EN 16006:2011

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.XQRDILQLWHWQLPRQVNLGHULYDWL]DFLMLFuttermittel - Bestimmung der Summe der Fumonisine B1 und B2 in Mischfutter mit Reinigung an einer Immunoaffinitätssäule und RP­HPLC­Verfahren mit Fluoreszenzdetektion nach Vor- oder NachsäulenderivatisierungAliments pour animaux - Dosage de la somme des fumonisines B1 et B2 dans les aliments pour animaux avec purification par immuno-affinité et RP-HPLC avec détection par fluorescence après dérivation pré- ou post-colonneAnimal feeding stuffs - Determination of the Sum of Fumonisin B1 & B2 in compound animal feed with immunoaffinity clean-up and RP-HPLC with fluorescence detection after pre- or post-column derivatisation65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16006:2011SIST EN 16006:2011en,fr,de01-oktober-2011SIST EN 16006:2011SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16006
August 2011 ICS 65.120 English Version
Animal feeding stuffs - Determination of the Sum of Fumonisin B1 & B2 in compound animal feed with immunoaffinity clean-up and RP-HPLC with fluorescence detection after pre- or post-column derivatisation
Aliments pour animaux - Dosage de la somme des fumonisines B1 et B2 dans les aliments pour animaux avec purification par immuno-affinité et RP-HPLC avec détection par fluorescence après dérivation pré- ou post-colonne
Futtermittel - Bestimmung der Summe der Fumonisine B1 und B2 in Mischfutter durch Reinigung an einer Immunoaffinitätssäule und RP-HPLC mit Fluoreszenzdetektion nach Vor- oder Nachsäulenderivatisierung This European Standard was approved by CEN on 25 June 2011.
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 without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC 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 translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, 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 STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16006:2011: ESIST EN 16006:2011

Precision data . 18Annex B (informative)
Examples of a chromatogram . 21Bibliography . 23 SIST EN 16006:2011

EN ISO 3696, Water for analytical laboratory use  Specification and test methods (ISO 3696:1987) 3 Principle FB1 and FB2 are extracted from the test material with a solution of 50% methanol in phosphate-buffered saline (PBS). The extract is then diluted with PBS and cleaned up using immunoaffinity columns (IAC). FB1 and FB2 are eluted from the IAC using methanol and then water. After volume adjustment, the eluate is directly injected into the HPLC and FB1 and FB2 are detected by their fluorescence after either pre- or post column derivatisation. 4 Reagents and materials During the analysis, unless otherwise stated, use only reagents of recognized analytical grade. Solvents shall be of HPLC or better quality and only double-distilled water or water of at least grade 2 as defined in
EN ISO 3696 shall be used. 4.1 Double distilled or deionised water (EN ISO 3696) 4.2 Methanol, CH3OH WARNING — Methanol is hazardous and handling should be carried out inside a fume cupboard. Appropriate safety equipment (lab coat, goggles, gloves) should be worn. 4.3 Acetonitrile, CH3CN WARNING — Acetonitrile is hazardous and handling should be carried out inside a fume cupboard. Appropriate safety equipment (lab coat, goggles, gloves) should be worn. SIST EN 16006:2011

PBS tablets, Phosphate buffered saline tablets one tablet dissolved in 200 ml of water (4.1) yields 0,01 mol/l phosphate buffer, 0,002 7 mol/l potassium chloride and 0,137 mol/l sodium chloride, pH 7,4, at 25°C (e.g. Sigma P4417). 4.17 Diluent Mix 50 parts per volume methanol (4.2) with 50 parts per volume water (4.1). 4.18 Extraction solvent Mix 50 parts per volume methanol (4.2) with 50 parts per volume of PBS (4.16). SIST EN 16006:2011

This will result in 2,0 ml of a solution containing ca. 4 µg/ml FB1 & FB2 each in mostly methanol /water (50/50, v/v). 4.22 Calibration solutions From the diluted stock solution for calibration (4.21) prepare five levels of calibration solutions by adding the volumes of diluted stock solution listed in the following table to a volumetric flask (5.13) of the indicated volume and make up to the mark with diluent (4.17).
Calculate the concentrations of FB1 & FB2 for the different calibration levels by dividing the certified or calculated concentrations of the stock solution (4.20) by the final dilution stated below. Should you observe saturation of the detector signal at the highest calibration level dilute 250 µl of diluted stock solution into 2,0 ml for a final dilution of 100. Table 1 — Recommended calibration solutions (4.22) for the determination of the sum of Fumonisin B1 & B2 Calibrant Diluted stock solution (4.21)
(µl) Volumetric flask (5.13)
(ml) Final dilution of stock solution (4.20) Approx. concentration of FB1 & FB2 each
(ng/ml) 1 50 20,0 5 000 10 2 125 10,0 1 000 50 3 125 5,0 500 100 4 500 2,0 50 1 000 5 1 000 2,0 25 2 000 SIST EN 16006:2011

4.23 IAC (Immunoaffinity column) The immunoaffinity columns must contain a stationary phase with immobilized monoclonal antibodies specific to, at least, Fumonisin B1 and B2. To be suitable for this method they must meet the requirements stated below: An aliquot of more than 5 ml of an extract of a fumonisin-free representative compound animal feed material is spiked with FB1 & FB2 in equal parts at either 920 (high) ng/ml or 110 (low) ng/ml for the sum of both. Then dilute 5,0 ml of that spiked extract to a total volume of 50,0 ml (see 6.2). Following the procedures described in 6.3 and 6.4 this will result in expected concentrations in the injection solutions of either 460 ng/ml or 55 ng/ml for the sum of FB1 & FB2. After measuring (Clause 7) these solutions the observed concentrations of FB1 & FB2 can be calculated with Equation (1) and Equation (2) of Clause 8. Dividing the sum of the observed concentrations of FB1 & FB2 by the expected concentrations will result in the yield of the immunoaffinity columns. These yields must be 99 % ± 18 % (U, k=2) at the high level and 118 % ± 18 % (U, k=2) at the low level. The above column test should be performed for each level on at least three randomly selected columns of every new batch of immunoaffinity columns which will be used. Should the tested batch not meet the above requirements either a new batch which does should be obtained or the conditions described in 6.3 need to be adjusted such that the requirements are met (the user instructions supplied with the columns are a good starting point). Any changes to the clean-up procedures will necessitate a revalidation of the clean-up and all subsequent steps (chromatography). SIST EN 16006:2011
...