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1、BRITISH STANDARD BS EN 14524:2004 Foodstuffs Determination of okadaic acid in mussels HPLC method with solid phase extraction clean-up, derivatization and fluorimetric detection The European Standard EN 14524:2004 has the status of a British Standard ICS 67.120.30 BS EN 14524:2004 This British Stand
2、ard was published under the authority of the Standards Policy and Strategy Committee on 18 August 2004 BSI 18 August 2004 ISBN 0 580 44310 8 National foreword This British Standard is the official English language version of EN 14524:2004. The UK participation in its preparation was entrusted to Tec
3、hnical Committee AW/-/3, Horizontal analysis, which has the responsibility 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
4、 may be found in the BSI Catalogue under the section entitled “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. Use
5、rs are responsible for its correct application. Compliance with 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
6、, and keep the UK interests informed; monitor related international 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 15 and a back cover. The BSI copyright notice displayed in this do
7、cument indicates when the document was last issued. Amendments issued since publication Amd. No. DateComments EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 14524 August 2004 ICS 67.120.30 English version Foodstuffs - Determination of okadaic acid in mussels - HPLC method with solid phase extr
8、action clean-up, derivatization and fluorimetric detection Produits alimentaires - Dosage de lacide okadaque dans les moules - Mthode par CLHP avec purification par extraction sur phase solide, drivation et dtection fluorimtrique Lebensmittel - Bestimmung von Okadasure in Muscheln - HPLC-Verfahren m
9、it Reinigung durch Festphasenextraktion, Derivatisierung und fluorimetrischer Bestimmung This European Standard was approved by CEN on 21 May 2004. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of
10、 a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A versio
11、n in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia
12、, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMI
13、TEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2004 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14524:2004: E EN 14524:2004 (E) 2 Contents Page Foreword3 1 Scope 4 2 Normative references4 3 Principle4 4 R
14、eagents and materials 4 5 Apparatus .6 6 Sampling.7 7 Procedure .7 8 Precision.10 9 Test report 11 Annex A (informative) Typical chromatogram12 Annex B (informative) Precision data14 Bibliography15 EN 14524:2004 (E) 3 Foreword This document (EN 14524:2004) has been prepared by Technical Committee CE
15、N/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 latest by February 2005, and conflicting national standards shall be w
16、ithdrawn 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: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hun
17、gary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. WARNING The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to
18、 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 regulatory limitations prior to use. EN 14524:2004 (E) 4 1 Scope This document specifies a method fo
19、r the quantitative determination of the content of okadaic acid in mussels and mussel products. The content of okadaic acid is determined as free extractable acid of mussel hepatopancreas. Okadaic acid, a fat-soluble toxin from dinophysis algae, is a main component of dinophysis toxins. The method h
20、as been validated in an interlaboratory study according to ISO general principles on assessing accuracy of measurement methods and results. The limit of determination of this method (signal/noise = 10) is 100 g/kg for okadaic acid in mussel hepatopancreas. The method has been validated for okadaic a
21、cid in cooked mussels at levels of 441 g/kg to 1 467 g/kg. Laboratory experiences have shown that this method can also be used to determine other dinophysis toxins, e.g. dinophysis toxins 1, 2 and 3 (DTX-1, DTX-2 and DTX-3), see 1, 2, 3, 4, 5 and 6 2 Normative references The following referenced doc
