1、March 2017 English price group 11No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.120.30!%bo*“2637607www.din.deDI
2、N EN 14176Foodstuffs Determination of domoic acid in raw shellfish, raw finfish and cooked mussels by RPHPLC using UV detection;English version EN 14176:2017,English translation of DIN EN 14176:2017-03Lebensmittel Bestimmung von Domoinsure in rohen Schalentieren, rohen Fischen und gekochten Miesmusc
3、heln mit RPHPLC und UVDetektion;Englische Fassung EN 14176:2017,Englische bersetzung von DIN EN 14176:2017-03Produits alimentaires Dosage de lacide domoque dans les coquillages crus, les poissons crus et les moules cuites par CLHP en phase inverse couple la dtection UV;Version anglaise EN 14176:2017
4、,Traduction anglaise de DIN EN 14176:2017-03SupersedesDIN EN 14176:200403www.beuth.deDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.Document comprises 19 pages 02.17 2 A comma is used as the decimal marker. National foreword This d
5、ocument (EN 14176:2017) has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany), Working Group 14 “Marine biotoxins”. The responsible German body involved in its preparation was DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produk
6、te (DIN Standards Committee Food and Agricultural Products), Working Group NA 057-01-03-01 AK Algentoxine. The DIN Standards corresponding to the International Standards referred to in this document are as follows, whereby EN ISO Standards are only listed below if these have not been published as DI
7、N EN ISO Standards with the same number: ISO 3696 DIN ISO 3696 ISO 13528 DIN ISO 13528 Amendments This standard differs from DIN EN 14176:2004-03 as follows: a) the title of the standard has been modified; b) the extraction procedure in 6.2 has been revised; c) the chromatographic conditions in 6.3
8、have been revised; d) the method has been re-validated, and the validation data in Annex A have been revised. Previous editions DIN EN 14176: 2004-03 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 13528, Statistic
9、al methods for use in proficiency testing by interlaboratory comparisons DIN EN 14176:2017-03 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 14176 January 2017 ICS 67.120.30 Supersedes EN 14176:2003English Version Foodstuffs - Determination of domoic acid in raw shellfish, raw finfish and cook
10、ed mussels by RP-HPLC using UV detection Produits alimentaires - Dosage de lacide domoque dans les coquillages crus, les poissons crus et les moules cuites par CLHP en phase inverse couple la dtection UV Lebensmittel - Bestimmung von Domoinsure in rohen Schalentieren, rohen Fischen und gekochten Mie
11、smuscheln mit RP-HPLC und UV-Detektion 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. U
12、p-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 tran
13、slation 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, Finla
14、nd, 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 STANDA
15、RDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR 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 worldwide for CEN national Members. Ref. No. EN 14176:2017 EEN 14176:2017 (E) 2 Contents
16、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
17、 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 DIN EN 14176:2017-03 EN 14176:2017 (E) 3 European
18、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 endorsem
19、ent, 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 p
20、atent rights. This document supersedes EN 14176:2003. EN 14176:2017 includes the following significant technical changes with respect to 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
21、 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, For
22、mer 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. DIN EN 14176:2017-03 EN 14176:20
23、17 (E) 4 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
24、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 chrom
25、atography 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
26、 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-ste
27、p 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 sta
28、ndard. 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
29、, 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, opera
30、tions 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 a
31、nd regulatory requirements for this purpose. DIN EN 14176:2017-03 EN 14176:2017 (E) 5 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
32、/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
33、 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 comple
34、te 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 referen
35、ces, 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
36、 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
37、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 av
38、ailable) 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 q
39、uality 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) DIN EN 14176:2017-03 EN 14176:2017 (E) 6 4.7 Eluents 4.7.1 Eluent 1 (isocr
40、atic 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 adju
41、st 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 4.8.1 Domoic acid as certified calibration solution1)Sealed ampoules should be stored in the dark in a refr
42、igerator (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 analys
43、is 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 Marine Biosciences, National Research Council of Canada, H
44、alifax, Nova Scotia-Canada1)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. Bo
45、th 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 g/ml. Closed vials should be stored in the dark in a refrigerator (at approximately +4
46、 C). 4.9.3 Standard solution Dilute the stock solution (4.9.2) with methanol to a final concentration of 50 g/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 http:/aes
47、an.msssi.gob.es/en/CRLMB/web/home.shtml and 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
48、to in the websites or available from Canada. Equivalent products may be used if they can be shown to lead to the same results. DIN EN 14176:2017-03 EN 14176:2017 (E) 7 4.9.4 Calibration solutions Prepare calibration solutions of appropriate mass concentrations of DA and epi-DA, either by diluting th
49、e 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 g/ml to 25 g/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 g/ml to 10 g/ml by diluting
