1、BRITISH STANDARD Foodstuffs Determination of fumonisins B 1 and B2 in maize HPLC method with solid phase extraction clean-up The European Standard EN 13585:2001 has the status ofa British Standard ICs 67.060 BS EN 1 3 585 :2002 Wk present to the responsible European committee any enquiries on the in
2、terpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. - A list of organizations represented on this committee can be obtained on request to its secretary. Cr oss-r e fer enc e s The British St
3、andards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Stan
4、dard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. This British Standard. having - been prepared under the
5、 direction of the Consumer Summary of pages This document comprises a front cover, an inside front cover, the EN title page, Products and Services Sector Policyand Strategycommittee, was published under the authority of the Standards Policy and Strategy Committee on 04 March 2002 pages 2 to 14, and
6、an inside back cover and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. O BSI 04 March 2002 ISBN O 580 39204X EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 13585 December 2001 ICs 67.060 English version Foodstuffs - Determination o
7、f fumonisins BI and B2 in maize - HPLC method with solid phase extraction clean-up Produits alimentaires - Dosage des fumonisines BI et 82 dans le mas - Mthode CLHP avec purification par extraction en phase solide Lebensmittel - Bestimmung von Fumonisin BI und 82 in Mais - HPLC-Verfahren mit Reinigu
8、ng durch Festphasenextraktion This European Standard was approved by CEN on 2 November 2001 CEN members are bound to comply with the CENKENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date l
9、ists and bibliographical references concerning such national standards may be obtained on application to the 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 respo
10、nsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Nethe
11、rlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION EUROPISCHES KOMITEE FR NORMUNG COMIT EUROPEN DE NORMALISATION Management Centre: rue de Stassart, 36 B-1050 Brussels O 2001 CEN All rights of exploitation in any form and by any means rese
12、rved worldwide for CEN national Members. Ref. No. EN 13585:2001 E EN 13585:2001 (E) Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagents and materials . 3 5 Apparat us . 4 6 7 Preparation of the test sample . 5 8 Procedure . 6 9 Calculation 6 10 Precision . 7 11 Test
13、report 8 Annex A (informative) Typical chromatogram 10 Annex B (informative) Recovery and relative standard deviation 11 Annex C (informative) Precision data 12 Bibliography 15 Sampling . 5 Foreword This European Standard has been prepared by Technical Committee CEN /TC 275. “Food analysis . Horizon
14、tal 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 June 2002. and conflicting national standards shall be withdrawn at the latest by June 2002
15、 The annexes A. B and C are informative . According to the CENKENELEC Internal Regulations. the national standards organizations of the following countries are bound to implement this European Standard: Austria. Belgium. Czech Republic. Denmark. Finland. France. Germany. Greece. Iceland. Ireland. I
16、taly. Luxembourg. Netherlands. Norway. Portugal. Spain. Sweden. Switzerland and the United Kingdom . 2 EN 13585:2001 (E) 1 Scope This European Standard specifies a method for the determination of fumonisin BI (FBI) and fumonisin B2 (FB2) in maize using high performance liquid chromatography (HPLC).
