1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16007:2011Animal feeding stuffs Determination of OchratoxinA in animal feed byimmunoaffinity column clean-up and High PerformanceLiquid Chromatography withfluorescence dete
2、ctionBS EN 16007:2011 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16007:2011.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can beobtained on requ
3、est to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2011ISBN 978 0 580 66994 1ICS 65.120Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard
4、 was published under the authority of theStandards Policy and Strategy Committee on 31 August 2011.Amendments issued since publicationDate Text affectedBS EN 16007:2011EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16007 August 2011 ICS 65.120 English Version Animal feeding stuffs - Determinat
5、ion of Ochratoxin A in animal feed by immunoaffinity column clean-up and High Performance Liquid Chromatography with fluorescence detection Aliments pour animaux - Dosage de lochratoxine A dans les aliments pour animaux par purification sur colonne dimmuno-affinit et chromatographie liquide haute pe
6、rformance avec dtection par fluorescence Futtermittel - Bestimmung von Ochratoxin A in Tierfutter durch Reinigung an einer Immunoaffinittssule und Hochleistungs-Flssig-Chromatographie mit Fluoreszenzdetektion This European Standard was approved by CEN on 25 June 2011. CEN members are bound to comply
7、 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 M
8、anagement 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
9、 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, Por
10、tugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any mean
11、s reserved worldwide for CEN national Members. Ref. No. EN 16007:2011: EBS EN 16007:2011EN 16007:2011 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents and materials 45 Apparatus .76 Sample preparation .87 Measurements . 108 Determination of concentrations 119 Pr
12、ecision 1110 Test report . 12Annex A (informative) Example of a chromatogram . 14Annex B (informative) Results of the interlaboratory study 15Bibliography . 18BS EN 16007:2011EN 16007:2011 (E) 3 Foreword This document (EN 16007:2011) has been prepared by Technical Committee CEN/TC 327 “Animal feedin
13、g 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 2
14、012. 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 Comm
15、ission 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 appro
16、priate 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, Cro
17、atia, 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. BS EN 16007:2011EN 16007:20
18、11 (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 g/kg to 338 g/kg OTA. 2 No
19、rmative 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 glassw
20、are 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
21、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
22、 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 chl
23、oride, 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 Toluen
24、e, C6H5CH3, analytical grade. BS EN 16007:2011EN 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. Dissol
25、ve 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 (
26、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 25C (e.g. Sigma P4417). 4.15 3% aqueous sodium hydro
27、gen 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 carbon
28、ate 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
29、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 solut
30、ion of 20 g/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-BS EN 16007:2011EN 16007:2011 (
31、E) 6 spectrophotometer (5.21). Identify the wavelength for maximum absorption. Calculate the mass concentration of OTA, ota, in micrograms per millilitre using Equation (1): bMA=100maxota(1) where Amax is the absorption determined at the maximum of the absorption curve (here: at 333 nm); M is the mo
32、lar mass, in grams per mol, of OTA (M = 403,8 g/mol); 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
33、 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 l of the OTA stock standard solution into a volumetric cylinder, evaporate the solution under a slight stream of nitro
34、gen (4.21) at 40 C 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 solutio
35、n 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): BS EN 16007:2011EN 16007:2011 (E) 7 Table 1 recommended calibration soluti
36、ons (4.23) for the determination of OTA Calibrant OTA diluted stock solution for calibration (4.22) (l) 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
37、 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 S
38、piking 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 l. The solution can be made from the OTA Stock Solution (4.20)
39、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
40、 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 m particle retention 5.7 Screw-cap flasks, 250 ml and 500 ml BS EN 16007:2011EN 16007:2011 (E) 8 5.8 Glass funnels, 9 cm ID 5.9 Reservoirs,
41、 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 l, 500 l and 1 000 l (variable volume) 5.12 Solvent vacuum filtration system, suitable for 47 mm filter 5.13 Glass microfibre filter, 1,6 m particle r
42、etention 5.14 Vortex mixer 5.15 HPLC syringe filter cartridges, Nylon with 0,45 m pore size 5.16 Ultrasonic bath 5.17 Amber glass vials, ca 2 ml capacity and crimp caps or equivalent 5.18 HPLC system The HPLC system comprises the following: pump, pulse free, flow capacity 0,5 ml/min to 1,5 ml/min; i
43、njector system, manual or autosampler, with loop suitable for 100 l to 300 l injections; fluorescence detector, suitable for measurements with excitation wavelengths 333 nm and emission at 467 nm; integrator, or PC workstation. 5.19 Analytical reversed phase HPLC column, C18 RP-column suitable to al
44、low a sufficient separation of OTA from other interfering components; Fully End Capped with column dimensions preferably 250 mm X 4,6 mm ID stationary phase with particle size 5 m. 5.20 Pre-column (guard-column), with preferably the same stationary phase material as the analytical column and interna
45、l diameter of 4,0 mm, stationary phase with particle size 5 m. 5.21 UV-Spectrophotometer. 6 Sample preparation 6.1 Extraction of OTA Weigh 25,0 g, to the nearest 0,1 g, of the test sample into a large enough container with lid, e.g. 500 ml screw-cap flask (5.67), NOTE 1 The materials for the interla
46、boratory study 1 were milled to a particle size of ca. 0,5 mm. BS EN 16007:2011EN 16007:2011 (E) 9 add 200,0 ml of extraction solvent (4.16), cap, and shake vigorously by hand for a few seconds; so that the material disperses evenly (check visually), put on a shaker (5.3) for 40 min. Choose a speed
47、such that the material is mixed well without collecting in the top of the flask. A possible alternative is to ultrasonificate for 15 minutes; in this case shortly shake the extract by hand for a few seconds, allow the extracted sample to settle after extraction, filter the extract through folded fil
48、ter paper (5.6) and collect the filtered extract in a screw cap flask of 250 ml (5.7), and transfer approx. 10 ml of the filtered extract into the vacuum system and filter through glass microfibre filter (5.13) by applying a slight vacuum (5.12). Discard this volume and filter again the remaining ex
49、tract in another screw cap flask of 250 ml (5.7) to obtain a clear extract for further analysis. Proceed immediately with the IAC clean-up procedure (6.2). NOTE 2 Do not apply a strong vacuum in the beginning of the filtration process as this can lead to turbid filtered extract after filtration. 6.2 Clean up and test solution Take one IAC (4.24) per extract; attach a reservoir (5.9), do not empty storage solution from column; transfer 4,00 ml of double
