EN 16204:2012

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 (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.

Lebensmittel - Bestimmung der lipophilen Algentoxine (Okadasäuregruppen-Toxine, Yessotoxine, Azaspirosäuren, Pectenotoxine) in Schalentieren und Schalentiererzeugnissen mit LC-MS/MS

Diese Europäische Norm legt ein Multireferenzverfahren zum Nachweis und zur Bestimmung lipophiler Algentoxine (fettlösliche Algentoxine, die von einigen Dinoflagellaten produziert werden) in rohen Schalentieren und Schalentiererzeugnissen und gekochten Muscheln mit Flüssigchromatographie gekoppelt mit Tandem-Massenspektrometrie LC-MS/MS [1], [2], [3] fest. Dieses Verfahren wurde in einem aus drei Teilen bestehenden Ringversuch durch die Untersuchung sowohl natürlich kontaminierter Miesmuschelhomogenate als auch aufgestockter Extrakte von Miesmuscheln, Austern und Venusmuscheln validiert. Hinsichtlich weiterer Informationen siehe Anhang A. In anderen Studien wurden auch weitere Matrices untersucht [4], [5]. Die Nachweisgrenze (LOD) wurde für die Toxine der Okadasäuregruppe, Azaspirosäuren und Pectenotoxine mit 6 µg/kg Schalentierfleisch und für Yessotoxine mit 10 µg/kg Schalentierfleisch bestimmt. Die quantitative Bestimmung von Okadasäure (OA), Pectenotoxin-2 (PTX-2), Azaspirosäure-1 (AZA-1) und Yessotoxin (YTX) kann aufgrund kommerziell erhältlicher Standardsubstanzen direkt durchgeführt werden. Unter der Annahme eines gleichen Responsefaktors wird Okadasäure für die indirekte quantitative Bestimmung der beiden Dinophysistoxine Dinophysistoxin-1 (DTX-1), Dinophysistoxin-2 (DTX-2) verwendet; ebenso wird Azaspirosäure-1 (AZA-1) für die indirekte quantitative Bestimmung von Azaspirosäure-2 (AZA-2) und Azaspirosäure-3 (AZA-3) verwendet, während YTX für Homo-Yessotoxin (Homo-YTX) und 45-OH-Yessotoxin und 45-OH-homo-Yessotoxin verwendet wird, und PTX-2 für Pectenotoxin-1 (PTX-1). Als Bestimmungsgrenze (LOQ) ergab sich für die Toxine der Okadasäuregruppe, Azaspirosäuren und Pectenotoxine ein Wert von 20 µg/kg Schalentierfleisch und für Yessotoxine ein Wert von 35 µg/kg Schalentierfleisch. Mit Hilfe eines Hydrolyseschrittes [6] können auch die Ester von Okadasäure, DTX-1 und DTX-2 als freie Säuren quantitativ gemessen werden.

Produits alimentaires - Dosage des toxines algales lipophiles (toxines du groupe acide okadaïque, Yessotoxines, Azaspiracides, Pecténotoxines) dans les coquillages et les produits à base de coquillages par CL-SM/SM

Le présent document spécifie une méthode multiréférence de dosage des toxines lipophiles (toxines algales liposolubles produites par les dinoflagellés) dans les coquillages et produits à base de coquillages par CL SM/SM. Cette méthode a été validée dans le cadre d’une étude interlaboratoires fondée sur l’analyse des homogénats naturellement contaminés et des extraits dopés. Pour de plus amples informations sur la validation, voir l’Annexe A.
La limite de détection des toxines DSP, azaspiracides et pecténotoxines a été déterminée à une valeur de 6 µg/kg de chair de coquillage et pour les yessotoxines de 10 µg/kg de chair de coquillage.
Le dosage quantitatif de l’acide okadaïque, pecténotoxine2 (PTX2), azaspiracide1 (AZA1) et yessotoxine (YTX) peut être réalisé directement en utilisant des substances étalons disponibles dans le commerce. Selon l’hypothèse d’un facteur de réponse égal, l’acide okadaïque est utilisé pour le dosage quantitatif indirect des deux dinophysistoxines, la dinophysistoxine-1 (DTX1) et la dinophysistoxine-2 (DTX2). De même, l’azaspiracide1 (AZA1) est utilisée pour le dosage quantitatif indirect de l’azaspiracide2 (AZA2) et de l’azaspiracide3 (AZA3), la YTX est utilisée pour l’homo-yessotoxine, l’OH-yessotoxine et l’OH-homo-yessotoxine, et PTX2 est utilisée pour PTX1.
La limite de quantification des toxines DSP, azaspiracides et pecténotoxines a été déterminée à une valeur de 20 µg/kg de chair de crustacé et pour les yessotoxines de 35 µg/kg de chair de crustacé.
L’hydrolyse permet également de déterminer de manière quantitative les esters d’acide okadaïque, de DTX1 et de DTX2.