22、uments 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 for analytical laboratory use - Specification and test met
23、hods (ISO 3696:1987). 3 Principle Mussel hepatopancreas is separated from cooked mussels and homogenized. The toxins are extracted using methanol, derivatized with 9-anthryldiazomethane and the extract is cleaned up using a solid phase extraction (SPE) cartridge with silica gel. Chromatographic sepa
24、ration is performed on a HPLC-gradient system, followed by fluorescence measurement of the 9-anthryldiazomethyl ester of the toxin at 412 nm with excitation at 365 nm. Determination of okadaic acid is performed using external standards. 4 Reagents and materials 4.1 General During the analysis, unles
25、s otherwise stated, use only reagents of recognized analytical grade and water according to grade 1 of EN ISO 3696. EN 14524:2004 (E) 5 4.2 Sodium sulfate, anhydrous 4.3 Nitrogen gas, volume fraction 99,999 % 4.4 Acetone 4.5 Acetonitrile 4.6 Chloroform, stabilised with 2-methyl-2-butene 4.7 Ethyl ac
26、etate 4.8 Dichloromethane, stabilised with 2-methyl-2-butene 4.9 Methanol 4.10 Methanol solution, = 80 % 4.11 n-hexane 4.12 9-anthryldiazomethane (ADAM) Weigh solid ADAM in discrete portions (e.g. 1 mg), store at -18 C and dissolve in ethyl acetate just before use. 4.13 Derivatization solution, mass
27、 concentration = 1,5 g/l in ethyl acetate (4.7) 4.14 Solvent mixture of n-hexane and dichloromethane Mix 1 part per volume of n-hexane (4.11) with 1 part per volume of dichloromethane (4.8). 4.15 Solvent mixture of dichloromethane and acetone Mix 9 parts per volume of dichloromethane (4.8) with 1 pa
28、rt per volume of acetone (4.4). 4.16 Solvent mixture of acetonitrile and dichloromethane Mix 5 parts per volume of acetonitrile (4.5) with 1 part per volume of dichloromethane (4.8). 4.17 Mixture of methanol and ethyl acetate Mix 1 part per volume of methanol (4.9) with 1 part per volume of ethyl ac
29、etate (4.7). 4.18 HPLC mobile phase solvent A Mix 800 ml of methanol (4.9) with 200 ml of ethyl acetate (4.7). 4.19 HPLC mobile phase solvent B Mix 700 ml of acetonitrile (4.5) with 300 ml of water. EN 14524:2004 (E) 6 4.20 Okadaic acid WARNING Okadaic acid is toxic. Gloves and safety glasses shall
30、be worn at all times, and all standard and sample preparation stages shall be carried out in a fume cupboard. 4.21 Okadaic acid stock solution in methanol Dilute okadaic acid with methanol (4.9) to a concentration of 1,0 g/ml. This solution can be stored at -18 C for at least 6 months. 4.22 Okadaic
31、acid methylanthrylester NOTE For the availability of this substance, contact your National Standardization Institute. It can for example be obtained from SIGMA-ALDRICH 1). 4.23 Solution of okadaic acid methylanthrylester in methanol Dilute okadaic acid methylanthrylester with methanol (4.9) to a con
32、centration of 0,1 g/ml. This corresponds to 1 600 g/kg mussel hepatopancreas (in samples). Store the solution in a brown bottle. At -18 C the solution is stable for at least 6 months. 4.24 Mussels free of okadaic acid and related compounds 5 Apparatus 5.1 General Usual laboratory apparatus, and in p
33、articular: 5.2 Instrument for separation of hepatopancreas, e.g. scalpel 5.3 Blender or homogenizer 5.4 Centrifuge, capable of a centrifugal force up to 3 000 g, with suitable tubes, e.g. 20 ml 5.5 Pear shaped flask, e.g. 10 ml and 25 ml capacity 5.6 Glass sample tubes with stoppers, e.g. 10 ml capa
34、city 5.7 One-mark volumetric flask, 20 ml capacity 5.8 Analytical balance, accurate to the nearest 0,1 mg 5.9 Solid phase extraction (SPE) cartridges, (3 ml) filled with 650 mg silica gel with solvent reservoir (approx. 10 ml), commercially available 5.10 Suitable pipettes of various volumina 1) Thi
35、s information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN. Equivalent products may be used if they can be shown to lead to the same results. EN 14524:2004 (E) 7 5.11 Apparatus for solvent removal using nitrogen gas 5.12 HPLC apparatus
36、, comprising the following 5.12.1 HPLC pump, for gradient elution at e.g. 0,8 ml/min constant flow rate 5.12.2 Injection system, capable to deliver an injection volume of e.g. 20 l 5.12.3 Analytical reverse-phase separating column which ensures a baseline resolution of the peaks of the derivatives f
