1、BS EN 14176:2017Foodstuffs Determinationof domoic acid in raw shellfish,raw finfish and cooked musselsby RP-HPLC using UV detectionBSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN 14176:2017 BRITISH STANDARDNational forewordThis British Standard is the UK implem
2、entation of EN 14176:2017. It supersedes BS EN 14176:2003 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained on request to its secretary.This
3、publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2017.Published by BSI Standards Limited 2017ISBN 978 0 580 89118 2 ICS 67.120.30 Compliance with a British Standard cannot confer i
4、mmunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 January 2017.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS EN 14176:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 1417
5、6 January 2017 ICS 67.120.30 Supersedes EN 14176:2003English Version Foodstuffs - Determination of domoic acid in raw shellfish, raw finfish and cooked mussels by RP-HPLC using UV detection Produits alimentaires - Dosage de lacide domoque dans les coquillages crus, les poissons crus et les moules cu
6、ites par CLHP en phase inverse couple la dtection UV Lebensmittel - Bestimmung von Domoinsure in rohen Schalentieren, rohen Fischen und gekochten Miesmuscheln 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/CENE
7、LEC 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 o
8、r 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 off
9、icial 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
10、, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All r
11、ights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14176:2017 EBS EN 14176:2017EN 14176:2017 (E) 2 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
12、. 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 (inf
13、ormative) Typical chromatogram 16 Figure B.1 Typical chromatogram of MUS1b reference material 16 Bibliography . 17 BS EN 14176:2017EN 14176:2017 (E) 3 European foreword This document (EN 14176:2017) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secreta
14、riat 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
15、 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. EN 14176:2017 includes the following significant technical changes with respect to EN
16、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 th
17、e 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, Netherl
18、ands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 14176:2017EN 14176:2017 (E) 4 Introduction The amnesic shellfish poisoning (ASP) toxin, domoic acid (DA), belongs to a group of amino acids, called the kainoids, whic
19、h 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 includ
20、e (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
21、 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 oth
22、er 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 extracti
23、on (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.
24、 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 absorban
25、ce 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 u
26、ser 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. BS EN 14176:2017EN 14176:2017 (E) 5 1 Scope This European Standard specifies methods for the quantit
27、ative 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 matr
28、ices 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
29、 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 normat
30、ively 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 - Specificatio
31、n 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.
32、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 accordin
33、g 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 conce
34、ntration. 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), spectrophotometri
35、c grade 99 % (Method A) 4.6 Formic acid, mass concentration 98 % (Method B) BS EN 14176:2017EN 14176:2017 (E) 6 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 w
36、ater, 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 formi
37、c 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 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 opene
38、d, 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 conta
39、ined as a minor component in the certified calibration solution from the Institute for Marine Biosciences, National Research Council of Canada, Halifax, 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
40、 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 flas
41、k 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 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 solu
42、tion by comparing with certified calibration solutions (4.8.1). 1) Information on suitable calibration solutions are e. g. available on http:/aesan.msssi.gob.es/en/CRLMB/web/home.shtml and http:/aesan.msssi.gob.es/en/CRLMB/web/estandares_materiales_referencia/materiales_referencia.shtml. This inform
43、ation 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. BS EN 14176:2017EN 14176:201
44、7 (E) 7 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 soluti
45、ons 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 the standar
46、d 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 material2)Mussel tiss
47、ue 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 immediate
48、ly. 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 Analytic
49、al 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) 5.4 Mechanical mixer, high speed at 8 000 min1to 45 000 min1(e.g. Ultra turrax) 5.5 Centrifuge, capable of effectively separating the liquid and solid phase, e.g. 3 000 g 3)(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 m or 0,45 m, e.g. surfactant-free