Živila - Določevanje lipofilnih toksinov alg (skupina toksinov okadaične kisline, jesotoksini, azaspiracidi, pektenotoksini) v školjkah in njihovih proizvodih z uporabo LC-MS/MS (tekočinska kromatografija s tandemsko masno spektrometrijo)

Ta evropski standard določa večreferenčno metodo za določevanje lipofilnih toksinov (toksini alg, topni v maščobah, ki jih proizvajajo nekateri dinoflageti) v surovih školjkah in njihovih proizvodih, vključno s kuhanimi školjkami, s tekočinsko kromatografijo s tandemsko masno spektrometrijo (LC-MS/MS) [1], [2], [3]. Ta metoda je bila validirana z medlaboratorijsko študijo v treh delih prek analize obeh naravno kontaminiranih homogenatov užitne klapavice in primešanih ekstraktov užitne klapavice, ostrige in venernice. Za več informacij o validaciji glejte dodatek A. Druge matrice so bile preiskane z drugimi študijami (glejte [4], [5]). Meja detekcije skupine toksinov okadaične kisline, azaspiracidov in pektenotoksinov je bila določena na 6 μg/kg mesa školjk, meja jesotoksinov pa na 10 μg/kg mesa školjk. Kvantitativno določevanje okadaične kisline (OA), pektenotoksina-2 (PTX-2), azaspiracida-1 (AZA-1) in jesotoksina (YTX) se lahko izvede neposredno s standardnimi snovmi, ki so komercialno dostopne. Okadaična kislina se ob predpostavljanju enakega faktorja odziva uporablja za posredno kvantitativno določevanje obeh dinofizistoksinov, in sicer dinofizistoksina-1 (DTX-1) ter dinofizistoksina-2 (DTX-2); podobno se azaspiracid-1 (AZA-1) uporablja za posredno kvantitativno določevanje azaspiracida-2 (AZA-2) in azaspiracida-3 (AZA-3), jesotoksin za homo-jesotoksin, 45-OH-jesotoksin in 45-OH-homo-jesotoksin, pektenotoksin-2 pa za pektenotoksin-1 (PTX-1). Meja kvantifikacije skupine toksinov okadaične kisline, azaspiracidov in pektenotoksinov je 20 μg/kg mesa školjk, meja jesotoksinov pa 35 μg/kg mesa školjk. Estra okadaične kisline, tj. dinofizistoksin-1 in dinofizistoksin-2, se lahko kot ustrezni prosti kislini kvantitativno določita tudi s hidrolizo [6].

General Information

• Status: Published
• Publication Date: 15-May-2012
• Withdrawal Date: 29-Nov-2012
• ICS: 67.120.30 - Fish and fishery products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 14 - Marine Biotoxins
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Completion Date: 29-Jan-2024
• Ref Project: SIST EN 16204:2012 - Foodstuffs - Determination of lipophilic algal toxins (okadaic acid group toxins, yessotoxins, azaspiracids, pectenotoxins) in shellfish and shellfish products by LC-MS/MS

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.VSHNWURPHWULMRLebensmittel - Bestimmung der lipophilen Algentoxine (Okadasäuregruppen-Toxine, Yessotoxine, Azaspirosäuren, Pectenotoxine) in Schalentieren und Schalentiererzeugnissen mit LC-MS/MSProduits alimentaires - Dosage des toxines algales lipophiles (toxines du groupe acide okadaïque, yessotoxines, azaspiracides, pecténotoxines) dans les coquillages et les produits à base de coquillages par CL-SM/SMFoodstuffs - Determination of lipophilic algal toxins (okadaic acid group toxins, yessotoxins, azaspiracids, pectenotoxins) in shellfish and shellfish products by LC-MS/MS67.120.30Ribe in ribji proizvodiFish and fishery products67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 16204:2012SIST EN 16204:2012en,fr,de01-september-2012SIST EN 16204:2012SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16204
May 2012 ICS 67.120.30 English Version
Foodstuffs - Determination of lipophilic algal toxins (okadaic acid group toxins, yessotoxins, azaspiracids, pectenotoxins) in shellfish and shellfish products by LC-MS/MS
Produits alimentaires - Dosage des toxines algales lipophiles (toxines du groupe acide okadaïque, yessotoxines, azaspiracides, pecténotoxines) dans les coquillages et les produits à base de coquillages par CL-SM/SM
Lebensmittel - Bestimmung der lipophilen Algentoxine (Okadasäuregruppen-Toxine, Yessotoxine, Azaspirosäuren, Pectenotoxine) in Schalentieren und Schalentiererzeugnissen mit LC-MS/MS This European Standard was approved by CEN on 20 April 2012.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
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Precision data . 15A.1Details on the inter-laboratory study . 15A.2Recovery . 28Annex B (informative)
Examples for suitable MS detection conditions . 29B.1Examples suitable for SCIEX API 4000 or API 4000 Q-Trap . 29B.2Examples suitable for Waters (Micromass) TSQ Ultima . 31B.3Examples suitable for Thermo Fisher TSQ Quantum Ultra . 33B.4Examples suitable for Agilent 6410 or 6460 QQQ . 35Annex C (informative)
Typical chromatogram . 37Bibliography . 38 SIST EN 16204:2012

2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable 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) 3 Principle Remove the shellfish meat from the shell and homogenize the total shellfish meat. Extraction is carried out with aqueous methanol (3 = 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 using triple quadrupole technology. The concentration of lipophilic toxins is determined by means of external calibration. 4 Reagents If 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 according to EN ISO 3696:1995. Since the use of this method involves reagents harmful to health, appropriate precautionary and protective measures such as avoiding skin contact and using an extractor hood shall be taken. 4.1 Aqueous methanol (3= 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 SIST EN 16204:2012

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 mmol/l solution) ammonium formate (4.6) and 2 ml (to give a 50 mmol/l 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 µm membrane 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/l solution) of ammonium hydrogen carbonate (4.8) in 1 000 ml of water. If necessary, filter the eluent using a 0,45 µm
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