1、Licensed Copy: Wang Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy, (c) BSIBRITISH STANDARD BS EN 14526:2004 Foodstuffs Determination of saxitoxin and dc-saxitoxin in mussels HPLC method using pre-column derivatization with peroxide or periodate oxidation T
2、he European Standard EN 14526:2004 has the status of a British Standard ICS 67.120.30 Licensed Copy: Wang Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy, (c) BSIBS EN 14526:2004 This British Standard was published under the authority of the Standards Policy
3、 and Strategy Committee on 19 August 2004 BSI 19 August 2004 ISBN 0 580 44311 6 National foreword This British Standard is the official English language version of EN 14526:2004. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Horizontal analysis, which has the r
4、esponsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled
5、 “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance wi
6、th a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related interna
7、tional and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 17 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendment
8、s issued since publication Amd. No. Date Comments Licensed Copy: Wang Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy, (c) BSIEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN14526 August2004 ICS67.120.30 Englishversion FoodstuffsDeterminationofsaxitoxinandd
9、csaxitoxinin musselsHPLCmethodusingprecolumnderivatizationwith peroxideorperiodateoxidation ProduitsalimentairesDterminationdelateneuren saxitoxineetendcsaxitoxinedanslesmoulesMthode parCLHPavecdrivationprcolonneetparoxydationau peroxideouauperiodate LebensmittelBestimmungvonSaxitoxinundDCSaxitoxin
10、inMuschelnHPLCVerfahrenmit VorsulenderivatisierungmitPeroxidoder Periodatoxdation ThisEuropeanStandardwasapprovedbyCENon21May2004. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration
11、.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheCentralSecretariatortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoit
12、sownlanguageandnotifiedtotheCentralSecretariathasthesamestatusast heofficial versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France, Germany,Greece,Hungary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Polan
13、d,Portugal, Slovakia, Slovenia,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2004CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMem
14、bers. Ref.No.EN14526:2004:E Licensed Copy: Wang Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy, (c) BSIEN 14526:2004 (E) 2 Contents Page Foreword3 Introduction .4 1 Scope 5 2 Normative references 5 3 Principle5 4 Reagents.6 5 Apparatus .9 6 Procedure .11 7
15、HPLC determination 13 8 Calibration graph .13 9 Identification.14 10 Calculation14 11 Precision.14 12 Test report 15 Annex A (informative) Performance data16 Bibliography 17 Licensed Copy: Wang Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy, (c) BSIEN 14526
16、:2004 (E) 3 Foreword This document (EN 14526:2004) 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
17、 by endorsement, at the latest by February 2005, and conflicting national standards shall be withdrawn at the latest by February 2005. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Aus
18、tria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. WARNING The use of this standar
19、d 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 establish appropriate safety and health practices and determine the applicability of regula
20、tory limitations prior to use. Licensed Copy: Wang Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy, (c) BSIEN 14526:2004 (E) 4 Introduction This document is based on a procedure described by Lawrence and Mnard 1. In this original procedure, the C 18solid pha
21、se extraction is followed by a solid phase extraction with a weak cation exchanger. In the latter step, three different groups of shellfish toxins are being separated by elution with mobile phases of different ionic strength and polarity. The different groups of toxins are then derivatized with peri
