SIST EN 14176:2017

Foodstuffs - Determination of domoic acid in raw shellfish, raw finfish and cooked mussels by RP-HPLC using UV detection

This European Standard specifies methods for the quantitative determination of domoic acid in raw bivalve molluscs and finfish as well as in cooked mussels. The limit of detection is about 10 ng/ml to 80 ng/ml (0,05 mg/kg to 0,4 mg/kg), depending on the UV detector sensitivity. The limit of quantification for DA by these methods is at least 2,7 mg/kg. Method A has been validated for the determination of DA in different raw matrices such as mussels, clams, scallops and anchovies, spiked and/or naturally contaminated at levels ranging from 2,7 mg/kg to 85,1 mg/kg. Method B has been validated for the determination of DA at levels ranging from 5 mg/kg to 12,9 mg/kg in cooked blue mussels.
For further information on validation data, see Clause 8 and Annex A.
Laboratory experience has shown that this standard can also be applied to other shellfish species, however, no complete validation study according to ISO 5725 has been carried out so far.

Lebensmittel - Bestimmung von Domoinsäure in rohen Schalentieren, rohen Fischen und gekochten Miesmuscheln mit RP-HPLC und UV-Detektion

Diese Europäische Norm legt die Verfahren für die quantitative Bestimmung von Domoinsäure in rohen Muscheln und Fischen als auch in gekochten Miesmuscheln fest. Abhängig von der Empfindlichkeit des UV Detektors liegt die Nachweisgrenze bei etwa 10 ng/ml bis 80 ng/ml (0,05 mg/kg bis 0,4 mg/kg). Verfahren A wurde in Bezug auf die Bestimmung von DA in verschiedenen rohen Matrices, beispielsweise Miesmuscheln, Venusmuscheln, Jakobsmuscheln und Anchovis (Sardellen), aufgestockt und/oder natürlich kontaminiert bei Gehalten validiert, die im Bereich von 2,7 mg/kg bis 85,1 mg/kg liegen. Verfahren B wurde für die Bestimmung von DA bei Gehalten validiert, die im Bereich von 5 mg/kg bis 12,9 mg/kg in gekochten Miesmuscheln liegen.
Hinsichtlich weiterer Informationen zu den Validierungsdaten siehe Abschnitt 8 und Anhang A.
Laborerfahrungen haben gezeigt, dass die vorliegende Norm auch für andere Schalentier-Arten angewendet werden kann, jedoch wurde bisher noch kein vollständiger Ringversuch nach ISO 5725 durchgeführt.

Produits alimentaires - Dosage de l’acide domoïque dans les coquillages crus, les poissons crus et les moules cuites par CLHP en phase inverse couplée à la détection UV

La présente Norme européenne spécifie des méthodes pour le dosage de l’acide domoïque dans les mollusques bivalves et les poissons crus, ainsi que dans les moules cuites. La limite de détection s’étend d’environ 10 ng/ml à 80 ng/ml (0,05 mg/kg à 0,4 mg/kg), en fonction de la sensibilité du détecteur UV. La Méthode A a été validée pour la détermination de l’acide domoïque dans différentes matrices crues telles que les moules, les palourdes, les coquilles Saint-Jacques et les anchois, supplémentés artificiellement (dopés) et/ou contaminés naturellement à des concentrations allant de 2,7 mg/kg à 85,1 mg/kg. La Méthode B a été validée pour le dosage de l’acide domoïque à des concentrations allant de 5 mg/kg à 12,9 mg/kg dans la moule commune cuite.
Pour de plus amples informations sur les données de validation, voir l’Article 8 et l’Annexe A.
L’expérience des laboratoires a montré que la présente norme peut également être appliquée à d’autres espèces de fruits de mer ; cependant, aucune étude de validation complète conformément à l’ISO 5725 n’a été menée jusqu’à ce jour.