17、The method has been successfully validated in an interlaboratory study according to AOAC Guidelines for Collaborative Studies I on maize containing 405 pg/kg to 6732 pg/kg fumonisin BI and 152 pg/kg to 2619 pg/kg fumonisin B2. The method works well with maize or minimally processed maize (e.g. fresh
18、 dried and milled maize), but does not provide reliable results with most maize-based processed products. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in t
19、he text, and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to
20、 applies (including amendments). EN IS0 3696 Water for analytical laboratory use - Specification and test methods (IS0 3696: 1987). 3 Principle Fumonisins are extracted from the sample of maize with a mixture of methanol and water. The filtered extract is purified on a strong-anion-exchange (SAX) so
21、lid-phase extraction (SPE) cartridge, and the fumonisins are eluted with a mixture of acetic acid and methanol. The extract is evaporated and the residue is redissolved in methanol and o-phthaldialdehyde/2-mercaptoethanol (OPAIMCE) is added to form fluorescent fumonisin derivatives. The derivatives
22、are analysed by reverse-phase high performance liquid chromatography (HPLC) with fluorescence detection. WARNING - Fumonisins are hepatoxic, nephrotoxic and carcinogenic to rats and mice. Effects on humans are not fully known. Observe appropriate safety precautions for handling fumonisins. Any labor
23、atory spills should be washed with a 5 % solution of sodium hypochlorite. 4 Reagents and materials 4.1 General During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilled water or water of grade 1 as defined in EN IS0 3696. Solvent shall be of qu
24、ality for HPLC analysis. 4.2 Methanol, abs. 4.3 Methanol solution, volume fraction dCH30H) = 75 % Mix 75 parts per volume of methanol (4.2) with 25 parts per volume of water 4.4 Acetonitrile solution, dCH3CN) = 50 % Mix 50 parts per volume of acetonitrile with 50 parts per volume of water. 4.5 o-pho
25、sphoric acid, dH3P04) = 85 % 4.6 Acetic acid-methanol solution, dCH3COOH) = 1 % for eluting the SPE column. Mix 1 part per volume of glacial acetic acid with 99 parts per volume of methanol (4.2). 3 EN 13585:2001 (E) 4.7 o-phthaldialdehyde (OPA) 4.8 2-mercaptoethanol (MCE) 4.9 Sodium dihydrogen phos
26、phate solution, substance concentration c(NaH2P04.2H20) = 0,l mol/l Dissolve 15,6 g of NaH2P04.2H20 in 1 I of water. 4.10 Disodium tetraborate solution, c(Na2B407.10 H20) = 0,l mol/l Dissolve 3,8 g of Na2B407.1 OH20 in 1 O0 ml of water. 4.1 1 Sodium hydroxide solution, c(Na0H) = 0,l mol/l Dissolve 0
27、4 g of NaOH in 100 ml of water. 4.12 Mobile phase Mix 77 volume parts of methanol (4.2) with 23 volume parts of sodium dihydrogen phosphate solution (4.9). Adjust to pH 3,35 with o-phosphoric acid (4.5). Filter the solution through a 0,45 pm membrane (5.7) filter. The mobile phase composition may h
28、ave to be adjusted to conform with individual HPLC column characteristics. 4.1 3 Derivatization reagent Dissolve 40 mg of OPA (4.7) in 1 ml of methanol (4.2) and dilute with 5 ml of disodium tetraborate solution (4.10). Add 50 pl of MCE (4.8) and mix. The reagent solution is stable for up to one wee
29、k at room temperature in a dark and capped amber vial. 4.14 FBI and FB2 standard solution Prepare individual stock solutions of FB, and FB2 at mass concentrations of 250 pg/ml in acetonitrile solution (4.4). Commercially available standard solutions may be used. Transfer 100 pl aliquots of each stoc
30、k solution to a glass vial and add 300 pl of acetonitrile solution to yield 500 pl of a standard solution containing both fumonisins at individual mass concentration of 50 pg/mI. If a calibration curve is made, mix different aliquots of standard solutions (.e. 20 pI, 50 pI, 100 pl and 200 pi) of ind
31、ividual fumonisins and dilute to 500 pl with acetonitrile solution to obtain the relevant calibration solutions. Fumonisin standard solutions are stable up to at least six months when stored at approximately 4 C. 5 Apparatus Usual laboratory equipment and, in particular, the following: 5.1 HPLC appa
32、ratus, comprising the following 5.1.1 equipped with an injection system capable to deliver e.g. 10 pI. High performance liquid chromatograph, isocratic pump set to deliver e.g. 1 ml/min constant flow rate, 5.1.2 Analytical reverse-phase separating column, e.g. octyldecylsilane (ODs), which ensures a
33、 baseline resolution of the fumonisin peaks from all other peaks, with the following characteristics: - stainless steel; 4 EN 13585:2001 (E) - a length of 150 mm; - an inner diameter of 4,6 mm; - a stationary phase with particle size of 5 pm; - a suitable corresponding reverse-phase guard column Col
34、umns of other dimensions can also be used. 5.1.3 wavelength of h = 440 nm. Fluorescence detector with the capability of using excitation wavelength of h = 335 nm and emission 5.1.4 Data system 5.2 Blender, homogenizer, or mixer. 5.3 Fluted filter paper 5.4 Strong-anion-exchanging solid phase extract
