DIN EN 14526-2017 Foodstuffs - Determination of saxitoxin-group toxins in shellfish - HPLC method using pre-column derivatization with peroxide or periodate oxidation German versio.pdf

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1、April 2017 English price group 24No 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!%dTn“2654975www.din.deDI

2、N EN 14526Foodstuffs Determination of saxitoxingroup toxins in shellfish HPLC method using precolumn derivatization with peroxide or periodate oxidation;English version EN 14526:2017,English translation of DIN EN 14526:2017-04Lebensmittel Bestimmung von Toxinen der Saxitoxingruppe in Schalentieren H

3、PLCVerfahren mit Vorsulenderivatisierung mit Peroxid oder Periodatoxidation;Englische Fassung EN 14526:2017,Englische bersetzung von DIN EN 14526:2017-04Produits alimentaires Dtermination de la teneur en toxines du groupe de la saxitoxine dans les coquillages Mthode par CLHP avec drivation prcolonne

4、 et par oxydation au peroxyde ou au periodate;Version anglaise EN 14526:2017,Traduction anglaise de DIN EN 14526:2017-04SupersedesDIN EN 14526:200411www.beuth.deDocument comprises 67 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritat

5、ive.04.17 DIN EN 14526:2017-04 2 A comma is used as the decimal marker. National foreword This document (EN 14526:2017) has been prepared by CEN Technical Committee TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany), after thorough preparatory work by Working Group 14 “Marine biot

6、oxins”. The responsible German body involved in its preparation was DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Group NA 057-01-03-01 AK “Algal toxins”. Amendments This standard differs from DIN EN 14526:2004-11

7、as follows: a) the title has been changed; b) the applicability has been extended as the procedure has been tested on several samples during interlaboratory studies; c) the extraction procedure in 6.2 has been revised; d) chromatographic conditions in Clause 7 have been revised; e) guidelines for th

8、e calculation in presence of several toxins have been included in Table 7; f) the method has been additionally validated in several interlaboratory studies and the precision data in Annex A have been revised. Previous editions DIN EN 14526: 2004-11 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM E

9、N 14526 January 2017 ICS 67.120.30 Supersedes EN 14526:2004English Version Foodstuffs - Determination of saxitoxin-group toxins in shellfish - HPLC method using pre-column derivatization with peroxide or periodate oxidation Produits alimentaires - Dtermination de la teneur en toxines du groupe de la

10、 saxitoxine dans les coquillages - Mthode par CLHP avec drivation pr-colonne et par oxydation au peroxyde ou au periodate Lebensmittel - Bestimmung von Toxinen der Saxitoxingruppe in Schalentieren - HPLC-Verfahren mit Vorsulenderivatisierung und Peroxid- oder Periodatoxidation This European Standard

11、 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 suc

12、h 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

13、 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, G

14、reece, 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 COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE

15、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 14526:2017 EContents Page European foreword . 3 Introduction 4 1 Scope 5 2 Normative references 6 3 Pr

16、inciple . 6 4 Reagents . 9 5 Apparatus 12 6 Procedure 14 7 HPLC determination 18 8 Calibration curve 20 9 Identification . 20 10 Calculation 20 11 Precision 35 12 Test report 35 Annex A (informative) Precision data . 36 Bibliography . 64 DIN EN 14526:2017-04 EN 14526:2017 (E) 2 European foreword Thi

17、s document (EN 14526:2017) 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

18、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

19、. This document supersedes EN 14526:2004. EN 14526:2017 includes the following significant technical changes with respect to EN 14526:2004: the applicability is greater as more samples were tested in interlaboratory studies; the extraction procedure in 6.2 has been revised; the chromatographic condi

20、tions in Clause 7 have been revised; guidelines for calculation in presence of several toxins were introduced; the method has been additionally validated in several interlaboratory studies, and the precision data in Annex A have been revised. According to the CEN-CENELEC Internal Regulations, the na

21、tional 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, L

22、ithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 14526:2017-04 EN 14526:2017 (E) 3 Introduction Paralytic shellfish poisoning (PSP) toxins are derivatives of saxitoxin. These tox