Živila - Določevanje domojske kisline v surovih lupinarjih in plavutonožcih ter kuhanih školjkah z uporabo RP-HPLC in ultravijolično (UV) detekcijo

Ta evropski standard določa metode za kvantitativno določevanje domojske kisline v surovih dvolupinskih školjkah in plavutonožcih ter kuhanih školjkah. Meja detekcije je približno od 10 ng/ml do 80 ng/ml (0,05 mg/kg do 0,4 mg/kg), odvisno od občutljivosti UV detektorja. Mejna vrednost kvantifikacije za domojsko kislino na podlagi teh metod je vsaj 2,7 mg/kg. Metoda A je bila potrjena za določevanje domojske kisline v različnih surovih matricah (kot so klapavice, školjke, pokrovače in sardoni), obogatenih in/ali naravno kontaminiranih z razponom od 2,7 mg/kg do 85,1 mg/kg. Metoda B je bila potrjena za določevanje domojske kisline v razponu od 5 mg/kg do 12,9 mg/kg v kuhanih užitnih klapavicah.
Za več informacij glede podatkov o potrditvi glej točko 8 in dodatek A.
Izkušnje v laboratoriju kažejo, da se lahko ta standard uporablja tudi za druge vrste školjk, vendar do zdaj ni bila izvedena nobena popolna potrditvena študija v skladu s standardom ISO 5725.

General Information

• Status: Published
• Public Enquiry End Date: 30-Jul-2015
• Publication Date: 19-Feb-2017
• ICS: 67.120.30 - Fish and fishery products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 27-Jan-2017
• Due Date: 03-Apr-2017
• Completion Date: 20-Feb-2017
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: EN 14176:2017 - Foodstuffs - Determination of domoic acid in raw shellfish, raw finfish and cooked mussels by RP-HPLC using UV detection

Relations

• Replaces: SIST EN 14176:2004 - Foodstuffs - Determination of domoic acid in mussels by HPLC
• Effective Date: 01-Mar-2017

Overview

EN 14176:2017 (CEN) specifies validated analytical procedures for the quantitative determination of domoic acid (DA) - the amnesic shellfish poisoning (ASP) toxin - in raw bivalve molluscs, raw finfish and cooked mussels. The methods use reversed-phase high-performance liquid chromatography (RP‑HPLC) with UV detection (HPLC‑UV), exploiting DA’s strong absorbance at 242 nm. The standard defines sample extraction, chromatographic conditions (isocratic and gradient), calibration and reporting requirements, and gives validated performance data.

Key detection limits:

• Limit of detection (LOD): about 10–80 ng/ml (≈ 0.05–0.4 mg/kg), depending on UV detector sensitivity.
• Limit of quantification (LOQ): at least 2.7 mg/kg.

EN 14176:2017 supersedes EN 14176:2003 and includes revised extraction and chromatographic conditions and updated validation data.

Key Topics

• Two procedural variants
  • Method A: for unsalted raw matrices (mussels, clams, scallops, anchovies) - uses single‑step extraction with 50% aqueous methanol and isocratic RP‑HPLC.
  • Method B: for cooked blue mussels - uses single‑step extraction (optional heating step noted) and binary gradient RP‑HPLC.
• Chromatography & detection
  • C18 reversed‑phase columns; detector set to 242 nm (diode array recommended).
  • Eluents include acetonitrile/water with acid modifiers (TFA for isocratic; formic acid for gradient).
• Standards & calibration
  • Uses certified domoic acid calibration solutions or prepared standards; external standard calibration is specified.
• Validation & precision
  • Method A validated across 2.7–85.1 mg/kg; Method B validated across 5–12.9 mg/kg. Annex A and Clause 8 contain detailed precision data.
• Practical notes
  • Optional strong anion exchange (SPE) clean‑up historically available but is not described as mandatory in this revision.
  • Heating can assist decanting but may degrade DA; labs should follow the standard’s guidance.
• Safety warning: ASP toxins are neurotoxins - apply appropriate laboratory safety and handling.

Applications

• Routine monitoring of shellfish and fish for food safety and regulatory compliance.
• Seafood quality control in public health laboratories, food safety authorities, accredited testing labs, research institutes, and seafood processors.
• Supporting outbreak investigations and proficiency testing (EURLMB referenced).

