DIN EN 15792-2009 Animal feeding stuffs - Determination of zearalenone in animal feed - High performance liquid chromatographic method with fluorescence detection and immunoaffinit.pdf

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1、English price group 10No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 65.120!$8Y“1562154www.din.deDDIN EN 15792Animal

2、feeding stuffs Determination of zearalenone in animal feed High performance liquid chromatographic method with fluorescencedetection and immunoaffinity column clean-upEnglish version of DIN EN 15792:2009-12Futtermittel Bestimmung von Zearalenon in Futtermitteln Hochleistungsflssigchromatographisches

3、 Verfahren mit Fluoreszenznachweis undReinigung an einer ImmunoaffinittssuleEnglische Fassung DIN EN 15792:2009-12www.beuth.deDocument comprises pages17December 2009National foreword This standard has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and anal

4、ysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-03-03 AA Futtermittel. The DIN Standard corresponding

5、to the International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods 2 DIN EN 15792:2009-12 EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15

6、792September 2009ICS 65.120English 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 zaralnone dans lesaliments des animaux - Mtho

7、de de chromatographieliquide haute performance avec dtection par fluorescenceet purification sur colonne dimmuno-affinitFuttermittel - Bestimmung von Zearalenon in Futtermitteln -Hochleistungsflssigchromatographisches Verfahren mitFluoreszenznachweis und Reinigung an einerImmunoaffinittssuleThis Eur

8、opean 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 conc

9、erning 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

10、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,

11、Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All righ

12、ts of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15792:2009: E2 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 . 1

13、1 10 Test report 11 Annex A (informative) Precision data 13 Bibliography 15 EN 15792:2009 (E) DIN EN 15792:2009-12 3 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 sha

14、ll 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 documen

15、t 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 Interna

16、l 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

17、, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. EN 15792:2009 (E) DIN EN 15792:2009-12 4 1 Scope This Standard is applicable to the determination of zearalenone in animal feed at concentrations from 30 g/kg to 3 000 g/kg. 2 Nor

18、mative 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 3696, Water for analytic

19、al laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle Zearalenone is extracted from the commodity using organic solvent. The solvent extract is then diluted with phosphate buffered saline to give an aqueous extract which is applied to an immunoaffinity column containing anti

20、bodies specific for zearalenone. The analyte is isolated, purified and concentrated on the column and removed from the antibodies with elution solvent. Zearalenone is quantitatively determined by high performance liquid chromatography (HPLC) with fluorescence detection. 4 Reagents During the analysi

21、s, unless otherwise stated, use only reagents of recognised analytical grade and only distilled water or water of grade 1 as defined in EN ISO 3696. Solvents shall be of quality for HPLC analysis. 4.1 Acetonitrile WARNING Acetonitrile is hazardous and handling shall be carried out inside a fume cupb

22、oard. Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. 4.2 Methanol, technical grade WARNING Methanol is hazardous and handling shall be carried out inside a fume cupboard. Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. Samples shall be blended using a

23、n explosion proof blender. 4.3 Methanol, HPLC grade WARNING Methanol is hazardous and handling shall be carried out inside a fume cupboard. Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. Samples shall be blended using an explosion proof blender. 4.4 Sodium chloride 4.5 Disod

24、ium hydrogen orthophosphate 4.6 Potassium dihydrogen phosphate EN 15792:2009 (E) DIN EN 15792:2009-12 5 4.7 Potassium chloride 4.8 Hydrochloric acid (32%) WARNING Hydrochloric acid is hazardous and handling shall be carried out with the necessary precaution inside a fume cupboard. Appropriate safety

25、 equipment (lab coat, goggles, gloves) shall be worn. 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 wi

26、th 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/PB

27、S = 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+2

28、5 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

29、standard and sample preparation stages shall be carried out in a fume cupboard. 4.15 Zearalenone (ZON) stock solution 10 g 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/m

30、l. Dilute 800 l of the 1,25 mg/ml standard solution to 5,0 ml with acetonitrile (4.1) for a standard solution of 200 g/ml. Dilute 250 l of the 200 g/ml standard solution to 5,0 ml of acetonitrile (4.1) to create the stock solution of 10 g/ml. To determine the exact concentration record the absorptio

31、n curve of this 10 g/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 = 274 nm. Calculate the mass concentration of zearalenone, zon, in micrograms per mi

32、llilitre using equation 1: dMAzon=100max(1) EN 15792:2009 (E) DIN EN 15792:2009-12 6 where: Amaxis 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); is the molar absorption coefficient of zearalenone in ace

33、tonitrile (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. 4.1

34、7 ZON working solution Transfer an aliquot of the calibrated stock solution (4.15), equivalent to 10 g 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 g/ml working solution. This solution is stable for 2 months if stored at below 18 C.

