1、BS EN 14526:2017Foodstuffs Determinationof saxitoxin-group toxins inshellfish HPLC method usingpre-column derivatizationwith peroxide or periodateoxidationBSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN 14526:2017 BRITISH STANDARDNational forewordThis British S
2、tandard is the UK implementation of EN 14526:2017. It supersedes BS EN 14526:2004 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods.A list of organizations represented on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 reques
3、t to its secretary.This 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 89117 5 ICS 67.120.30 Compliance with a British
4、Standard cannot confer immunity 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 Text affectedBS EN 14526:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCH
5、E NORM EN 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 grou
6、pe de la 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
7、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 concer
8、ning 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
9、 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, Ge
10、rmany, 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 COMIT EUROPEN DE NORMALISATION EUROPISCHES
11、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 14526:2017 EBS EN 14526:2017EN 14526:2017 (E) 2 Contents Page European foreword . 3 Introducti
12、on 4 1 Scope 5 2 Normative references 6 3 Principle . 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 BS EN 14526:2017EN
13、 14526:2017 (E) 3 European foreword This 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 id
14、entical 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 id
15、entifying any or all such patent rights. 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
16、been revised; the chromatographic conditions 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 C
17、EN-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, Hung
18、ary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 14526:2017EN 14526:2017 (E)BS EN 14526:2017EN 14526:2017 (E) 2 Contents Page European forew
19、ord . 3 Introduction 4 1 Scope 5 2 Normative references 6 3 Principle . 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
20、BS EN 14526:2017EN 14526:2017 (E) 3 European foreword This 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 pu
21、blication 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
22、responsible for identifying any or all such patent rights. 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 pro
23、cedure in 6.2 has been revised; the chromatographic conditions 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.
24、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, Ger
25、many, 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. BS EN 14526:2017EN 14526:2017 (E)BS EN 14526:2017EN 14526:2017 (E) 4 Introducti
26、on Paralytic shellfish poisoning (PSP) toxins are derivatives of saxitoxin. These toxins 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
27、to fatal respiratory paralysis. This document describes an analytical method for the 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
28、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 personn
29、el prior to application of the standard, and fulfil statutory and regulatory requirements for this purpose. BS EN 14526:2017EN 14526:2017 (E) 5 1 Scope This European standard specifies a method 1 for the quantitative determination of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), neosaxitoxin (NEO)
30、, decarbamoyl neosaxitoxin (dcNEO), gonyautoxin 1 and 4 (GTX1,4; sum of isomers), 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 (
31、C1,2; sum of isomers) and (depending on the availability of certified reference 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
32、 shellfish products. The method described was validated in an interlaboratory study 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 ad
33、ditional interlaboratory studies 7, 8. The lowest validated levels 4, 5, 8, are 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) 5 114
34、b0,29bgonyautoxin 5 (GTX5, B1) 5 27c 0,07c dc-saxitoxin (dcSTX) 5 8c 0,03c neosaxitoxin (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 834b
35、0,08 2,11bdc-gonyautoxin 2,3 (dcGTX2,3) 8 271a0,77adc-neosaxitoxin (dcNEO) 8 594b2,18balowest 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 stu
36、dy but several laboratories detected this toxin directly after solid phase extraction 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 av
37、ailability of the standard substance. Currently it is possible to determine GTX6 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
38、was included in the first interlaboratory study. The present method is applicable to quantify C3,4 directly, depending on the availability of the standard substance. BS EN 14526:2017EN 14526:2017 (E)BS EN 14526:2017EN 14526:2017 (E) 4 Introduction Paralytic shellfish poisoning (PSP) toxins are deriv
39、atives of saxitoxin. These toxins 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
40、 an analytical method for the 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 material
41、s, 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 sta
42、tutory and regulatory requirements for this purpose. BS EN 14526:2017EN 14526:2017 (E) 5 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
43、 (GTX1,4; sum of isomers), 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 availabilit
44、y of certified reference 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
45、 in an interlaboratory study 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 valid
46、ated levels 4, 5, 8, are 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
47、 (dcSTX) 5 8c 0,03c neosaxitoxin (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-n
48、eosaxitoxin (dcNEO) 8 594b2,18balowest 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 directl
49、y after solid phase extraction 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 after a hydrolysis of Fraction 2 of the SPE-COOH clean-up, described in 6.4 as NEO. The indirect q