Keywords: EN 14176:2017, domoic acid, RP‑HPLC, UV detection, HPLC‑UV, cooked mussels, raw shellfish, finfish, limit of detection, limit of quantification, Method A, Method B.

Related Standards

• EN ISO 3696 - Water for analytical laboratory use.
• ISO 5725 - Accuracy (trueness and precision) of measurement methods (referenced for full validation methodology).

Standards Content (Sample)

SLOVENSKI STANDARD
01-marec-2017
1DGRPHãþD
SIST EN 14176:2004
äLYLOD'RORþHYDQMHGRPRMVNHNLVOLQHYVXURYLKOXSLQDUMLKLQSODYXWRQRåFLKWHU
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Foodstuffs - Determination of domoic acid in raw shellfish, raw finfish and cooked
mussels by RP-HPLC using UV detection
Lebensmittel - Bestimmung von Domoinsäure in rohen Schalentieren, rohen Fischen und
gekochten Miesmuscheln mit RP-HPLC und UV-Detektion
Produits alimentaires - Dosage de l’acide domoïque dans les coquillages crus, les
poissons crus et les moules cuites par CLHP en phase inverse couplée à la détection UV
Ta slovenski standard je istoveten z: EN 14176:2017
ICS:
67.120.30 Ribe in ribji proizvodi Fish and fishery products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 14176
EUROPEAN STANDARD
NORME EUROPÉENNE
January 2017
EUROPÄISCHE NORM
ICS 67.120.30 Supersedes EN 14176:2003
English Version
Foodstuffs - Determination of domoic acid in raw shellfish,
raw finfish and cooked mussels by RP-HPLC using UV
detection
Produits alimentaires - Dosage de l'acide domoïque Lebensmittel - Bestimmung von Domoinsäure in rohen
dans les coquillages crus, les poissons crus et les Schalentieren, rohen Fischen und gekochten
moules cuites par CLHP en phase inverse couplée à la Miesmuscheln mit RP-HPLC und UV-Detektion
détection UV
This European Standard was approved by CEN on 7 November 2016.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14176:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Principle . 5
4 Reagents . 5
5 Apparatus . 7
6 Procedure. 8
7 Evaluation of results . 11
8 Precision . 11
9 Test report . 12
Annex A (informative) Precision data . 13
A.1 Precision data for Method A using isocratic elution. 13
A.2 Precision data for Method B using gradient elution . 14
A.3 Precision data from EURLMB Proficiency Testing Schemes . 15
Annex B (informative) Typical chromatogram . 16
Figure B.1 — Typical chromatogram of MUS1b reference material . 16