35、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 Calibr

36、ation solution Volume of ZON working solution (4.17) l Mass concentration of calibration solution ng/ml 1 50 102 250 50 3 450 904 650 130 5 850 170The procedures above for standard preparation can be performed either by the use of pipettes or calibrated glassware as available. 4.19 Immunoaffinity co

37、lumn 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). EN 15792:2009 (E) DIN EN 15792:2

38、009-12 7 5 Apparatus Usual laboratory equipment and in particular the following: 5.1 Analytical balance, with d=0,001 g or better 5.2 Horizontal or vertical shaker 5.3 Homogeniser/ High Speed Blender 5.4 Vortex Mixer, or equivalent 5.5 pH meter 5.6 Mill (various screens) 5.7 Tumble mixer 5.8 Glass v

39、ials, various sizes 5.9 Graduated pipettes, with volumes of 5 ml and 50 ml 5.10 Graduated cylinders with and without stoppers, with volumes of 5 ml and 250 ml 5.11 Volumetric flasks, with volumes of 3 ml, 5 ml and 10 ml 5.12 Beaker, 250 ml 5.13 Conical or screw cap flasks, with volumes of 100 ml and

40、 250 ml to 500 ml 5.14 Glass funnels, of appropriate size 5.15 Folded filters, cellulose (ca. 30 m pore size) for the glass funnels (5.14) 5.16 Filter disks, binder-free glas microfibre ( 2 m pore size) of appropriate size for the solvent vacuum filtration system (5.22) 5.17 Pipettors or gas-tight g

41、las syringes, with a volume of 100 l, 500 l and 1 000 l 5.18 Vacuum manifold or Automated SPE Vacuum System, capable of accommodating the immunoaffinity columns 5.19 Reservoirs, of appropriate volume with attachments to fit the immunoaffinity columns 5.20 Plastic syringes, 5 ml 5.21 Vacuum pump, cap

42、able of generating sufficient vacuum for the solvent vacuum filtration system (5.22) 5.22 Solvent vacuum filtration system, fitted with glass microfibre filter (5.16) EN 15792:2009 (E) DIN EN 15792:2009-12 8 5.23 HPLC syringe filter unit, polyamide (nylon) with 0,45 m pore size 5.24 Ultrasonic bath

43、5.25 HPLC apparatus, comprising the following: 5.25.1 Injection system, manual or autosampler, with loop suitable for 100 l to 300 l injections 5.25.2 Pump, isocratic, pulsation-free, capable of maintaining a volume flow rate of 0,5 ml/min to 1,5 ml/min 5.25.3 Analytical reversed phase HPLC column,

44、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 m particle size, 250 pore size, or Spherisorb ODS2-Excel (250 mm x 4,6 mm i.d.), 5 m particle size, 250 pore size

45、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

46、.26 UV spectrophotometer, for checking the concentration of the stock solution (4.15) 6 Procedures 6.1 Sample preparation It is important that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport or storage. Samples should be taken and p

47、repared in accordance with European legislation where applicable 2. Samples should be finely ground and thoroughly mixed using a mill (5.6) and a tumble mixer (5.7) or another process that has been demonstrated to give complete homogenisation before a test portion is removed for analysis. In all ins

48、tances if the sample has been frozen allow it to thaw completely before sampling. Stir the sample thoroughly before removing an analytical test portion. 6.2 Extraction Weigh 20,00 g (recorded to 2 decimal places) test portion into a screw cap flask of 250 ml to 500 ml (5.13). Add 150 ml extraction s

49、olvent (4.10). Mix briefly by hand to obtain a homogeneous suspension then either shake for 1 h on a shaker (5.2), or sonicate for 15 min in an ultrasonic bath (5.24) and shake on a shaker (5.2) for another 15 min. Filtrate extract through folded filter paper (5.15) and collect the extract in a screw cap flask of 100 ml (5.13). Transfer exactly 30 ml (or 3 ml in case of results above 500 g/kg, see section 8) of the filtered extract into a 250 ml graduated cylinder with stopper (5.10). Dilute the extra