SIST EN 16007:2011

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Bestimmung von Ochratoxin A in Tierfutter mit Reinigung an einer Immunoaffinitätssäule und Hochleistungs-Flüssig-Chromatographie mit FluoreszenzdetektionAliments pour animaux - Dosage de l'ochratoxine A dans les aliments pour animaux par purification sur colonne d'immuno-affinité et chromatographie liquide à haute performance avec détection par fluorescenceAnimal feeding stuffs - Determination of Ochratoxin A in animal feed by immunoaffinity column clean-up and High Performance Liquid Chromatography with fluorescence detection65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16007:2011SIST EN 16007:2011en,fr,de01-oktober-2011SIST EN 16007:2011SLOVENSKI
STANDARD



SIST EN 16007:2011



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16007
August 2011 ICS 65.120 English Version
Animal feeding stuffs - Determination of Ochratoxin A in animal feed by immunoaffinity column clean-up and High Performance Liquid Chromatography with fluorescence detection
Aliments pour animaux - Dosage de l'ochratoxine A dans les aliments pour animaux par purification sur colonne d'immuno-affinité et chromatographie liquide à haute performance avec détection par fluorescence
Futtermittel - Bestimmung von Ochratoxin A in Tierfutter durch Reinigung an einer Immunoaffinitätssäule und Hochleistungs-Flüssig-Chromatographie mit Fluoreszenzdetektion 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.
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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 16007:2011: ESIST EN 16007:2011



EN 16007:2011 (E) 2 Contents Page Foreword .31Scope .42Normative references .43Principle .44Reagents and materials .45Apparatus .76Sample preparation .87Measurements . 108Determination of concentrations . 119Precision . 1110Test report . 12Annex A (informative)
Example of a chromatogram . 14Annex B (informative)
Results of the interlaboratory study . 15Bibliography . 18 SIST EN 16007:2011



EN 16007:2011 (E) 3 Foreword This document (EN 16007:2011) 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 February 2012, and conflicting national standards shall be withdrawn at the latest by February 2012. 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 protocol involves hazardous materials, operations and equipment. This protocol does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this protocol 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, 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 the United Kingdom. SIST EN 16007:2011



EN 16007:2011 (E) 4 1 Scope This European Standard specifies a method for the determination of Ochratoxin A (OTA) in cereal based animal feed using immunoaffinity for clean-up followed by liquid-chromatography with fluorescence detection.
NOTE The validated mass fraction range was 39 µg/kg to 338 µg/kg OTA. 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 1042, Laboratory glassware  One-mark volumetric flasks (ISO 1042:1998)
EN ISO 3696, Water for analytical laboratory use  Specification and test methods (ISO 3696:1987) 3 Principle OTA is extracted from the test material with a mixture of methanol – 3% aqueous sodium bicarbonate solution. The extract is filtered, diluted with PBS and purified using immunoaffinity columns (IAC). The purified OTA is eluted from the IAC using first methanol and then water, brought to a defined volume with water and quantified by HPLC with fluorescence detection. 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. 4.1 Methanol, CH3OH, technical grade. 4.2 Methanol, CH3OH, HPLC grade. 4.3 Water, water (EN ISO 3696 grade 1 (HPLC grade)) and water (EN ISO 3696 grade 3), or equivalent. 4.4 Potassium chloride, KCI. 4.5 Sodium chloride, NaCl. 4.6 Disodium hydrogenphosphate dodecahydrate, Na2HPO4*12 H2O . 4.7 Acetonitrile, CH3CN, HPLC grade. 4.8 Glacial acetic acid, CH3COOH, 96% minimum. 4.9 Solution of acetonitrile/ glacial acetic acid, acetonitrile (4.7) and glacial acetic acid (4.8) in proportion of 99/1 (v/v). 4.10 Toluene, C6H5CH3, analytical grade. SIST EN 16007:2011



