Studies On Detection Of Multiple Shellfish Toxins By High Pefrormance Liquid Chromatography-tandem Mass Spectrometry

With rapid development of national coastal aquaculture industry, marineenvironmental problems become increasingly serious and red tides occur frequently.Poisoning events of humans eating marine shellfish in coastal areas have appeared tobe an upward trend. Shellfish toxins are highly poisonous and reply quickly withsudden and widespread. What is more, there is no appropriate antidote. So theseafood safety problem is directly related to health and safety of consumers.Therefore, governments around the world have been paying more and more attentionon the shellfish toxins contamination problem and have carried on lots of furtherresearches on the methods to determine shellfish toxins. But currently the detectionand analysis ability of shellfish toxins is low in our country. Such new detectiontechnologies as liquid chromatography-mass spectrometry technology (LC-MS) israrely applied in the detection and analysis of shellfish toxins. However, technicalcharacteristics of LC-MS itself makes its great potential for application in thedetection and analysis of shellfish toxins. In this study, such shellfish toxins as DSP,PSP and ASP, as well as residue of shellfish toxins in samples are detected using highperformance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)technology.This paper include three parts as follows．1. Detection of diarrhetic shellfish poisoning (DSP) in aquatic products withHPLC-MS/MSExperimental conditions including extraction, purification, LC and MS werestudied. A sensitive method of the HPLC-MS/MS to detect DSPs residues in aquaticproducts was established. Methods are as follows: Toxins were extracted withmethanol-water (80/20, V/V) by ultrasonic. The supernatant was purificated byWaters OASIS HLB solid phase extraction column to purification after centrifugation,then, evaporate to dryness, constant volume and through the filter. The final filtratethus obtained was subjected to high performance liquid chromatography-tandem massspectrometry analysis. Under the optimized chromatographic and mass spectrometry conditions, five DSPs showed a good linear relationship when the concentration oftoxins were between10.0μg/L to160.0μg/L (R2＞0.998). Under the addedconcentration of the toxins between2.0μg/kg to200.0μg/kg level, the averagerecovery of five DSPs was between60.7%to94.0%, and the coefficient of variationwas between2.0%to6.0%. The detection limit of GYM, SPX1, PTX2, OA andAZA1were0.2μg/kg,0.1μg/kg,1.0μg/kg,0.1μg/kg and0.2μg/kg.2. Detection of paralytic shellfish poisoning (PSP) in aquatic products withHPLC-MS/MSExperimental conditions including extraction, purification, LC and MS werestudied. The MS conditions of six PSPs and a sensitive method of the HPLC-MS/MSto detect dc-STX and STX-e residues in aquatic products were established. Methodsare as follows: Toxins were extracted with0.1mol/L HCl by heating at100℃. Thesupernatant was purificated by Waters OASIS HLB solid phase extraction column topurification after centrifugation. Then, let the liquid supermatant through the filter.The final filtrate thus obtained was subjected to high performance liquidchromatography-tandem mass spectrometry analysis. Under the optimizedchromatographic and mass spectrometry conditions, two PSPs showed a good linearrelationship when the concentration of toxins between10.0μg/L to500.0μg/L (R2＞0.998). Under the added concentration of the toxin between50.0μg/kg to500.0μg/kglevel, the average recovery of dc-STX was between53.4%to62.5%, and thecoefficient of variation is between2.5%to4.6%. In the add concentration of the toxinbetween50.0μg/kg to300.0μg/kg level, the average recovery of STX-e was between59.8%to68.0%, and the coefficient of variation was between1.6%to2.4%. Thedetection limit of dc-STX and STX-e was20.0μg/kg and30.0μg/kg.3. Detection of acids shellfish poisoning (ASP) in aquatic products withHPLC-MS/MSExperimental conditions including extraction, purification, LC and MS werestudied. A sensitive method of the HPLC-MS/MS to detect ASP residues in aquaticproducts was established. Methods are as follows: Toxin were extracted withmethanol-water (50/50, V/V) by ultrasonic. The supernatant was purificated bySupelclean LC-SAX solid phase extraction column to purification after centrifugation, then, through the filter. The final filtrate thus obtained was subjected to highperformance liquid chromatography-tandem mass spectrometry analysis. Under theoptimized chromatographic and mass spectrometry conditions, DA-f showed a goodlinear relationship when the concentration of the toxin between20.0μg/L to1000.0μg/L (R2＞0.999). Under the added concentration of the toxin between20.0μg/kg to100.0μg/kg level, the average recovery of DA-f was between71.3%to84.1%, andthe coefficient of variation was between4.4%to4.6%. The detection limit of DA-fwas5.0μg/kg.4. Detection DSP、PSP and ASP residue in samplesThe method established in this paper was applied to toxin detection work ofDonggang Entry-Exit Inspection and Quarantine Center of2012. Based on themethod of high sensitivity and low detection limit, three shellfish toxins, DSP, PSPand ASP, were determined in the sample of saxidomus purpurtus, meretrix meretrixlinnaeus, chinese venus, short necked clam and mussel, et al. Three batches ofsamples were detected, and there were different levels to be detected, but the contentwere far below the standard limit, which showed the sample test pass rate was100%.Overall, the study in this paper provides specific methods and technical specificationsfor the detection of toxins in aquatic products, which has important theoreticalresearch value and application prospect.