23、ins have been detected in filter-feeding bivalve molluscs in various parts of the world. Paralytic shellfish poisoning is characterized by symptoms varying from slight tingling sensation or numbness around the lips to fatal respiratory paralysis. This document describes an analytical method for the

24、quantification of these PSP toxins by extraction from shellfish tissue followed by several clean-up steps and a separation by high performance liquid chromatography (HPLC) with fluorescence detection (FLD). WARNING The use of this standard can involve hazardous materials, operations and equipment. T

25、his 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 requirem

26、ents for this purpose. DIN EN 14526:2017-04 EN 14526:2017 (E) 4 1 Scope This European standard specifies a method 1 for the quantitative determination of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), neosaxitoxin (NEO), decarbamoyl neosaxitoxin (dcNEO), gonyautoxin 1 and 4 (GTX1,4; sum of isomers)

27、, gonyautoxin 2 and 3 (GTX2,3; sum of isomers), gonyautoxin 5 (GTX5 also called B1), gonyautoxin 6 (GTX6 also called B2), decarbamoyl gonyautoxin 2 and 3 (dcGTX2,3; sum of isomers), N-sulfocarbamoyl-gonyautoxin 1 and 2 (C1,2; sum of isomers) and (depending on the availability of certified reference

28、materials (CRMs) N-sulfocarbamoyl-gonyautoxin 3 and 4 (C3,4; sum of isomers) in (raw) mussels, oysters, scallops and clams. Laboratory experience has shown that it is also be applicable in other shellfish 2, 3 and cooked shellfish products. The method described was validated in an interlaboratory st

29、udy 4, 5 and was also verified in a EURL-performance test aiming the total toxicity of the samples 6. Toxins which were not available in the first interlaboratory study 4, 5 as dcGTX2,3 and dcNEO were validated in two additional interlaboratory studies 7, 8. The lowest validated levels 4, 5, 8, are

30、given in g toxin (free base)/kg shellfish tissue and also as mol/kg shellfish tissue and are listed in Table 1. Table 1 Lowest validated levels Toxin g/kg mol/kg saxitoxin (STX) 5 22c 0,07c gonyautoxin 2,3 (GTX2,3) 5 114b0,29bgonyautoxin 5 (GTX5, B1) 5 27c 0,07c dc-saxitoxin (dcSTX) 5 8c 0,03c neosa

31、xitoxin (NEO) 5 33c 0,10c gonyautoxin 1,4 (GTX1,4) 5 61,4c 0,15c N-sulfocarbamoyl-gonyautoxin 1,2 (C1,2) 5 93c 0,20c N-sulfocarbamoyl-gonyautoxin 3,4 (C3,4) 5 725b1,48bgonyautoxin 6 (GTX6, B2) Direct 4 Indirect 9 30 834b0,08 2,11bdc-gonyautoxin 2,3 (dcGTX2,3) 8 271a0,77adc-neosaxitoxin (dcNEO) 8 594

32、b2,18balowest spiked level; mean recovery: 58 % 8 blowest concentration tested clowest concentration tested with a HorRat 2 4, 5 A quantitative determination of GTX6 (B2) was not included in the first interlaboratory study but several laboratories detected this toxin directly after solid phase extra

33、ction with ion-exchange (SPE-COOH) clean-up and reported a mass concentration of 30 g/kg or higher in certain samples. For that reason, the present method is applicable to quantify GTX6 (B2) directly, depending on the availability of the standard substance. Currently it is possible to determine GTX6

34、 after a hydrolysis of Fraction 2 of the SPE-COOH clean-up, described in 6.4 as NEO. The indirect quantification of GTX6 was validated in two additional interlaboratory studies 7, 8. A quantitative determination of C3,4 was included in the first interlaboratory study. The present method is applicabl

35、e to quantify C3,4 directly, depending on the availability of the standard substance. DIN EN 14526:2017-04 EN 14526:2017 (E) 5 If no standard substances are available, C3,4 can only be quantified as GTX1,4 if the same hydrolysis protocol used for GTX6 (6.4) is applied to Fraction 1 of the SPE-COOH c

