to strengthen the continuous monitoring and risk assessment of DA in the coastal aquaculture areas in this region.A rapid and automatic online SPE-LC-MS/MS method was developed for the simultaneous determination of 14 common LMATs in seawater,in order to realize the efficient and automatic detection of various LMATs in seawater.LMATs in seawater were enriched on an online reversed-phase C8 column,and was detected by LC-MS/MS in multiple reactive monitoring(MRM)mode.The optimal experimental conditions were determined by optimizing conditions of online SPE and LC-MS/MS.Under the optimum conditions,all target compounds have good recoveries(62.0%-117.0%)at three spiked levels in the blank seawater samples.In addition to the LOD of BTX1,BTX2 and BTX3 are 30.00 ng/L,LODs of other compounds were all?3.000 ng/L,with good precision(RSD?11.3%)and linear relationship(R~2>0.9916),which realized the efficient detection of 6 typical LMATs in seawater.Compared with offline SPE,online SPE has the advantages of high automation,low test cost,higher accuracy and repeatability,and can significantly reduce the amount of seawater sample consumption,which can significantly reduce the workload of seawater sample collection,storage and transportation.Finally,the proposed method was applied to detect LMATs in the Bohai,Southern Yellow Seas in summer 2019 and the Bohai,the Northern Yellow Seas in autumn 2020.Okadaic acid(OA)and pectenotoxin 2(PTX2)were detected in all samples,and dinophysistoxin 1(DTX1)was detected in a few samples.In this study area,the total LMAT concentration(?LMATs)in the Bohai Sea and the Southern Yellow Sea in summer is higher than that in autumn.Therefore,it is necessary to carry out regular monitoring of LMATs in coastal mariculture zones to prevent shellfish pollution,especially in summer.