Bibliography . 17
European foreword
This document (EN 14176:2017) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by July 2017, and conflicting national standards shall be
withdrawn at the latest by July 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 14176:2003.
— the extraction procedure in 6.2 has been revised;
— the chromatographic conditions in 6.3 have been revised;
— the method has been re-validated, and the validation data in Annex A have been revised.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Introduction
The amnesic shellfish poisoning (ASP) toxin, domoic acid (DA), belongs to a group of amino acids, called
the kainoids, which are classed as neuroexcitants or excitoxins that interfere with the
neurotransmission mechanisms in the brain. The toxin can be accumulated in shellfish feeding on a
number of toxic Pseudonitzschia species. Ingestion of seafood contaminated with DA can lead to an
intoxication which symptoms include (among others) abdominal cramps, vomiting, disorientation and
memory loss (amnesia) and can become severe in certain cases.
High performance liquid chromatography with ultraviolet detection (HPLC-UV) was the first chemical
analytical method for DA and is still the most commonly used for monitoring shellfish. DA detection is
possible by its strong absorbance at 242 nm [1].
This European Standard is based on two, comparable procedures. One procedure for the quantitative
determination of DA and its isomers e.g. epi-domoic acid (epi-DA) in unsalted raw seafood (Method A)
is described in [2]. The other procedure for the quantitative determination of DA and its isomers e.g.
epi-DA in cooked mussel (Method B) is described in [3].
Method A uses a single-step extraction with 50 % aqueous methanol and an optional selective clean-up
and concentration step with strong anion exchange solid phase extraction (SPE). Taking into account
results of the validation procedure, the optional clean-up step of Method A as published under [2] is not
described in this standard. Analytes are determined by high performance liquid chromatography
(HPLC) under isocratic conditions with ultraviolet absorbance detection.
Method B uses a single-step extraction with 50 % aqueous methanol and an optional heating step which
allows a better decanting of the supernatant. However, it has been observed that heating can degrade
DA and epi-DA. DA and epi-DA are determined by HPLC with binary gradient and ultraviolet absorbance
detection.
Both methods can be applied for the quantitative determination of DA.
WARNING — The use of this standard can involve hazardous materials, operations and equipment. This
standard does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this standard to take appropriate measures to ensure the safety and health
of personnel prior to application of the standard, and fulfil statutory and regulatory requirements for
this purpose.
1 Scope
This European Standard specifies methods for the quantitative determination of domoic acid in raw
bivalve molluscs and finfish as well as in cooked mussels. The limit of detection is about 10 ng/ml to
80 ng/ml (0,05 mg/kg to 0,4 mg/kg), depending on the UV detector sensitivity. Method A has been
validated for the determination of DA in different raw matrices such as mussels, clams, scallops and
anchovies, spiked and/or naturally contaminated at levels ranging from 2,7 mg/kg to 85,1 mg/kg.
Method B has been validated for the determination of DA at levels ranging from 5 mg/kg to 12,9 mg/kg
in cooked blue mussels.
For further information on validation data, see Clause 8 and Annex A.
Laboratory experience has shown that this standard can also be applied to other shellfish species,
however, no complete validation study according to ISO 5725 has been carried out so far.
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, Water for analytical laboratory use - Specification and test methods (ISO 3696)
3 Principle
DA and epi-DA are extracted from sample tissue with a mixture of methanol and water. The extract is
filtered through a membrane filter and measured using HPLC equipment with isocratic (Method A) or
gradient (Method B) elution and detection by UV absorption. The amount of DA is calculated by the
method of external standard calibration.
WARNING — ASP toxins are neurotoxins which can be taken up by inhalation or orally. Therefore,
adequate protection measures are to be applied.
4 Reagents
During the analysis, unless otherwise stated, use only water according to grade 1 of EN ISO 3696.
If not otherwise indicated, all chemicals shall be of pro analysis (p. a.) quality.
Reference materials (certified, if available) and standard substances originating from other sources as
indicated may also be used if well-characterized and with a well-defined mass concentration.
If not already specified, stability of solutions should be determined by the laboratory.
4.1 Methanol, HPLC quality
4.2 Acetonitrile, HPLC quality
4.3 Extraction solvent, methanol/water 50:50 v/v
4.4 Acetonitrile/water, 10:90 v/v (Method A)
4.5 Trifluoroacetic acid (TFA), spectrophotometric grade ≥ 99 % (Method A)
4.6 Formic acid, mass concentration ≥ 98 % (Method B)
4.7 Eluents
4.7.1 Eluent 1 (isocratic conditions)
Aqueous 10 % v/v acetonitrile (4.4) with 0,1 % v/v TFA (4.5). For single pump systems, mix 100 ml
acetonitrile with approximately 400 ml water, add 1,0 ml TFA, and dilute to 1 l with water.
4.7.2 Eluent 2 (gradient conditions)
Mix 100 ml acetonitrile (4.2) with 900 ml water and adjust pH to 2,5 using formic acid (4.6).
4.7.3 Eluent 3 (gradient conditions)
Mix 300 ml acetonitrile (4.2) with 700 ml water and adjust pH to 2,5 using formic acid (4.6).
4.8 Standard substances
1)
4.8.1 Domoic acid as certified calibration solution
Sealed ampoules should be stored in the dark in a refrigerator (at approximately +4 °C). Do not freeze
the solution. Prior to opening, each ampoule should be at room temperature. Once the ampoule has
been opened, accurate aliquots should be removed using calibrated volumetric equipment and
transferred to other amber glass vial for dilution and/or analysis as soon as possible. Closed vials
should be stored in the dark in a refrigerator (at approximately +4 °C) for no more than 3 months.
NOTE Epi-DA is contained as a minor component in the certified calibration solution from the Institute for
1)
Marine Biosciences, National Research Council of Canada, Halifax, Nova Scotia-Canada
4.8.2 Domoic acid, as crystalline powder, purity of > 95 %
4.9 Standard solutions
4.9.1 General
Either use commercially available certified calibration solutions (4.8.1) or prepare calibration solutions
by dissolving crystalline DA powder (4.8.2) and subsequently dilute. Both procedures have been proven
to lead to successful validation data.
4.9.2 Stock solution
Weigh (5.1) DA crystalline powder (4.8.2) into a volumetric flask and dissolve in methanol to a final
concentration of 500 µg/ml. Closed vials should be stored in the dark in a refrigerator (at approximately
+4 °C).
4.9.3 Standard solution
Dilute the stock solution (4.9.2) with methanol to a final concentration of 50 µg/ml. Check the mass
concentration of this solution by comparing with certified calibration solutions (4.8.1).

