EN 16204:2012

Foreword
Introduction
1 Scope
2 Normative references
3 Principle
4 Reagents
5 Apparatus
6 Procedure
7 HPLC-MS/MS analysis
8 Calculation
9 Precision
10 Test report
Annex A (informative) - Precision data
Annex B (informative) - Examples for suitable
        MS detection conditions
Annex C (informative) - Typical chromatogram
Bibliography

This European Standard specifies a multi-reference method for the determination of lipophilic algal toxins (fat-soluble algal toxins produced by some dinoflagellates) in raw shellfish and shellfish products including cooked shellfish, by liquid chromatography coupled to tandem mass spectrometry LC-MS/MS [1], [2], [3]. This method has been validated in an inter-laboratory study consisting of three parts via the analysis of both naturally contaminated homogenates of blue mussel and spiked extracts of blue mussel, oyster and clam. For further information on the validation, see Annex A. Additional studies have investigated further matrices (see [4], [5]).The detection limit for toxins of the okadaic acid group, azaspiracids and pectenotoxins was determined to be 6 µg/kg shellfish meat and for yessotoxins 10 µg/kg 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, 45-OH-yessotoxin and 45-OH-homo-yessotoxin, and PTX-2 for pectenotoxin-1 (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 hydrolysis [6], the esters of okadaic acid, DTX-1 and DTX-2 can also be determined quantitatively as the corresponding free acids.