BS EN 16204:2012 pdf download – Foodstuffs — Determination of lipophilic algal toxins (okadaic acid group toxins, yessotoxins, azaspiracids, pectenotoxins) in shellfish and shellfish products by LC-MS/MS
This European Standard specifies a multi-reference method for the determination of lipophilic algal toxins (fatsoluble algal toxins produced by some dinoflagellates) in raw shellfish and shellfish products including cookedshellfish, by liquid chromatography coupled to tandem mass spectrometry LC-MSIMS [1],[2],[3].This methodhas been validated in an inter-laboratory study consisting of three parts via the analysis of both naturallycontaminated homogenates of blue mussel and spiked extracts of blue mussel, oyster and clam.For furtherinformation 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 be6 ug/kg shelfish meat and for yessotoxins 10 ug/kg shellfish meat.
Quantitative determination of okadaic acid (OA), pectenotoxin-2(PTX-2), azaspiracid-1(AZA-1) andyessotoxin (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 twodinophysistoxins dinophysistoxin-1(DTX-1) and dinophysistoxin-2(DTX-2); likewise azaspiracid-1(AZA-1) isused for the indirect quantitative determination of azaspiracid-2(AZA-2) and azaspiracid-3(AZA-3), while YTXis 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 wasdetermined to be 20 ug/kg shellfish meat and for yessotoxins 35 ug/kg shellfish meat.
By means of hydrolysis [6], the esters of okadaic acid，DTX-1 and DTX-2 can also be determinedquantitatively as the corresponding free acids.
2Normative references
The following documents，in whole or in part,are normatively referenced in this document and areindispensable for its application.For dated references, only the edition cited applies.For undated references,the latest edition of the referenced document (including any amendments) applies.
EN ISO 3696:1995, Water for analytical laboratory use —Specification and test methods (ISo 3696:1987)
3Principle
Remove the shellfish meat from the shell and homogenize the total shelfish meat. Extraction is carried outwith aqueous methanol (g = 80 % ).Separation is performed on a HPLC reverse-phase column provided with abinary gradient and detection is carried out by means of tandem mass spectrometry using triple quadrupoletechnology.The concentration of lipophilic toxins is determined by means of external calibration.
4Reagents
lf not otherwise specified, reagents of analytical grade and solvents suitable for LC-MS/MS shall be used.Water shall be distilled in glass vessels or demineralised before use, or shall be of equivalent purity accordingto EN ISO 3696:1995， Since the use of this method involves reagents harmful to health， appropriateprecautionary and protective measures such as avoiding skin contact and using an extractor hood shall betaken.
4.1 Aqueous methanol (φ= 80 %).
NOTE The validation data of this method have been elaborated with 80 % aqueous methanol. However, it has been shown (see [4], [5]) that equivalent results can be obtained when using 100 % methanol.
4.2 Acetonitrile
4.3 Sodium hydoxide, c(NaOH) = 2,5 mol/l.
4.4 Hydrochloric acid, c(HCl) = 2,5 mol/l.
4.5 Formic acid, 98 % to 100 % w/w.
4.6 Ammonium formate
4.7 Nitrogen, gaseous, min purity: 5,0.
4.8 Ammonium hydrogen carbonate
4.9 HPLC mobile phase 1 (chromatography under acidic conditions)
4.9.1 Eluent A1
Dissolve 126 mg (to give a 2 mmol/l solution) of ammonium formate (4.6) and 2 ml (to give a 50 mmol/l solution) of formic acid (4.5) in 50 ml of water and fill up to 1 000 ml with water. If necessary, filter the eluent using a 0,45 µm membrane filter.
4.9.2 Eluent B1
Dissolve 126 mg (to give a 2 mmoll solution) ammonium formate (4.6) and 2 ml (to give a 50 mmoll solution)of formic acid (4.5) in 50 ml of water. Add 950 ml of acetonitrile (4.2) and filter the eluent using a 0,45 ummembrane filter, if required.
4.10 HPLC mobile phase 2 (chromatography under basic conditions)4.10.1 Eluent A2
Dissolve 395 mg (to give a 5 mmol solution) of ammonium hydrogen carbonate (4.8) in 1 000 ml of water. fnecessary, filter the eluent using a 0,45 um membrane filter.
4.10.2 Eluent B2
Dissolve 395 mg (to give a 5 mmoll solution) of ammonium hydrogen carbonate (4.8) in 50 ml water.Add950 ml of acetonitrile (4.2) in portions of about 100 ml. Shake vigorously after each portion added. lfnecessary, filter the eluent using a 0,45 um membrane filter.
4.11 Toxin-free shellfish homogenate
To estimate and determine matrix effects，standard solutions in matrix are prepared. The shelfishhomogenate required for this purpose is prepared from shelfishes that have been proved free from toxins.4.12 Reference substances1)
NOTE During the validation study, the following reference substances were available. If other certified referencesubstances should become available in the future, the use of these substances is recommended.