35、ion column, e.g. Bond-Elut SAX-cartridges, containing 500 mg of sorbent, or similar has been found to be suitable. 5.5 SPE extraction manifold 5.6 Solvent evaporator, with heating module, or similar. 5.7 Membrane filter, for aqueous solutions, with a pore size of 0,45 pm. 6 Sampling It is important
36、that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport or storage. 7 Grind the sample to pass through a 1 ,O mm sieve and homogenize the sample. Preparation of the test sample 8 Procedure 8.1 Extraction Place 50 g of the test sample i
37、nto a suitable glass or plastic container (e.g. a 250 ml plastic centrifuge bottle). Extract for 3 min with 100 ml of methanol solution (4.3) using the blender (5.2) at approximately 10 O00 min-. The time needed for complete extraction can vary with the type of equipment used. Centrifuge the blended
38、 extract for 10 min at 500 g and filter the supernatant through a fluted filter paper (5.3). Check the pH of the eluate and adjust, if necessary, with sodium hydroxide solution (4.1 1) to between pH 5,8 and pH 6,5. Bond-Elut is an example of a suitable product available commercially. This informatio
39、n is given for the convenience of users 5 of this European Standard and does not constitute an endorsement by CEN of this product. EN 13585:2001 (E) 8.2 Clean up Attach the SPE cartridge (5.4) to the SPE manifold (5.5) and condition by washing successively with 5 ml of methanol (4.2) and then with 5
40、 ml of methanol solution (4.3). While maintaining a flow rate of no more than 2 ml/min, apply a 10 ml aliquot of the filtered sample extract to the SPE cartridge. Wash the SPE cartridge with 8 ml of methanol-water solution (4.3), followed by 3 ml of methanol (4.2). Do not allow the cartridge to run
41、dry. Discard the washings. Elute the fumonisins with 10 ml of methanolic acetic acid 1 % (4.6) at a flow rate not more than 1 ml/min. Collect the eluate in a suitable vial. Transfer the eluate in the collection vial to a suitable vial and evaporate the eluate to dryness by using the solvent evaporat
42、or (5.6) under nitrogen at approximately 60 OC. Wash the collection vial with 1 ml of methanol (4.2) and add the washing to the suitable vial. Evaporate the additional methanol to dryness under nitrogen, ensure that all the acetic acid has evapored, and cap the vial. Retain the dried residue at appr
43、oximately 4 OC until HPLC analysis, that should be performed within two weeks. 8.3 Derivatization and determination 8.3.1 Standard derivative solution Transfer 25 pl of the fumonisin standard solution (4.14) to the base of a small test tube. Add 225 pl derivatization reagent (4.13), mix the solution
44、s vigorously, and inject an aliquot (e.g. 10 pl = 0,050 pg FB, and FB2) of the derivatized solution onto the HPLC column (5.1.2) at a reproducible time within 3 min after addition of the derivatization reagent. If a single point calibration is used, adjust the sensitivity of the fluorescence detecto
45、r (5.1.3) to at least an 80 % recorder response. 8.3.2 Maize test solution Redissolve the dried residue (see 8.2) in 200 pl of methanol (4.2). Acetonitrile solution (4.4) may also be used. Transfer 25 pl of this solution to the base of a small test tube and add 225 pl of the derivatization reagent (
46、4.13). Mix the solutions and inject an aliquot, e.g. 10 pl of the derivatized solution onto the HPLC column (5.1.2) at a reproducible time within 3 min after addition of the derivatization reagent (4.1 3). If fumonisin chromatographic peaks exceed those of fumonisin standard solution or exceed the h
47、ighest point of the calibration curve (4.14), make additional dilutions of the sample extracts with methanol (4.2) and repeat derivatization. NOTE 1 It is critical to adhere to reproducible times between the addition of the reagent (4.13) and the injection onto the HPLC column, because of progressiv
48、e decay in fluorescence of the fumonisin-derivatives that occurs after periods in excess to 4 min. NOTE 2 Limits of detection and quantification vary considerably according to the sensitivity of the detector used. Limits of detection (signal/noise = 3) as low as 5 pg/kg can be reached with the new g
49、eneration of fluorometric detector. NOTE 3 A typical chromatogram is given in annex A. 9 Calculation Calculate the mass of each fumonisin m, in micrograms, injected onto the HPLC column using equation (1): where pt is the fumonisin peak of the test sample, in units of height or area; f, is the fumonisin peak of the standard solution, in units of height or area; 6 EN 13585:2001 (E) m, is the mass of each fumonisin FBI standard in micrograms, injected onto the HPLC column, based on the concentration in the fumonisin standard solution (here: 0,050 pg of each fumonisin); Calcul
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