EN 16007:2011 (E) 5 4.11 Toluene/Acetic acid solution, Toluene (4.10) / Glacial acetic acid (4.8) in proportion of 99/1 (v/v). 4.12 Sodium hydrogen carbonate, NaHCO3, minimum 99% purity. 4.13 PBS concentrate, Phosphate buffered saline concentrate. Dissolve the following in 1 800 ml of water (EN ISO 3696 grade 1):
 4 g KCl (4.4);  160 g NaCl (4.5);  72 g Na2HPO4*12 H2O (4.6). 4.14 PBS Ready to use. Dilute 100 ml of PBS concentrate (4.13) to 1 000 ml with water (EN ISO 3696 grade 1). Adjust to pH 7,4 with 1 mol/l HCl and make up to 2 000 ml with water (EN ISO 3696 grade 1), or
PBS tablets, Phosphate buffered saline tablets, One tablet dissolved in 200 ml of water (EN ISO 3696 grade 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). 4.15 3% aqueous sodium hydrogen carbonate solution. Add 30 g of sodium hydrogen carbonate (4.12) to 1 000 ml of water (EN ISO 3696 grade 3). 4.16 Extraction solvent, methanol / 3% aqueous sodium hydrogen carbonate solution in proportion of 50/50 (v/v). Add 500 ml of methanol (4.1) to 500 ml of 3% aqueous sodium hydrogen carbonate solution (4.15). Mix well. 4.17 Aqueous solution of glacial acetic acid. Add 30 ml of glacial acetic acid (4.8) to 870 ml of water (EN ISO 3696 grade 1) and filter. 4.18 HPLC mobile phase, acetonitrile / methanol / aqueous solution of glacial acetic acid in proportion of 35/35/30 (v/v/v). Mix 1 050 ml of methanol (4.2) with 1 050 ml of acetonitrile (4.77) and with 900 ml of aqueous solution of glacial acetic acid (4.1717). Mix well and degas.
4.19 OTA, ochratoxin A. OTA in pure form as crystals, dried film, powder or in solution. 4.20 OTA Stock Solution. Prepare from the OTA (4.19) a solution of 20 µg/ml in toluene/acetic acid solution (4.11). To determine the exact concentration, record the absorption curve of this solution between a wavelength of 300 nm and 370 nm in a 1 cm quartz cell with toluene/acetic acid solution (4.11) as reference using the UV-SIST EN 16007:2011



EN 16007:2011 (E) 6 spectrophotometer (5.21). Identify the wavelength for maximum absorption. Calculate the mass concentration of OTA, ρota, in micrograms per millilitre using Equation (1): bMA×××=ερ100maxota (1) where Amax is the absorption determined at the maximum of the absorption curve (here: at 333 nm); M is the molar mass, in grams per mol, of OTA (M = 403,8 g/mol); 0 is the molar absorption coefficient, in square metres per mol, of OTA in toluene/acetic acid solution (4.11), (here: 544 m2/mol); b is the optical path length, in centimetres, of the quartz cell. 4.21 Nitrogen. 4.22 OTA diluted stock solution for calibration. Prepare 10 ml of OTA diluted stock solution for calibration by diluting 100 times the OTA stock standard solution (4.20) with mobile phase (4.18). For this pipette 100 µl of the OTA stock standard solution into a volumetric cylinder, evaporate the solution under a slight stream of nitrogen (4.21) at 40 ºC and take up with mobile phase (4.18).
NOTE To ease complete dissolution of the OTA in mobile phase, first fill the volumetric cylinder to approximately 1/3, let the OTA dissolve, and then fill up to the mark and shake. 4.23 Calibration solutions. From the OTA diluted stock solution for calibration (4.22) prepare six levels of calibration solutions by adding the volumes of diluted stock solution listed below to a volumetric flask of the indicated volume and make up to the mark with mobile phase (4.18): SIST EN 16007:2011



EN 16007:2011 (E) 7 Table 1 — recommended calibration solutions (4.23) for the determination of OTA CalibrantOTA diluted stock solution for calibration (4.22)
(µl) Volumetric flask (5.5)
(ml) Concentration OTA
(ng/ml) 1 50 10,0 1 2 250 10,0 5 3 500 10,0 10 4 750 10,0 15 5 1 000 10,0 20 6 1 250 10,0 25
4.24 IAC with antibodies specific to OTA. The IAC contains antibodies raised against OTA. The column shall have a total capacity of not less than 100 ng of OTA. OTA recovery shall not be less than 85 % when 5 ng OTA is applied in 50 ml of a mixture of 4 parts per volume of extraction solvent (4.16) and 96 parts per volume of PBS Ready to use (4.14). 4.25 Spiking solution. OTA in a solution of acetonitrile/ glacial acetic acid in proportion of 99/1 (v/v). The OTA concentration of the spiking solution will depend on the spiking level required. The spiking volume should be approximately 500 µl. The solution can be made from the OTA Stock Solution (4.20) in a similar manner as the OTA diluted stock solution for calibration (4.22). 5 Apparatus Usual laboratory equipment and, in particular, the following: 5.1 Common laboratory glassware, such as graduated cylinders, beakers, volumetric pipettes 5.2 Analytical balance, capable of weighing to 0,1 mg 5.3 Horizontal or vertical shaker 5.4 Automated SPE Vacuum System 5.5 Volumetric flasks (class A, EN ISO 1042), 5 ml, 10 ml, 100 ml 5.6 Filter paper pre-folded, 30 µm particle retention 5.7 Screw-cap flasks, 250 ml and 500 ml SIST EN 16007:2011



EN 16007:2011 (E) 8 5.8 Glass funnels, 9 cm ID 5.9 Reservoirs, polypropylene, suitable for attachment to top of IAC, 50 ml to 75 ml size 5.10 Plastic syringes, 5 ml 5.11 Calibrated displacement micropipette, 100 µl, 500 µl and 1 000 µl (variable volume) 5.12 Solvent vacuum filtration system, suita
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