SIST EN 13585:2002

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Fumonisin B1 und B2 in Mais - HPLC-Verfahren mit Reinigung durch FestphasenextraktionProduits alimentaires - Dosage des fumonisines B1 et B2 dans le mais - Méthode CLHP avec purification par extraction en phase solideFoodstuffs - Determination of fumonisins B1 and B2 in maize - HPLC method with solid phase extraction clean-up67.060QMLKCereals, pulses and derived productsICS:Ta slovenski standard je istoveten z:EN 13585:2001SIST EN 13585:2002en01-junij-2002SIST EN 13585:2002SLOVENSKI
STANDARD



SIST EN 13585:2002



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 13585December 2001ICS 67.060English versionFoodstuffs - Determination of fumonisins B1 and B2 in maize -HPLC method with solid phase extraction clean-upProduits alimentaires - Dosage des fumonisines B1 et B2dans le maïs - Méthode CLHP avec purification parextraction en phase solideLebensmittel - Bestimmung von Fumonisin B1 und B2 inMais - HPLC-Verfahren mit Reinigung durchFestphasenextraktionThis 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 EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards 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 translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2001 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 13585:2001 ESIST EN 13585:2002



EN 13585:2001 (E)2ContentspageForeword.21Scope.32Normative references.33Principle.34Reagents and materials.35Apparatus.46Sampling.57Preparation of the test sample.58Procedure.69Calculation.610Precision.711Test report.8Annex A (informative)
Typical chromatogram.10Annex B (informative)
Recovery and relative standard deviation.11Annex C (informative)
Precision data.12Bibliography.15ForewordThis European Standard has been prepared by Technical Committee CEN /TC 275, "Food analysis - Horizontalmethods", 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 orby endorsement, at the latest by June 2002, and conflicting national standards shall be withdrawn at the latest byJune 2002.The annexes A, B and C are informative.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden,Switzerland and the United Kingdom.SIST EN 13585:2002



EN 13585:2001 (E)31 ScopeThis European Standard specifies a method for the determination of fumonisin B1 (FB1) and fumonisin B2 (FB2) inmaize using high performance liquid chromatography (HPLC).The method has been successfully validated in an interlaboratory study according to AOAC Guidelines forCollaborative Studies [1] on maize containing 405 µg/kg to 6732 g/kg fumonisin B1 and 152 g/kg to 2619 g/kgfumonisin 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-based processed products.2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications. Thesenormative references are cited at the appropriate places in the text, and the publications are listed hereafter. Fordated references, subsequent amendments to or revisions of any of these publications apply to this EuropeanStandard only when incorporated in it by amendment or revision. For undated references the latest edition of thepublication referred to applies (including amendments).EN ISO 3696Water for analytical laboratory use - Specification and test methods (ISO 3696:1987).3 PrincipleFumonisins are extracted from the sample of maize with a mixture of methanol and water. The filtered extract ispurified on a strong-anion-exchange (SAX) solid-phase extraction (SPE) cartridge, and the fumonisins are elutedwith a mixture of acetic acid and methanol. The extract is evaporated and the residue is redissolved in methanoland o-phthaldialdehyde/2-mercaptoethanol (OPA/MCE) is added to form fluorescent fumonisin derivatives. Thederivatives are analysed by reverse-phase high performance liquid chromatography (HPLC) with fluorescencedetection.WARNING - Fumonisins are hepatoxic, nephrotoxic and carcinogenic to rats and mice. Effects on humansare not fully known. Observe appropriate safety precautions for handling fumonisins. Any laboratory spillsshould be washed with a 5 % solution of sodium hypochlorite.4 Reagents and materials4.1 GeneralDuring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilledwater 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 (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 % for eluting the SPE column.Mix 1 part per volume of glacial acetic acid with 99 parts per volume of methanol (4.2).SIST EN 13585:2002



EN 13585:2001 (E)44.7 o-phthaldialdehyde (OPA)4.8 2-mercaptoethanol (MCE)4.9 Sodium dihydrogen phosphate solution, substance concentration c(NaH2PO4·2H2O) = 0,1 mol/lDissolve 15,6 g of NaH2PO4·2H2O in 1 l of water.4.10 Disodium tetraborate solution, c(Na2B4O7·10 H2O) = 0,1 mol/lDissolve 3,8 g of Na2B4O7·10H2O in 100 ml of water.4.11 Sodium hydroxide solution, c(NaOH) = 0,1 mol/lDissolve 0,4 g of NaOH in 100 ml of water.4.12 Mobile phaseMix 77 volume parts of methanol (4.2) with 23 volume parts of sodium dihydrogen phosphate solution (4.9). Adjustto pH 3,35 with o-phosphoric acid (4.5). Filter the solution through a 0,45 m membrane (5.7) filter.The mobile phase composition may have to be adjusted to conform with individual HPLC column characteristics.4.13 Derivatization reagentDissolve 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 stable for up to one week at room temperature in a darkand capped amber vial.4.14 FB1 and FB2 standard solutionPrepare individual stock solutions of FB1 and FB2 at mass concentrations of 250 µg/ml in acetonitrile solution (4.4).Commercially available standard solutions may be used. Transfer 100 l aliquots of each stock solution to a glassvial and add 300 l of acetonitrile solution to yield 500 l of a standard solution containing both fumonisins atindividual 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 and 200 l) of individual fumonisins and dilute to 500 l with acetonitrile solution to obtainthe relevant calibration solutions.Fumonisin standard solutions are stable up to at least six months when stored at approximately 4 °C.5 ApparatusUsual laboratory equipment and, in particular, the following:5.1 HPLC apparatus, comprising the following5.1.1 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 (ODS), which ensures a baselineresolution of the fumonisin peaks from all other peaks, with the following characteristics: stainless steel;SIST EN 13585:2002



EN 13585:2001 (E)5 a length of 150 mm; an inner 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 emissionwavelength of
= 440 nm.5.1.4
Data system5.2 Blender, homogenizer, or mixer.5.3 Fluted filter paper5.4 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 manifold5.6 Solvent evaporator, with heating module, or similar.5.7 Membrane filter, for aqueous solutions, with a pore size of 0,45 m.6 SamplingIt is important that the laboratory receives a sample which is truly representative and has not been damaged orchanged during transport or storage.7 Preparation of the test sampleGrind the sample to pass through a 1,0 mm sieve and homogenize the sample.8 Procedure8.1 ExtractionPlace 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 approximately 10 000 min-1. Thetime 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 solution (4.11) to between pH 5,8 andpH 6,5.
1 Bond-Elut is an example of a suitable product available commercially. This information is given for the convenience of usersof this European Standard and does not constitute an endorsement by CEN of this product.SIST EN 13585:2002



EN 13585:2001 (E)68.2 Clean upAttach the SPE cartridge (5.4) to the SPE manifold (5.5) and condition by washing successively with 5 ml
...