EN 15791:2009
(Main)Animal feeding stuffs - Determination of Deoxynivalenol in animal feed - HPLC method with UV detection and immunoaffinity column clean-up
Animal feeding stuffs - Determination of Deoxynivalenol in animal feed - HPLC method with UV detection and immunoaffinity column clean-up
This Standard is applicable to the determination of deoxynivalenol (DON) in animal compound feed at concentrations of 150 μg/kg up to at least 4 000 μg/kg.
Futtermittel - Bestimmung von Deoxynivalenol in Futtermitteln - Hochleistungsflüssigkeitschromatografie (HPLC-) Verfahren mittels UV-Detektion und Reinigung an einer Immunoaffinitätssäule
Diese Norm ist für die Bestimmung von Deoxynivalenol (DON) in Futtermitteln in Konzentrationen von 150 µg/kg bis mindestens 4 000 µg/kg anwendbar.
Aliments des animaux - Dosage du déoxynivalénol dans les aliments pour animaux - Méthode de chromatographie liquide haute performance avec détection UV et purification sur colonne d'immuno-affinité
La présente norme s’applique au dosage du déoxynivalénol (DON) dans les aliments composés pour animaux à des concentrations comprises entre 150 et 4 000 µg/kg.
Krma - Določevanje deoksinivalenola v krmi - Metoda HPLC z imunoafinitetnim kolonskim čiščenjem
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.ILQLWHWQLPFuttermittel - Bestimmung von Deoxynivalenol in Futtermitteln - HPLC-Verfahren mit Reinigung an einer ImmunoaffinitätssäuleProduits alimentaires - Dosage du désoxynivalénol dans les aliments pour animaux - Méthode de chromatographie liquide haute performance avec détection UV et purification sur colonne d'immuno-affinitéFoodstuffs - Determination of Deoxynivalenol in animal feed - HPLC method with immunoaffinity column clean-up65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 15791:2009SIST EN 15791:2009en,fr,de01-december-2009SIST EN 15791:2009SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15791September 2009ICS 65.120 English VersionFoodstuffs - Determination of Deoxynivalenol in animal feed -HPLC method with immunoaffinity column clean-upProduits alimentaires - Dosage du désoxynivalénol dans lesaliments pour animaux - Méthode de chromatographieliquide haute performance avec détection UV et purificationsur colonne d'immuno-affinitéFuttermittel - Bestimmung von Deoxynivalenol inFuttermitteln - HPLC-Verfahren mit Reinigung an einerImmunoaffinitätssäuleThis European Standard was approved by CEN on 1 August 2009.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 CEN 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 CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15791:2009: ESIST EN 15791:2009
Page Foreword . 3 1 Scope . 4 2 Normative references . 4 3 Principle . 4 4 Reagents . 4 5 Apparatus. 6 6 Procedure. 7 7 HPLC determination . 8 8 Calculation . 9 9 Precision . 10 10 Test report. 10 Annex A (informative) Precision data . 12 Annex B (informative) Chromatogram . 14 Bibliography . 15
The aqueous extract is then cleaned up with an immunoaffinity column to remove impurities from the sample. Subsequently DON is quantitatively determined by HPLC with UV detection. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and only double-distilled water or water of grade 1 as defined in EN ISO 3696.
Solvents shall be of quality for HPLC analysis. 4.1 Acetonitrile WARNING — Acetonitrile is hazardous and handling shall be carried out inside a fume cupboard.
Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. 4.2 Deoxynivalenol (DON), with a minimum purity of 97 % WARNING — Deoxynivalenol is highly toxic. 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. 4.3 Methanol WARNING — Methanol is hazardous and handling shall be carried out inside a fume cupboard.
Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. 4.4 Glacial acetic acid WARNING — Glacial acetic acid is hazardous and handling shall be carried out inside a fume cupboard.
Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. 4.5
Mobile Phase Mix 15 parts per volume of methanol (4.3) with 84,9 parts per volume of water and 0,1 parts of glacial acetic acid (4.4). The exact amount of methanol used and whether acetic acid will have to be used depends on the HPLC column chosen for analysis and must be adjusted if necessary. Degas this solution before use. SIST EN 15791:2009
is the absorption determined at the maximum of the absorption curve (here: at 220 nm); M
is the molar mass of deoxynivalenol (M = 296,3 g/mol); κ is the molar absorption coefficient of deoxynivalenol in acetonitrile (4.1),
(here: 681 m2/mol ± 12,6
m2/mol [1]); d
is the optical path length of the quartz cell in centimetres (here: 1 cm).
Calculate the exact concentration of the 250 µg/ml stock solution by the following equation: ()()10/25~/250~×=mlgmlgDONDONµρµρ
(2)
Stock solution may be stored in the dark for up to 3 months at 4°C to 8°C or at least 6 months at below
-18 ºC. NOTE Stock solution preparation can be carried out gravimetrically by accurately weighing the DON standard material and the solvent used to dissolve it. 4.8 DON spiking solution Pipette an aliquot of the calibrated DON stock solution (4.7), equivalent to 500 µg DON, into a 5 ml volumetric flask (5.11). Make up to the mark with acetonitrile (4.1). This will result in the spiking solution of 100 µg/ml. 4.9 DON working solution Pipette an aliquot of the calibrated diluted DON stock solution (4.7), equivalent to 50 µg DON, into a 5 ml volumetric flask (5.11). Make up to the mark with acetonitrile (4.1). This will result in the DON working solution of 10 µg/ml.
Examples are:
Phenomenex ODS3-Prodigy (15 cm x 4,6 mm i.d.), 5 µm particle size, 100 Å pore size, Octadecylsilane (ODS) 250 mm x 4,6 mm I.D., 3 µm particle size, 80 Å pore size, Octadecyl (C18) 250 mm x 4,6 mm I.D., 5 µm particle size, 180 Å pore size 5.14.3 Pre-column (optional), appropriate for the analytical column used 5.14.4 Autosampler, capable of injecting appropriate volumes with sufficient repeatability 5.14.5 UV detector, capable of measuring at 220 nm 5.14.6 Data collection system 5.15 UV spectrophotometer, for checking the concentration of the DON stock solution (4.7) 5.16 Reservoirs, of appropriate size with adaptors to fit the immunoaffinity columns 5.17 Glass vials, of appropriate size for autosampler (5.14.4) but with minimum volume of 2,0 ml 5.18 Syringe filter unit, polyamide (nylon) with 0,45 µm pore size 5.19 Evaporator, capable of maintaining 50ºC with a steady stream of air or nitrogen 6 Procedure 6.1 Sample preparation It is important that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport or storage. Samples should be taken and prepared in accordance with European legislation where applicable [2]. Samples should be finely ground and thoroughly mixed using a mill (5.5) and a tumble mixer (5.6) or another process that has been demonstrated to give complete homogenisation before a test portion is removed for analysis. In all instances if the sample has been frozen allow it to thaw completely before sampling. Mix the sample thoroughly before removing an analytical test portion.
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