The Determination Method of the Lipophilic Marine Biotoxins in Bivalve Subject to EU Regulations Using a Triple Quadrupole Mass Spectrometer

User Benefits

- In compliance with procedures issued by the EU Reference Laboratory for Marine Biotoxins (EU-RL-MB), simultaneous analysis of five lipophilic marine biotoxins in bivalves is possible. - Yessotoxin (YTX) could be analyzed with high sensitivity using a basic mobile phase. - Matrix-matched calibration curves could be automatically created using the pre-processing function of the autosampler.

Introduction

Shellfish poisoning mainly occurs when bivalves feed on highly toxic marine dinoflagellates and these toxins accumulate in the body of the bivalve. Eating these poisonous shellfish can cause poisoning syndromes in humans. The EU has established limits for lipophilic marine biotoxins. Regulation (EC) 853/2004, Annex III, Section VIII, Chapter V prescribes a maximum concentration of lipophilic marine biotoxins in bivalves intended for human consumption of 160 µg/kg (okadaic acid equivalent) for okadaic acid, dinophysistoxins, and pectenotoxins, and 160 µg/kg (azaspiracid equivalent) for azaspiracids. In addition, Regulation (EU) 786/2013, Annex III reduced the permissible level of yessotoxin in live bivalves to 3.75 mg/kg (yessotoxin equivalent). Regulation (EU) No. 15/2011 recognizes an EU-RL LC-MS/MS method as the standard method for detecting lipophilic marine biotoxins in live bivalves. In Japan, safety standards and regulatory limits for shellfish toxins are established based on the Food Sanitation Act (Shokuan Notice No. 0306-1, dated March 6, 2015). For diarrheal shellfish toxins, the amount of toxin present in the edible parts of bivalves is restricted to 0.16 mg okadaic acid equivalent/kg and below. Shokuanki Notice No. 0306-3, dated March 6, 2015, specifies an instrument-based testing method for detecting okadaic acid and dinophysistoxins that uses an acidic mobile phase, which is the testing method in current use. This article describes an LC-MS/MS method that uses a basic mobile phase and an analysis of samples extracted from scallops spiked with known amounts of five standard toxins. The five toxins have been chosen to represent the five compounds/chemical groups of lipophilic marine biotoxins cited in EU regulations (okadaic acid, dinophysistoxins, pectenotoxins, azaspiracids, and yessotoxins). The procedures used during extraction and analysis follow the methods of the EU-Harmonised Standard Operating Procedure for determination of Lipophilic marine biotoxins in molluscs by LC- MS/MS Ver. 5. In order to determine total amounts of okadaic acid and dinophysistoxins, a hydrolysis step is needed after sample extraction. In this study, scallop extract solutions were prepared with and without this hydrolysis step, then matrix- matched calibration curves were prepared and quantitative analysis was performed on both types of samples.

July 28, 2022 GMT