SIST EN 15791:2009
(Main)Foodstuffs - Determination of Deoxynivalenol in animal feed - HPLC method with immunoaffinity column clean-up
Foodstuffs - Determination of Deoxynivalenol in animal feed - HPLC method with immunoaffinity column clean-up
This International 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 g/kg.
Futtermittel - Bestimmung von Deoxynivalenol in Futtermitteln - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule
Das folgende Verfahren wird angewendet, um Deoxynivalenol (DON) in Futtermitteln in Konzentrationen über 150 µg/kg zu bestimmen. Das Verfahren ist anwendbar auf Konzentrationen bis 16 000 µg/kg.
Produits alimentaires - Dosage du désoxynivalénol dans les aliments pour animaux - Méthode de chromatographie liquide haute performance avec détection UV et purification sur colonne d'immuno-affinité
La présente méthode est utilisée pour doser le désoxynivalénol (DON) dans les aliments des animaux à des concentrations supérieures à 150 g/kg. Elle est applicable jusqu’à 16 000 g/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
SIST EN 15791:2009
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
EN 15791:2009 (E) 2 Contents
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
SIST EN 15791:2009
EN 15791:2009 (E) 3 Foreword This document (EN 15791:2009) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, the secretariat of which is held by NEN. 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 March 2010, and conflicting national standards shall be withdrawn at the latest by March 2010. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. WARNING — The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to 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. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: 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 the United Kingdom.
SIST EN 15791:2009
EN 15791:2009 (E) 4 1 Scope 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. 2 Normative references The following referenced documents 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 methods (ISO 3696:1987) 3 Principle Deoxynivalenol (DON) is extracted from the commodity using water.
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
EN 15791:2009 (E) 5 4.6 Wash Solvent Mix 50 parts per volume of methanol (4.3) with 50 parts per volume of water. 4.7 DON stock solution 250 µg Deoxynivalenol per ml of Acetonitrile. May be prepared by the following: Add 4,0 ml of acetonitrile (4.1) to 5 mg of DON (4.2) for a solution of 1,25 mg/ml. Dilute 1 000 µl of the 1,25 mg/ml solution to 5,0 ml with acetonitrile for the stock solution of 250 µg/ml. Dilute 200 µl of the 250 µg/ml stock solution in a 2,0 ml volumetric flask (5.11) with acetonitrile to create a diluted stock solution of 25 µg/ml. To determine the exact concentration record the absorption curve of this 25 µg/ml diluted stock solution with the spectrophotometer (5.15) in the range of 200 nm to 270 nm in a 1 cm quartz cell with acetonitrile (4.1) as reference. Determine the absorption at 220 nm. Calculate the mass concentration of deoxynivalenol, ρDon, in micrograms per millilitre using equation 1: ()dMAmlgDON×××=κµρ100/25~max (1) where: Amax
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.
SIST EN 15791:2009
EN 15791:2009 (E) 6 4.10 DON Calibration solutions Calibration solutions are prepared from the 10 µg/ml DON working solution (4.9). For example, add the volumes of 10 µg/ml DON working solution (4.9) shown in the table below into 10 ml volumetric flasks (5.11). Fill the flasks up to the mark with mobile phase (4.5). Deviations are permissible as long as the lowest level is above the limit of detection, the highest level does not lead to saturation of the detector signal, and there are at least two more levels equidistantly in between. Table 1 — Preparation of standard solutions Calibration solution DON Working solution (4.9) (µµµµl) DON concentration ng/ml 1 450 450 2 375 375 3 300 300 4 225 225 5 150 150 6 75 75 4.11 DON immunoaffinity clean-up columns The immunoaffinity (IA) column contains antibodies raised against deoxynivalenol. The column shall have a capacity of not less than 2 500 ng of DON and shall give a recovery of not less than 70% when 25 ng of DON are applied in 1 ml to-2 ml of water (depending on manufacturer’s instructions). 5 Apparatus Usual laboratory equipment and in particular the following: 5.1 Analytical balance, with d=0,001g for sample weighing, with d=0,01mg for gravimetrical preparation of the DON stock solution (4.7) 5.2 Homogeniser/ High Speed Blender 5.3 Laboratory shaker 5.4 Vortex Mixer, or equivalent 5.5 Mill (various screens) 5.6 Tumble mixer 5.7 Screw cap flasks, with volumes of 250 ml and 500 ml 5.8 Funnels, of appropriate size 5.9 Filter, cellulose with ca. 30 µm pore size SIST EN 15791:2009
EN 15791:2009 (E) 7 5.10 Filter, binder-free glass microfiber with ca. 2 µm pore size 5.11 Volumetric
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