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1、BRITISH STANDARD BS EN 13585:2002 Foodstuffs Determination of fumonisins B1 and B2 in maize HPLC method with solid phase extraction clean-up The European Standard EN 13585:2001 has the status of a British Standard ICS 67.060 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Lice
2、nsed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:58 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS EN 13585:2002 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Policy and Strategy Committee, was published under the authority of the
3、 Standards Policy and Strategy Committee on 04 March 2002 BSI 04 March 2002 ISBN 0 580 39204 X National foreword This British Standard is the official English language version of EN 13585:2001. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizon
4、tal methods, 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 may be found in the BSI Standard
5、s Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for
6、their 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 European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informe
7、d; 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 14, and an inside back cover and a back cover. The BSI copyright date displayed in this document indi
8、cates when the document was last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:58 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 13585 December 2001 ICS 67.060 English ve
9、rsion Foodstuffs - Determination of fumonisins B1 and B2 in maize - HPLC method with solid phase extraction clean-up Produits alimentaires - Dosage des fumonisines B1 et B2 dans le mas - Mthode CLHP avec purification par extraction en phase solide Lebensmittel - Bestimmung von Fumonisin B1 und B2 in
10、 Mais - HPLC-Verfahren mit Reinigung durch Festphasenextraktion This European Standard was approved by CEN on 2 November 2001. 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
11、without any alteration. Up-to-date lists 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
12、 made by translation under the responsibility 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, Icelan
13、d, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2001 CENAll rights of exploitation
14、in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 13585:2001 E Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:58 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13585:2001 (E) 2 Contents page Foreword2 1Scope 3 2Normative references 3 3Principle3 4Reag
15、ents and materials.3 5Apparatus .4 6Sampling.5 7Preparation of the test sample .5 8Procedure .6 9Calculation6 10Precision.7 11Test report 8 Annex A (informative) Typical chromatogram10 Annex B (informative) Recovery and relative standard deviation11 Annex C (informative) Precision data12 Bibliograph
16、y15 Foreword This European Standard 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 endorseme
17、nt, at the latest by June 2002, and conflicting national standards shall be withdrawn at the latest by June 2002. The annexes A, B and C are informative. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this Eur
18、opean Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:58 GMT+00:00 2006, Uncontro
19、lled Copy, (c) BSI EN 13585:2001 (E) 3 1 Scope This European Standard specifies a method for the determination of fumonisin B1 (FB1) and fumonisin B2 (FB2) in maize using high performance liquid chromatography (HPLC). The method has been successfully validated in an interlaboratory study according t
20、o AOAC Guidelines for Collaborative Studies 1 on maize containing 405 g/kg to 6732 ?g/kg fumonisin B1 and 152 ?g/kg to 2619 ?g/kg fumonisin B2. The method works well with maize or minimally processed maize (e.g. fresh, dried and milled maize), but does not provide reliable results with most maize-ba
21、sed 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 the text, and the publications are listed hereafter. For dated references, subsequen
22、t 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 applies (including amendments). EN ISO 3696Water for analytical laboratory use - S
23、pecification and test methods (ISO 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) solid-phase extraction (SPE) cartridge, and the fumonisins are eluted with a mixture of
24、 acetic acid and methanol. The extract is evaporated and the residue is redissolved in methanol and o-phthaldialdehyde/2-mercaptoethanol (OPA/MCE) is added to form fluorescent fumonisin derivatives. The derivatives are analysed by reverse-phase high performance liquid chromatography (HPLC) with fluo
25、rescence 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 laboratory spills should be washed with a 5 % solution of sodium hypochlorite. 4 Reagents
26、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 ISO 3696. Solvent shall be of quality for HPLC analysis. 4.2 Methanol, abs. 4.3 Methanol solution, volume fraction ?(
27、CH3OH) = 75 % Mix 75 parts per volume of methanol (4.2) with 25 parts per volume of water. 4.4 Acetonitrile solution, ?(CH3CN) = 50 % Mix 50 parts per volume of acetonitrile with 50 parts per volume of water. 4.5 o-phosphoric acid, ?(H3PO4) = 85 % 4.6 Acetic acid-methanol solution, ?(CH3COOH) = 1 %