36、lean-up, see 10. 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 (in

37、cluding any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696) 3 Principle WARNING PSP toxins are neurotoxins which can be taken up by inhalation or orally. Therefore, adequate protection measures are to be applied. Paralytic Shellfish P

38、oisoning (PSP) toxins are extracted from shellfish tissue homogenate by heating with acetic acid. After centrifugation the supernatant is purified by solid phase extraction (SPE) using a C18 clean-up cartridge. It is analysed by HPLC after oxidation with periodate or peroxide with fluorescence detec

39、tion. Most toxins (STX, C1,2, GTX5 (B1), dcSTX, GTX2,3 and dcGTX2,3) can be quantified after SPE-C18 clean-up1). Oxidation of PSP toxins leads to several reaction products that are separated by reversed phase HPLC and detected by fluorescence detection. The obtained reaction products for PSP toxins

40、after oxidation with peroxide and periodate are listed in Table 2. Additionally, the corresponding chromatograms are shown in Figure 1. The gonyautoxins GTX2 and GTX3 as well as GTX1 and GTX4 and decarbamoyl gonyautoxins dcGTX2 and dcGTX3 and the N-sulfocarbamoyl-gonyautoxins C1 and C2 as well as C3

41、 and C4 are structural isomers and lead with both oxidation modes to the same reaction products. The amount of structural isomers is determined as sum of both toxins. STX reacts to a single specific oxidation product regardless of the kind of oxidation reaction (whether peroxide or periodate). The s

42、ame is valid for GTX2,3 as well as GTX5 (B1) and C1,2. In contrast, dcSTX and dcGTX2,3 produce each two different oxidation products in both oxidation reactions, see also Table 2. The toxin dcNEO is oxidized into two oxidation products only with the periodate oxidation. Each of the toxins NEO, GTX6

43、(B2), GTX1,4 and C3,4 produce three peaks after periodate oxidation but only the second eluting peak is used for quantification (peroxide oxidation cannot be used for quantification). Co-occurrence of different PSP toxins in shellfish can influence the analytical results, because some of the PSP tox

44、ins can (partially) lead to the same reaction products (see Table 2). So the chromatograms shall be carefully interpreted after a SPE C18 clean-up. 1) This document is based on a procedure described by Lawrence et al. 4 and was also published as AOAC Official Method 2005.06 1. DIN EN 14526:2017-04 E

45、N 14526:2017 (E) 6 Table 2 Reaction products after oxidation with periodate and peroxide Toxin Oxidation products and HPLC-eluting order Intensity Oxidation product at the same retention time as peroxide periodate peroxide periodate peroxide periodate STX one one + + NEOa3 bNEO 3; GTX6 (B2) 3 dc-STX

46、 first 1 first 1 + - dcNEO 1 second 2 second 2 + + NEOa2 NEO 2; GTX6 (B2) 2; dcNEO 2 NEO no first 1 + GTX6 (B2) 1 second 2 second 2 - + dcSTX 2 GTX6 (B2) 2; dcSTX 2; dcNEO 2 third 3 third 3 - + STX STX; GTX6 (B2) 3 C1,2 one one + + C3,4 no first 1 + GTX1,4 1 no second 2 + GTX1,4 2; dcGTX2,3 2 no thi

47、rd 3 + GTX1,4 3; GTX2,3 GTX1,4 no first 1 + C3,4 1 no second 2 + C3,4 2; dcGTX2,3 2 third 3 third 3 - + GTX2,3 C3,4 3; GTX2,3 GTX2,3 one one + + GTX1,4a3 C3,4 3; GTX1,4 3 GTX5 (B1) one one + - GTX6 (B2) no first 1 + NEO 1 no second 2 + NEO 2; dcSTX 2; dcNEO 2 no third 3 - NEO 3; STX dcGTX2,3 first 1 first 1 + + second 2 second 2 + + C3,4 2; GTX1,4 2 dcNEO first 1 first 1 - + dcSTX 1 second 2 second 2 - + dcSTX 2 dcSTX 2; N