1) Information on suitable calibration solutions are e. g. available on
and
http://aesan.msssi.gob.es/en/CRLMB/web/home.shtml
http://aesan.msssi.gob.es/en/CRLMB/web/estandares_materiales_referencia/materiales_referencia.shtml. This
information is given for the convenience of the users of this European Standard and does not constitute an
endorsement by CEN of the products referred to in the websites or available from Canada. Equivalent products
may be used if they can be shown to lead to the same results.
4.9.4 Calibration solutions
Prepare calibration solutions of appropriate mass concentrations of DA and epi-DA, either by diluting
the standard solution (4.9.3) or by diluting the certified calibration solution (4.8.1).
For the validation of Method A with isocratic elution, calibration solutions were prepared in the range
of 0,2 µg/ml to 25 µg/ml by diluting the certified calibration solution (4.8.1) with acetonitrile:water,
10:90 v/v (4.4).
For the validation of Method B with gradient elution, calibration solutions were prepared in the range
of 0,5 µg/ml to 10 µg/ml by diluting the standard solution (4.9.3) with a methanol/water solution (4.3).
Keep solutions in the dark and refrigerated (at approximately + 4 °C) when not in use. Do not store
them for more than 3 months. Do not freeze the solutions. Warm up solutions to room temperature
before use.
)
4.10 Reference material
Mussel tissue reference material should be stored according to the manufacturers specifications. Each
bottle should be allowed to warm to room temperature prior to opening and the contents thoroughly
mixed by vortexing for a minimum of 2 min. When a bottle is opened the entire contents should be used
immediately. The reference material can be used to test the accuracy of an existing analytical procedure.
Extraction of the reference material should be performed according to the procedure described in 6.2.1
or 6.2.2.
5 Apparatus
Use usual laboratory apparatus and, in particular, the following:
5.1 Analytical balance, capable of weighing to the nearest 0,1 mg
5.2 Balance, capable of weighing to the nearest 0,01 g
5.3 Homogenizer (e.g. grinding or blending machine)
−1 −1
5.4 Mechanical mixer, high speed at 8 000 min to 45 000 min (e.g. Ultra turrax)
3)
5.5 Centrifuge, capable of effectively separating the liquid and solid phase, e.g. 3 000 g
(refrigerated at + 4 °C, if possible)
5.6 Centrifuge tubes, nominal volume 30 ml to 50 ml, with screw caps
5.7 Membrane filter, methanol compatible with a pore size of 0,2 µm or 0,45 µm, e.g. surfactant-free
cellulose acetate with glass fibre pre-filter
5.8 Adjustable automatic pipettes, covering the range from 20 µl to 1000 µl

2)
Suitable reference material is e. g. available from the Institute for Marine Biosciences, National
Research Council of Canada, Halifax, Nova Scotia-Canada. Epi-DA is contained as a minor component in
this certified reference mat
...