28、for eluting the SPE column. Mix 1 part per volume of glacial acetic acid with 99 parts per volume of methanol (4.2). Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:58 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13585:2001 (E) 4 4.7 o-phthaldialdehyde (OPA) 4.8 2-mercaptoethanol (MCE)
29、 4.9 Sodium dihydrogen phosphate solution, substance concentration c(NaH2PO42H2O) = 0,1 mol/l Dissolve 15,6 g of NaH2PO42H2O in 1 l of water. 4.10 Disodium tetraborate solution, c(Na2B4O710 H2O) = 0,1 mol/l Dissolve 3,8 g of Na2B4O710H2O in 100 ml of water. 4.11 Sodium hydroxide solution, c(NaOH) =
30、0,1 mol/l Dissolve 0,4 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 ?m membrane (5.7) filter. The mobile pha
31、se composition may have to be adjusted to conform with individual HPLC column characteristics. 4.13 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 ?l of MCE (4.8) and mix. The reagent solution is stabl
32、e for up to one week at room temperature in a dark and capped amber vial. 4.14 FB1 and FB2 standard solution Prepare individual stock solutions of FB1 and FB2 at mass concentrations of 250 g/ml in acetonitrile solution (4.4). Commercially available standard solutions may be used. Transfer 100 ?l ali
33、quots of each stock solution to a glass vial and add 300 ?l of acetonitrile solution to yield 500 ?l of a standard solution containing both fumonisins at individual mass concentration of 50 ?g/ml. If a calibration curve is made, mix different aliquots of standard solutions (i.e. 20 ?l, 50 ?l, 100 ?l
34、 and 200 ?l) of individual fumonisins and dilute to 500 ?l 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 follo
35、wing: 5.1 HPLC apparatus, comprising the following 5.1.1 High performance liquid chromatograph, isocratic pump set to deliver e.g. 1 ml/min constant flow rate, equipped with an injection system capable to deliver e.g. 10 ?l. 5.1.2 Analytical reverse-phase separating column, e.g. octyldecylsilane (OD
36、S), which ensures a baseline resolution of the fumonisin peaks from all other peaks, with the following characteristics: ? stainless steel; Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:58 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13585:2001 (E) 5 ? a length of 150 mm; ? an inner
37、diameter of 4,6 mm; ? a stationary phase with particle size of 5 ?m; ? a suitable corresponding reverse-phase guard column. Columns of other dimensions can also be used. 5.1.3 Fluorescence detector with the capability of using excitation wavelength of ? = 335 nm and emission wavelength of ? = 440 nm
38、. 5.1.4 Data system 5.2 Blender, homogenizer, or mixer. 5.3 Fluted filter paper 5.4 Strong-anion-exchanging solid phase extraction column, e.g. Bond-Elut ? 1) SAX-cartridges, containing 500 mg of sorbent, or similar has been found to be suitable. 5.5 SPE extraction manifold 5.6 Solvent evaporator, w
39、ith heating module, or similar. 5.7 Membrane filter, for aqueous solutions, with a pore size of 0,45 ?m. 6 Sampling It is important that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport or storage. 7 Preparation of the test sample Gr
40、ind the sample to pass through a 1,0 mm sieve and homogenize the sample. 8 Procedure 8.1 Extraction Place 50 g of the test sample into 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
41、approximately 10 000 min-1. The time needed for complete extraction can vary with the type of equipment used. Centrifuge the blended 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
42、solution (4.11) to between pH 5,8 and pH 6,5. 1 Bond-Elut? is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. Licensed Copy: sheffieldun sheffieldu
43、n, na, Mon Oct 30 01:44:58 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13585:2001 (E) 6 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 ml of methanol solution (4.3). While maintaining a flow ra
44、te 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 dry. Discard the washings. Elute the fumonisins with 10 ml
45、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 evaporator (5.6) under nitrogen at approximately 60 C. Wash the col
46、lection 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 approximately 4 C until HPLC analysis, that should be performed
47、within two weeks. 8.3 Derivatization and determination 8.3.1 Standard derivative solution Transfer 25 ?l of the fumonisin standard solution (4.14) to the base of a small test tube. Add 225 ?l derivatization reagent (4.13), mix the solutions vigorously, and inject an aliquot (e.g. 10 ?l = 0,050 g FB1
48、 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 detector (5.1.3) to at least an 80 % recorder response. 8.3.2 Maize t
49、est solution Redissolve the dried residue (see 8.2) in 200 ?l of methanol (4.2). Acetonitrile solution (4.4) may also be used. Transfer 25 ?l of this solution to the base of a small test tube and add 225 ?l of the derivatization reagent (4.13). Mix the solutions and inject an aliquot, e.g. 10 ?l of the derivatized solution onto the HPLC column (5.1.2) at a reproducible time within 3 min after addition of the derivatization reagent (4.13). If fumonisin chromatographic peaks exceed those of fumonisin standard solution or exceed the highest point of the calibrati