EN 16007:2011

Animal feeding stuffs - Determination of Ochratoxin A in animal feed by immunoaffinity column clean-up and High Performance Liquid Chromatography with fluorescence detection

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.

Futtermittel - Bestimmung von Ochratoxin A in Tierfutter durch Reinigung an einer Immunoaffinitätssäule und Hochleistungs-Flüssig-Chromatographie mit Fluoreszenzdetektion

Diese Norm legt ein Verfahren für die Bestimmung von Ochratoxin A (OTA) in Futtermitteln auf Getreidebasis durch Hochleistungs-Flüssig-Chromatographie (HPLC) mit Fluoreszenzdetektion nach Reinigung an Immunoaffinitätssäulen fest. ANMERKUNG Der validierte Konzentrationsbereich liegt zwischen 39 μg/kg bis 338 μg/kg OTA.

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

La présente Norme européenne spécifie une méthode de dosage de l’ochratoxine A (OTA) dans les aliments
pour animaux à base de céréales en utilisant une purification sur colonne d'immuno-affinité suivie d’une
chromatographie liquide avec détection par fluorescence

Krma - Določevanje ohratoksina A v krmi z imunoafinitetnim kolonskim čiščenjem in s tekočinsko kromatografijo visoke ločljivosti s fluorescentno detekcijo

Ta evropski standard opredeljuje metodo za določevanje ohratoksina A (OTA) v krmi na osnovi žit, z uporabo imunoafinitetnega čiščenja, ki mu sledi tekočinska kromatografija s fluorescentno detekcijo.

General Information

• Status: Published
• Publication Date: 02-Aug-2011
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 1 - Minerals and Contaminants
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 01-Jan-2024
• Completion Date: 14-Apr-2025
• Directive: 70/373/EC - Council Directive 70/373/EEC of 20 July 1970 on the introduction of Community methods of sampling and analysis for the official control of feeding-stuffs
• Mandate: M/382 - Mandate for standardisation addressed to CEN in the field of Methods of analysis for animal Feedingstuffs
• Ref Project: SIST EN 16007:2011 - Animal feeding stuffs - Determination of Ochratoxin A in animal feed by immunoaffinity column clean-up and High Performance Liquid Chromatography with fluorescence detection

Overview

EN 16007:2011 (CEN) defines a standardized laboratory method for the determination of Ochratoxin A (OTA) in cereal-based animal feeding stuffs. The method uses immunoaffinity column (IAC) clean‑up followed by High Performance Liquid Chromatography (HPLC) with fluorescence detection. The method was validated over a mass-fraction range of 39 µg/kg to 338 µg/kg OTA and is intended for routine testing in feed safety and quality control.

Key Topics and Requirements

• Scope: Quantitative determination of OTA in cereal-based animal feed using IAC cleanup and HPLC‑FLD.
• Sample size and extraction: Typical test portion is 25.0 g; extraction solvent is methanol / 3% aqueous sodium hydrogen carbonate (50/50 v/v).
• Immunoaffinity columns (IAC): Antibodies specific for OTA; column capacity ≥ 100 ng OTA; required recovery ≥ 85% when 5 ng OTA is applied under specified conditions.
• Cleanup and elution: Dilute filtered extract with PBS, apply to IAC, elute with methanol and water, add acetic acid solution and make up to defined volume for HPLC analysis.
• HPLC conditions:
  • Mobile phase: acetonitrile / methanol / aqueous glacial acetic acid (35/35/30 v/v/v).
  • Column: reversed-phase C18 (recommended 250 × 4.6 mm, 5 µm).
  • Injection: 100 µl recommended.
  • Fluorescence detection: excitation 333 nm, emission 467 nm.
• Calibration and quantification: Prepare multi-level calibration from OTA stock and diluted standards; use linear regression and verify linearity/intercept.
• Performance and precision: Document includes interlaboratory study results and precision guidance.
• Safety and quality: Use analytical-grade solvents, HPLC-grade reagents and appropriate lab safety measures.

Applications and Users

EN 16007:2011 is practical for:

• Feed testing laboratories performing routine mycotoxin screening and quantification.
• Quality assurance/quality control (QA/QC) teams in feed manufacturing to monitor OTA contamination.
• Regulatory bodies and accredited labs verifying compliance with feed safety limits.
• Research laboratories studying OTA occurrence in cereal-based feeds. Practical uses include regulatory compliance testing, batch release decisions, surveillance programs, and interlaboratory method validation.

Related Standards

• EN ISO 1042 - Laboratory glassware (volumetric flasks)
• EN ISO 3696 - Water for analytical laboratory use These normative references are cited within EN 16007:2011 for equipment and reagent quality requirements.

Keywords: EN 16007:2011, Ochratoxin A, OTA, animal feed, immunoaffinity column, IAC, HPLC, fluorescence detection, feed safety, mycotoxin analysis.

Standards Content (Sample)

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
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.
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 16007:2011: ESIST EN 16007:2011

Example of a chromatogram . 14Annex B (informative)
Results of the interlaboratory study . 15Bibliography . 18 SIST EN 16007:2011

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

 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

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

(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
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