EN 15792:2009

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Bestimmung von Zearalenon in Futtermitteln - Hochleistungsflüssigchromatographisches Verfahren mit Fluoreszenznachweis und Reinigung an einer ImmunoaffinitätssäuleAliments des animaux - Dosage de la zéaralénone dans les aliments des animaux - Méthode de chromatographie liquide haute performance avec détection par fluorescence et purification sur colonne d'immuno-affinitéAnimal feeding stuffs - Determination of zearalenone in animal feed - High performance liquid chromatographic method with fluorescence detection and immunoaffinity column clean-up65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 15792:2009SIST EN 15792:2009en,fr,de01-december-2009SIST EN 15792:2009SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15792September 2009ICS 65.120 English VersionAnimal feeding stuffs - Determination of zearalenone in animalfeed - High performance liquid chromatographic method withfluorescence detection and immunoaffinity column clean-upAliments des animaux - Dosage de la zéaralénone dans lesaliments des animaux - Méthode de chromatographieliquide haute performance avec détection par fluorescenceet purification sur colonne d'immuno-affinitéFuttermittel - Bestimmung von Zearalenon in Futtermitteln -Hochleistungsflüssigchromatographisches Verfahren mitFluoreszenznachweis und 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 15792:2009: ESIST EN 15792:2009

Contents Page Foreword . 3 1 Scope . 4 2 Normative references . 4 3 Principle . 4 4 Reagents . 4 5 Apparatus . 7 6 Procedures . 8 7 HPLC determination . 9 8 Calculations . 10 9 Precision . 11 10 Test report . 11 Annex A (informative) Precision data . 13 Bibliography . 15
Foreword This document (EN 15792:2009) 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. 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.
4.9 Phosphate buffered saline (PBS) Dissolve 8 g sodium chloride (4.4), 1,2 g disodium hydrogen orthophosphate (4.5), 0,2 g potassium dihydrogen phosphate (4.6) and 0,2 g potassium chloride (4.7) in 1 l of distilled water. Adjust the pH to 7,4 with hydrochloric acid (4.8). NOTE Commercially available phosphate buffered saline tablets with equivalent properties may be used. 4.10 Extraction solvent, methanol/water = 75+25 parts by volume Mix 75 parts per volume methanol (4.2) with 25 parts per volume of water 4.11 Washing solvent, methanol/PBS = 15 + 85 parts by volume Mix 15 parts per volume methanol (4.3) with 85 parts per volume PBS (4.9). 4.12 Injection solvent for HPLC analysis, methanol/water = 50+50 parts by volume Mix 50 parts per volume methanol (4.3) with 50 parts per volume water. 4.13 HPLC mobile phase, methanol/water = 75+25 parts by volume Mix 75 parts per volume methanol (4.3) and 25 parts per volume water.
Mix well and degas. 4.14 Zearalenone, minimum purity of 98 % WARNING — Zearalenone is an oestrogenic compound and shall be treated with extreme caution. 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. 4.15 Zearalenone (ZON) stock solution 10 µg Zearalenone per millilitre of Acetonitrile. May be prepared by the following:
Add 4,0 ml of acetonitrile (4.1) to 5 mg of zearalenone (4.14) for a standard solution of 1,25 mg/ml. Dilute 800 µl of the 1,25 mg/ml standard solution to 5,0 ml with acetonitrile (4.1) for a standard solution of 200 µg/ml. Dilute 250 µl of the 200 µg/ml standard solution to 5,0 ml of acetonitrile (4.1) to create the stock solution of 10 µg/ml. To determine the exact concentration record the absorption curve of this 10 µg/ml stock solution with the spectrophotometer (5.26) in the range of 200 nm to 300 nm in a 1 cm quartz cell with acetonitrile (4.1) as reference. Determine the absorption of the second maximum at λ = 274 nm. Calculate the mass concentration of zearalenone, ρzon, in micrograms per millilitre using equation 1: dMAzon×××=κρ100max
(1) SIST EN 15792:2009
is the absorption determined at the second maximum of the absorption curve (here: at 274 nm) M
is the molar mass of zearalenone (M = 318,4 g/mol); κ
is the molar absorption coefficient of zearalenone in acetonitrile (4.1) (here: 1 262 m2/mol ± 1 m2/mol [1]);
d
is the optical path length of the quartz cell in centimetres (here: 1 cm). Store standard solutions at below −18 °C. 4.16 ZON spiking solution The calibrated stock solution, see (4.15). This solution is stable for 2 months if stored at below −18 °C. 4.17 ZON working solution Transfer an aliquot of the calibrated stock solution (4.15), equivalent to 10 µg of ZON, into a volumetric flask (5.11). Add acetonitrile (4.1) to make the total volume up to 5 ml. This is a 2 µg/ml working solution. This solution is stable for 2 months if stored at below −18 °C. 4.18 ZON Calibration solutions for HPLC Prepare 5 HPLC calibration solutions in separate 10 ml volumetric flasks (5.11) by pipetting the volumes shown in Table 1 below. Make up each standard to volume (10 ml) with injection solvent for HPLC (4.12). Table 1 — Preparation of calibration solutions Calibration solution Volume of ZON working solution (4.17) µl Mass concentration of calibration solution ng/ml 1 50 10 2 250 50 3 450 90 4 650 130 5 850 170
The procedures above for standard preparation can be performed either by the use of pipettes or calibrated glassware as available. 4.19 Immunoaffinity column The immunoaffinity (IA) column contains antibodies raised against zearalenone. The column shall have a capacity of not less than 1 500 ng of zearalenone and a recovery of not less than 70% when 75 ng of zearalenone are applied in 10 ml of washing solvent (4.11).
Generally every RP-column is suitable that allows a sufficient separation of zearalenone from other interfering components. For example Phenomenex ODS3-Prodigy (150 mm x 4,6 mm i.d.), 5 µm particle size, 250 Å pore size, or Spherisorb ODS2-Excel (250 mm x 4,6 mm i.d.), 5 µm particle size, 250 Å pore size have been found to be suitable. 5.25.4 Pre-column (optional), appropriate for the analytical column used 5.25.5 Fluorescence detector, fitted with a flow cell and suitable for measurements with excitation wavelength of 274 nm, and emission of 446 nm 5.25.6 Data system, integrator or PC workstation 5.26 UV spectrophotometer, for checking the concentration of the stock solution (4.15) 6 Procedures 6.1 Sample preparation It is important that
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