37、rom all other peaks, for example: RP-C18 a length of 250 mm; an inner diameter of 4 mm; a stationary phase with particle size of 5 m. Columns of other dimensions may also be used. 5.12.4 Fluorescence detector, fitted with a flow cell and set at 365 nm (excitation) and 412 nm (emission). Specific pro
38、perties of the detector shall be regarded. 5.12.5 Data system 5.13 Rotary evaporator, with evaporation flask and water bath 5.14 Laboratory shaker for tubes 5.15 Ultrasonic bath 5.16 Glass funnel, with paper filter, 90 mm diameter 6 Sampling It is important that the laboratory receives a sample whic
39、h is truly representative and has not been damaged or changed during transport or storage. Samples shall be deep frozen until use or shall be extracted immediately. NOTE Storage of mussel samples in frozen state at 18 C has no negative influence on the result of this method. 7 Procedure 7.1 Preparat
40、ion of test sample Transfer fresh mussels into boiling water and cook for 10 min. Separate 20 g to 30 g of hepatopancreas material from approximately 1 kg of mussels (including the shells). The hepatopancreas is the green or brown part of the mussel. If only smaller sample amounts of hepatopancreas
41、material are available, the whole material has to be prepared. Homogenize the hepatopancreas material with the homogenizer or blender (5.3) and start immediately with the preparation or otherwise store the homogenate at 18 C. EN 14524:2004 (E) 8 7.2 Extraction procedure Transfer 1,00 g 0,02 g of hom
42、ogenate to a centrifuge tube (5.4) and stir with 9 ml of the methanol solution (4.10) using the homogenizer (5.3). Centrifuge the mixture at approximately 3 000 g for 5 min. Transfer the supernatant to a one-mark volumetric flask (5.7). For a second extraction, add 9 ml of the methanol solution (4.1
43、0), stir and centrifuge the mixture and transfer the resulting supernatant to the volumetric flask (5.7). Fill the flask to the calibration mark with the methanol solution (4.10). 7.3 Removal of fat Pipette 5,0 ml of the sample extract as prepared in 7.2 into a centrifuge tube (5.4) and mix with 4 m
44、l of n- hexane (4.11). Centrifuge the mixture at approximately 3 000 g for 5 min. Remove and discard the hexane layer at the top. Add another 4 ml of n-hexane (4.11), mix and centrifuge as described. Remove and discard the hexane layer at the top. Repeat this procedure a third time. Add 2 ml of wate
45、r and 4 ml of chloroform (4.6) to the remaining water layer. Stir the mixture and centrifuge at 3 000 g for 5 min. Remove the chloroform layer at the bottom and transfer it to a conical flask. Again add 4 ml of chloroform, stir and centrifuge as described above. Remove the chloroform layer and add i
46、t to the chloroform of the first extraction. Dry the chloroform by adding approximately 1 g of sodium sulfate (4.2). Wait for 30 min and filter the chloroform using a glass funnel with paper filter (5.16). Collect the filtrate into a pear shaped flask (5.5). Wash the conical flask and the filter wit
47、h additional 6 ml of chloroform. Remove the chloroform with a rotary evaporator (5.13) at approximately 40 C under 250 hPa to dryness. Dissolve the residue in 10,0 ml of methanol (4.9). This solution can be stored in a refrigerator for at least 24 h at 4 oC to 8 oC. 7.4 Derivatization with 9-anthryl
48、diazomethane (ADAM) Pipette 0,50 ml of the methanolic solution prepared in 7.3 into a sample tube (5.6). Remove the solvent using a gentle stream of nitrogen (4.3). Dissolve the residue in 50 l of derivatization solution (4.13). Treat the mixture in an ultrasonic bath (5.15) for 2 min or with a shak
49、er (5.14) for 1 min. Add 150 l of methanol (4.9) and mix again with ultrasonic bath or shaker. The derivatization reaction is finished after 60 min at room temperature in the dark. Remove the solvent using a gentle stream of nitrogen (4.3). NOTE Okadaic acid methylanthryl ester is decomposed by light and forms a substance which shows a peak with a retention time only a few minutes later than that of okadaic acid methylanthryl ester, if chromatographic conditions described in this document are used. Therefore, it is