DIN EN 16204:2012-08

This document describes a multi-reference method for the detection and determination of lipophilic toxins (fat-soluble algal toxins produced by dinoflagellates) in shellfish and shellfish products by LC-MS/MS. This method has been validated in an inter-laboratory study consisting of three parts via the analysis of both naturally contaminated homogenates and spiked extracts. The limit of quantification (LOQ) for DTX toxins, azaspiracids and pectenotoxins was determined to be 6 µg/kg of shellfish meat and for yessotoxins 10 µg/kg of shellfish meat. Quantitative determination of okadaic acid (OA), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1) and yessotoxin (YTX) can be carried out directly by means of standard substances available commercially. Assuming an equal response factor, okadaic acid is used for the indirect quantitative determination of the two dinophysistoxins dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2); likewise azaspiracid-1 (AZA-1) is used for the indirect quantitative determination of azaspiracid-2 (AZA-2) and azaspiracid-3 (AZA-3), while YTX is used for homo-yessotoxin (Homo-YTX) and OH-yessotoxin (OH-YTX), and PTX-2 for PTX-1. The limit of quantification (LOQ) for toxins of the okadaic acid group, azaspiracids and pectenotoxins was determined to be 20 µg/kg shellfish meat and for yessotoxins 35 µg/kg shellfish meat. By means of a hydrolysis step, the esters of okadaic acid, DTX-1 and DTX-2 can also be determined quantitatively. Remove the shellfish meat from the shell and homogenize the total shellfish meat. Extraction is carried out with aqueous methanol (φ = 80 %). Separation is performed on a HPLC reverse-phase column provided with a binary gradient and detection is carried out by means of tandem mass spectrometry. The concentration of lipophilic toxins is determined by means of external calibration. This European Standard has been prepared by Technical Committee CEN/TC 275 "Food analysis - Horizontal methods" (secretariat: DIN) of the European Committee for Standardization (CEN) after in-depth preliminary work of Working Group 5 "Biotoxine (Mycotoxine und Phycotoxine)" ("Biotoxins (mycotoxins and phycotoxins)"). The responsible German committee is Working Committee NA 057-01-03 AA "Biotoxine" ("Biotoxins") of the Food and Agricultural Products Standards Committee (NAL) of DIN, the German Institute for Standardization e. V.