22、odate or peroxide depending on the peak responses of the different toxins to the respective derivatization reactions. The method described in this document is specifically for saxitoxin and decarbamoyl (dc)-saxitoxin, so only the C 18clean up is applied. The method described in this document offers
23、the option to choose between an extraction with acetic acid (HAc) or an extraction with hydrochloric acid (HCl). The analysts choice depends on his desires and final needs. HCl extraction with boiling leads to partial hydrolysis of the PSP toxins, leading to conversion of some of the PSP toxins (C-1
24、, C-2, GTX-5) into more toxic analogues (GTX-2, GTX-3, saxitoxin). This process also occurs more or less in the human stomach. This extraction can therefore be considered a reasonable approach to estimate potential toxicity. HAc extraction without boiling is a milder extraction procedure, which leav
25、es the toxin profile of the sample practically intact. This extraction procedure can be applied if it is considered more important to estimate the actual toxin profile. The HAc extraction has been applied in the development of (certified) reference material for PSP toxins 2 and 3. Licensed Copy: Wan
26、g Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy, (c) BSIEN 14526:2004 (E) 5 1 Scope This document specifies a method for the quantitative determination of saxitoxin (STX) and decarbamoyl saxitoxin (dc-STX) in mussels. It may also be applicable in other she
27、llfish, for example scallops. The limit of determination of this method (signal/noise = 10) is 0,006 mg/kg for saxitoxin and 0,02 mg/kg for dc-saxitoxin in mussel meat. The method has been tested for saxitoxin at levels at 0,4 mg/kg and 0,5 mg/kg and for dc- saxitoxin at levels at 0,4 mg/kg and 1,6
28、mg/kg. 2 Normative references The following referenced documents are indispensable for the application of this document. 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
29、for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle Paralytic Shellfish Poisoning (PSP) toxins are extracted from mussel meat homogenate with acetic acid (HAc) or with hydrochloric acid (HCl). After centrifugation the supernatant is purified by solid phase extr
30、action (SPE) over a C 18clean-up cartridge. Part of the extract is derivatized with hydrogen peroxide. WARNING PSP toxins are strong neurotoxins. Gloves and safety glasses shall be worn at all times, and all standard and sample preparation stages shall be carried out in a fume cupboard. Derivatizati
31、on of STX and dc-STX leads to several oxidation products that are separated by reverse phase HPLC with fluorescence detection. Table 1 gives the reaction products for STX and dc-STX after derivatization with hydrogen peroxide. Table 1 Reaction products after derivatization with hydrogen perioxide To
32、xin Eluting order Intensity Peak name of reaction product STX first + dc-STX oxid. (1) second + dc-STX oxid. (2) third + STX oxid. dc-STX first + dc-STX oxid. (1) second + dc-STX oxid. (2) The method described in this document is suitable for the quantitative determination of the PSP toxins STX and
33、dc-STX. The following should however be considered. Co-occurrence of different PSP toxins in mussels could influence the analytical results, because some of the PSP toxins could (partially) lead to the same reaction products. So the chromatograms shall be carefully interpreted. The analytical result
34、 obtained for STX with hydrogen peroxide oxidation is not influenced by the presence of other toxins. Licensed Copy: Wang Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy, (c) BSIEN 14526:2004 (E) 6 The analytical result obtained for dc-STX with hydrogen pero
35、xide oxidation is slightly influenced by the presence of STX, because the dc-STX oxid. (1) reaction product of STX is the same as the main reaction product of dc-STX (see Table 1). The contribution of STX to the dc-STX peak is approximately 4 % of the peak area of the STX peak 4 and 5. If oxidised w
