Liquid Chromatography - Mass Spectrometry Screening And Determination Of Seawater And Algae Algal Toxins In The Fat-soluble

Lipophilic algae toxins are secondary metabolites mostly produced by marinealgae, including okadaic acid(OA) series, pectenotoxin(PTX) series, yessotoxin(YTX)series, azaspiracid(AZA) series and so forth. Lipophilic algae toxins can beaccumulated or transformed in bivalves, and could cause severe intoxication ofvarious symptoms if human consume the bivalves polluted by these toxins. In recentyears, harmful algal blooms(HABs) induced by toxic algae that can produce lipophilicalgae toxins wreak havoc on the ecosystem and human health. So far, most study wereabout the lipophilic algae toxins in shellfish and algae, and little attention was paid tothe analysis of lipophilic algae toxins in seawater. However, to screen and determinatethe lipophilic algae toxins in seawater is of importance for monitoring seawaterquality after harmful algal blooms and studying the marine environmental behavior oflipophilic algae toxins in seawater. In this study, pretreatment method and liquidchromatography-mass spectrometry (LC-MS) for the screening and determination oflipophilic algae toxins in seawater and algae were developed, and real samples weredetected. Besides, the distribution of typical lipophilic algae toxins in coastal seawaterof Qingdao was analyzed, and enriching method of typical lipophilic algae toxins inseawater was explored. The details are as follows:1. The performance of determinating OA, YTX, PTX2by high performanceliquid chromatography-multi-stage mass spectrometry (HPLC-MS/MS) in alkalinesystem and acidic system were compared. The separation effect was better in alkalinesystem than in acidic system; during MS analysis the selected parent ions of OA andYTX were [M+Na]+(m/z827.7) and [M-H]-(m/z1141.8) respectively in both system,but the selected parent ions of PTX2were [M+NH4]+(m/z876.7) and [M+Na]+(m/z881.7) in alkaline and acidic system respectively; all calibration curves showed goodlinearity(R2>0.99), and the limit of detection(LOD) of YTX was0.79pg in bothsystem, but the LOD of OA and PTX2in alkaline system(0.68pg and0.13pgrespectively) was lower than that in acidic system(1.37pg and0.65pg respectively);the precision in acidic system was better than that in alkaline system. In all, the alkaline system was more applicative.2. New method to screen multiple lipophilic marine toxins in seawater and algaewas established. Solid-phase extraction (SPE) method was adopted to gather multiplelipophilic marine toxins in seawater. With comparison to traditional liquid-liquidextraction (LLE) method, SPE method could achieve better recovery (55.18%~69.94%) for different lipophilic marine toxins, have better repeatability, and consume lessorganic solvents, therefore it is more applicative for the enrichment of lipophilicmarine toxins in seawater. The ultrasonic cell disruption coupled withultrasonic-assisted extraction method had been developed to extract lipophilic marinetoxins in algae, with a good recovery (73.40%~79.77%) which is satisfactory for thescreening of multiple lipophilic marine toxins in algae. High performance liquidchromatography-high-resolution mass spectrometry method was used to detectsamples, after which screening of multiple lipophilic marine toxins was conducted byMasshunter data processing software based on database of accurate relative molecularmass. This whole method was applied to the screening of real seawater samples andlab-grown algae samples for lipophilic marine toxins. OA, PTX2were found inQingdao coastal seawater samples, and OA, DTX1, AZA2were found inProrocentrum lima algae samples. This proves that high performance liquidchromatography-high-resolution mass spectrometry method can be used for rapidscreening of multiple lipophilic marine toxins in seawater and algae.3. High performance liquid chromatography-multi-stage mass spectrometry todeterminate4typical lipophilic marine toxins in seawater and algae had beendeveloped, and using macro absorption resin to concentrate lipophilic marine toxins inseawater had been studied. The established detecting method has good sensitivity,repeatability (RSD<25.00%) and recovery (56.25%~80.20%), and could be used tothe detemination of4typical lipophilic marine toxins in real seawater and lab-grownalgae samples. The determinating results of8samples of Qingdao coastal seawaterand4samples from South China sea showed that there were OA and PTX2in8samples of Qingdao coastal seawater, while the concentration range were4.24ng/L~ 9.64ng/L and0.42ng/L~0.74ng/L respectively. There were OA and PTX2in4samples from the South China Sea too, but concentrations of them were all under thelimit of quantitation. The changing trends of OA and PTX2in4locations of Qingdaocoastal waters from Oct.2012to Mar.2013were studied, and the results showed thatthe changing trends of OA and PTX2were different, with concentration range of2.24ng/L~11.19ng/L and0.10ng/L~0.74ng/L respectively. OA and DTX1inProrocentrum lima algae samples were quantified, with concentration of21.6μg/gand304.4μg/g (wet weight) respectively. Under optimized conditions of macroabsorption resin method, the recovery of OA and PTX2in seawater could reach65.91%and75.89%respectively, and the weight of OA and PTX2gathered from10Lseawater once were11.05ng and0.82ng respectively. This indicates that macroabsorption resin can be used to gather OA and PTX2from seawater, and it has thepotential to be a new way to preparate standard substance of them.