Establishment Andapplication Of Theanalytical Method For Shellfish Poisoning With Liquid Chromatography-tandem Mass Spectrometry

Eutrophication of the Water Body has led to the frequent outbreaks of red tide along Chinese coast. Toxins accumulated in shellfish impaire the health of the consumers and hinder the shellfish exporting trade, which have become one of bottlenecks influencing the sustainable development of marine shellfish industry. In this paper, new shellfish poisoning (Azaspiracid, Cyclic imines), Diarrhentic shellfish poisoning (Okadaic acid and Pecenotoxin-2) and Paralytic shellfish poisoning were studied and the analytical methods with liquid chromatography-tandem mass spectrometry were developed. Lipophilic marine toxins in shellfishes from the main coast city were surveyed with the method that had been estabished. At the same time, An Alexandrium tamarens was cultived in the lab to conduct the study of toxin production. It has been showed that the methods we have developed can apply to the detection of the shellfish and alge sample, will provide great technical support and play an important role in the monitoring of red tide and shellfish safety.The main contents of this paper are as follows:1. Determination of azaspiracid (AZA1) in shellfish by liquid chromatography with tandem mass spectrometryA liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of Azaspiracids (AZA1) in shellfish was described. After being extracted using methanol and water (80/20,v/v), the extraction was cleaned by solid phase extraction of MAX column, then determined by using a reversed-phase HPLC isocratic program coupled with tandem mass spectrometry in selected-reaction-monitoring mode. And the extraction was eluted with acetonitrile/water (80/20, v/v) on a Atlantis dC18 column (150mm×4.6 mm , 5.0μm). Both mobile phase contained 50 mM formic acid and 2 mM ammonium formate. The method detection limit was 11 ng/kg. Calibration curve was linear in the range of 48.85~2 442 ng/L (R2=0.9981). Recoveries of the shellfish tissue extraction at three spiked levers (36.64、73.27、146.54 ng/kg) were 71.12~94.07% (n=6). Relative standard derivations were less than 10 percent.2. Determination of five lipophilic marine toxins in shellfish by liquid chromatography with tandem mass spectrometryA liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of five lipophilic marine toxins including Gymnodimine (GYM), Spirolide-1 (SPX1), Okadaic acid (OA) Pecenotoxin-2 (PTX2) and Azaspiracid-1 (AZA1) in shellfish was developed. After being extracted with methanol and water (80/20,v/v), the extraction was cleaned by solid phase extraction of MAX column, then determined by a reversed-phase HPLC gradient program coupled with tandem mass spectrometry in selected-reaction-monitoring mode. GYM、SPX1、OA、PTX2、AZA were analyzed in positive ion mode, while OA in negative ion mode. The calibration curves were good linear between LC peak area of the selected ion-pairs and the concentration of five lipophilic marine toxins with the correlation coefficient over 0.99. The average recoveries from spiked scallop muscle at the three concentrations ranged from 78.6% to 94.4% with relative standard deviation from 6.8% to 14.9%. The limits of detection were 0.10, 0.21, 2.00, 0.32, 0.04μg/kg for GYM, SPX1, OA, PTX2 and AZA1, respectively.3. Determination of Paralytic Shellfish Poisoning by liquid chromatography with tandem mass spectrometryAfter being extracted using actontrile and water (80/20,v/v), the extraction was cooled under -20℃for 2 hours. The lower aqueous phase was cleaned by solid phase extraction of HLB column, then determined by using a reversed-phase HPLC coupled with tandem mass spectrometry in selected-reaction-monitoring mode. And the extraction was gradiently eluted with acetonitrile/water on a Amide-80 column (150 mm×2 mm,3μm). Both mobile phase contained 3.6 mM formic acid and 2 mM ammonium formate. With this method, 13 components of PSP could be defined within 13 min Average recoveries of spiked scallop muscle ranged from 65.33% to 76.22% with relative standard deviation from 8.96% to 16.89%.4. Investigation on lipophilic marine toxins in shellfish purchased from main coastal city markets210 shellfish samples from Dalian, Qingdao, Yantai, Rizhao, Guangzhou, Xiamen were detected with the method for the five lipophilic marine toxins. GYM, SPX1, YTX, OA, PTX-2, AZA1 were found in shellfish. The positive rates were 23.80%, 23.33%, 19.05%, 20.95%, 10.95%, 1.90%, respectively. It was found that the content and species of toxins enriched in shellfish were related to the red tide and algae, in winter the concentration of toxins were lower than other seasons. Except the gland of some shellfish had high concentration of GYM and YTX, shellfish had low concentration toxins. There were differences for different tissues to enrich toxins. The Gland of shellfish had stronger ability to toxins than other tissues, and the capability of tissues enrich toxins were Gland> Gonad>Lamina. Different shellfishes have different ability to enrich different toxins. Crassostrea gigas could enrich more GYM than other shellfish, Chlamys farreri had stronger ability to enrich YTX, and Atrina pectinate had stronger ability to enrich AZA1.5. Preliminary study on growth and toxin production of Alexandrium tamarenAn Alexandrium tamaren was cultived through Synchronized Culture method. In the culture period,the toxin species and prodution of this algae were monitored with the method that had been optimized. It had been observed that this algae coud generate STX, dcSTX, GTX1, GTX2, GTX3 and GTX4, which the GTX 1 and GTX4 had maximum concentration. Besides that, difffernt toxins could reach the maximum concentration at different time, and the GTX 1 and GTX4 got the peak later than other toxins.