36、ith peroxide, the gonyautoxins GTX-2 and GTX-3 lead to the same reaction product, whereas GTX-1 and GTX-4 can not be detected. If oxidised with periodate, GTX-1, GTX-2, GTX-3 and GTX-4 lead to the same reaction products. GTX-5 can be detected with peroxide oxidation, neo-STX only with periodate oxid
37、ation. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only water according to grade 1 of EN ISO 3696. All chemicals shall be of pro analysis (p.a.) quality, unless otherwise indicated. Reference materials (calibrants of the toxins) originating from other sources than indica
38、ted may also be used if well-characterised and with a well-defined mass concentration. 4.2 Methanol 4.3 Acetonitrile, HPLC quality 4.4 Ammonium formate 4.4.1 Ammonium formate solution 1, substance concentration c = 0,3 mol/l Dissolve 18,9 g ammonium formate (4.4) in water and make up to 1 l with wat
39、er. 4.4.2 Ammonium formate solution 2, c = 0,1 mol/l Dilute 350 ml of ammonium formate solution 1 (4.4.1) with 700 ml of water. Adjust to pH 6 with acetic acid (pH paper) and filter over a membrane filter (5.15). Prepare fresh every 2 days of analysis. 4.5 Hydrochloric acid solution 4.5.1 General vo
40、lume fraction 25 % (acidimetric) 4.5.2 Hydrochloric acid solution 1, c = 5 mol/l Dilute 100 ml of hydrochloric acid solution (4.5) with 54 ml of water. 4.5.3 Hydrochloric acid solution 2, c = 0,1 mol/l Dilute 5 ml of hydrochloric acid solution 1 (4.5.2) with 245 ml of water. 4.6 Sodium hydroxide 4.6
41、.1 Sodium hydroxide solution 1, c = 5 mol/l Dissolve 20 g of sodium hydroxide (4.6) in 100 ml of water. Licensed Copy: Wang Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy, (c) BSIEN 14526:2004 (E) 7 4.6.2 Sodium hydroxide solution 2, c = 1 mol/l Dilute 1 ml
42、 of sodium hydroxide solution 1 (4.6.1) with 4 ml of water. 4.6.3 Sodium hydroxide solution 3, c = 0,1 mol/l Dilute 1 ml of sodium hydroxide solution 2 (4.6.2) with 9 ml of water. 4.7 Hydrogen peroxide solution, volume fraction = 10 % Dilute 1 ml of commercially available hydrogen peroxide solution,
43、 of mass fraction = 30 % with 2 ml of water. Store cool and in the dark. Prepare fresh every week. 4.8 Periodic acid solution, c = 0,03 mol/l Dissolve 0,68 g of periodic acid in 100 ml of water. 4.9 Disodium hydrogenphosphate solution, c = 0,3 mol/l Dissolve 0,53 g of disodium hydrogenphosphate dihy
44、drate in 10 ml of water. 4.10 Acetic acid, 100 % 4.10.1 Acetic acid solution 1, c = 1 mol/l Dilute 57,2 ml of acetic acid solution (4.10) to 1,0 l with water. 4.10.2 Acetic acid solution 2, c = 0,1 mol/l Dilute 10 ml of acetic acid solution 1 (4.10.1) with 90 ml of water. 4.10.3 Acetic acid solution
45、 3, c = 0,2 mol/l Dilute 200 ml of acetic acid solution 1 (4.10.1) with 800 ml of water. 4.10.4 Acetic acid solution 4, c = 0,03 mol/l Dilute 300 ml of acetic acid solution 2 (4.10.2) with 700 ml of water. 4.11 Helium 4.12 Peroxide derivatization reagent Dilute 400 l of hydrogen peroxide solution (4
46、.7) with 4 ml of sodium hydroxide solution 2 (4.6.2) and mix well. Prepare fresh every day of analysis. 4.13 Periodate derivatization reagent Mix 1 volume part of periodic acid solution (4.8) with 1 volume part of disodium hydrogenphosphate solution (4.9) and 1 volume part of ammonium formate soluti
47、on 1 (4.4.1). Bring the mixture to about pH 8 by dropwise adding sodium hydroxide solution 2 (4.6.2) and check the pH by using pH paper. Prepare fresh every day of analysis just before use. Licensed Copy: Wang Bin, ISO/Exchange China Standards Information Centre, 10 November 2004, Uncontrolled Copy,
48、 (c) BSIEN 14526:2004 (E) 8 4.14 Saxitoxin standard substance 4.14.1 Saxitoxin stock solution, mass concentration = 1,0 g/ml Prepare a stock solution containing 1,0 g/ml of saxitoxin in acetic acid solution 4 (4.10.4) by weighing with the analytical balance (5.3). Store the solution cool (approximately + 4 C) and in the dark. This solution is stable for at least 5 months (see 6). NOTE Ampoules containing saxitoxin (STX2HCl) standard are for example available from the National Research Council Canada, H
