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

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1、BS EN 15792:2009ICS 65.120NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal feeding stuffs Determinationof zearalenone inanimal feed Highperformance liquidchromatographicmethod withfluorescence detectionand immunoaffinitycolumn clean-upThis British Standard

2、was published underthe authority of theStandards Policy andStrategy Committee on 30September 2009 BSI 2009ISBN 978 0 580 61808 6Amendments/corrigenda issued since publicationDate CommentsBS EN 15792:2009National forewordThis British Standard is the UK implementation of EN 15792:2009.The UK participa

3、tion in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for

4、its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 15792:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15792September 2009ICS 65.120English VersionAnimal feeding stuffs - Determination of zearalenone in animalfeed - High performance

5、liquid chromatographic method withfluorescence detection and immunoaffinity column clean-upAliments des animaux - Dosage de la zaralnone dans lesaliments des animaux - Mthode de chromatographieliquide haute performance avec dtection par fluorescenceet purification sur colonne dimmuno-affinitFuttermi

6、ttel - Bestimmung von Zearalenon in Futtermitteln -Hochleistungsflssigchromatographisches Verfahren mitFluoreszenznachweis und Reinigung an einerImmunoaffinittssuleThis European Standard was approved by CEN on 1 August 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations wh

7、ich stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Sta

8、ndard 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 language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national stan

9、dards bodies of Austria, Belgium, Bulgaria, 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 King

10、dom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15792:2009: EBS EN 15792:

11、2009EN 15792:2009 (E) 2 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 . 11 10 Test report 11 Annex A (informative) Precision data 13 Bibliography 15 BS EN 15792:2009EN

12、 15792:2009 (E) 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 shall be given the status of a national standard, either by publication of an identical text or by end

13、orsement, 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 document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identif

14、ying 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 Internal Regulations, the national standards organizations of the following countries are bound to impleme

15、nt this European Standard: Austria, Belgium, Bulgaria, 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 an

16、d the United Kingdom. BS EN 15792:2009EN 15792:2009 (E) 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 Normative references The following referenced documents are indispensable for the application of this docum

17、ent. 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 analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle Zearalenone is extracted

18、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 antibodies specific for zearalenone. The analyte is isolated, purified and concentrated on the column and re

19、moved from the antibodies with elution solvent. Zearalenone is quantitatively determined by high performance liquid chromatography (HPLC) with fluorescence detection. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and only distilled water or

20、 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 cupboard. Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. 4.2 Methanol, technical gr

21、ade 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. 4.3 Methanol, HPLC grade WARNING Methanol is hazardous and handling shall be

22、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 Disodium hydrogen orthophosphate 4.6 Potassium dihydrogen phosphate BS EN 15792:2009EN 15792:2009 (E) 5 4.7 P

23、otassium 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 equipment (lab coat, goggles, gloves) shall be worn. 4.9 Phosphate buffered saline (PBS) Dissolve 8 g sodium

24、 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 with hydrochloric acid (4.8). NOTE Commercially available phosphate buffered saline tablets with equivalent pro

25、perties 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/PBS = 15 + 85 parts by volume Mix 15 parts per volume methanol (4.3) with 85 parts per volume PBS (4.9). 4.12 I

26、njection 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+25 parts by volume Mix 75 parts per volume methanol (4.3) and 25 parts per volume water. Mix well and degas. 4

27、.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 standard and sample preparation stages shall be carried out in a fume cupboard. 4.15 Zearalenone (ZON) stock

28、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/ml. Dilute 800 l of the 1,25 mg/ml standard solution to 5,0 ml with acetonitrile (4.1) for a standard solution

29、 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 absorption curve of this 10 g/ml stock solution with the spectrophotometer (5.26) in the range of 200 nm to 300 nm in

30、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 millilitre using equation 1: dMAzon=100max(1) BS EN 15792:2009EN 15792:2009 (E) 6 where: Amaxis the absorption

31、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 acetonitrile (4.1) (here: 1 262 m2/mol 1 m2/mol 1); d is the optical path length of the quartz cell in centimetres (h

32、ere: 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. 4.17 ZON working solution Transfer an aliquot of the calibrated stock solution (4.15), equivalent to 10 g of ZON, int

33、o 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. 4.18 ZON Calibration solutions for HPLC Prepare 5 HPLC calibration solutions in separate 10 ml volumetric flasks (

34、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 Calibration solution Volume of ZON working solution (4.17) l Mass concentration of calibration solution ng/ml 1 50 102 2

35、50 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 column The immunoaffinity (IA) column contains antibodies raised against zearalenone. The column shall have a capaci

36、ty 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). BS EN 15792:2009EN 15792:2009 (E) 7 5 Apparatus Usual laboratory equipment and in particular the following: 5.1 Analytical balance, with d=0,001 g

37、 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 vials, various sizes 5.9 Graduated pipettes, with volumes of 5 ml and 50 ml 5.10 Graduated cylinders with and without st

38、oppers, 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 250 ml to 500 ml 5.14 Glass funnels, of appropriate size 5.15 Folded filters, cellulose (ca. 30 m pore size) for the g

39、lass 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 glas syringes, with a volume of 100 l, 500 l and 1 000 l 5.18 Vacuum manifold or Automated SPE Vacuum System, capable of

40、 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, capable of generating sufficient vacuum for the solvent vacuum filtration system (5.22) 5.22 Solvent vacuum filtration sys

41、tem, fitted with glass microfibre filter (5.16) BS EN 15792:2009EN 15792:2009 (E) 8 5.23 HPLC syringe filter unit, polyamide (nylon) with 0,45 m pore size 5.24 Ultrasonic bath 5.25 HPLC apparatus, comprising the following: 5.25.1 Injection system, manual or autosampler, with loop suitable for 100 l

42、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, Generally every RP-column is suitable that allows a sufficient separation of zearalenone from other interfering components.

43、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 have been found to be suitable. 5.25.4 Pre-column (optional), appropriate for the analytical column used 5.25.5 Fluorescence

44、 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 5.26 UV spectrophotometer, for checking the concentration of the stock solution (4.15) 6 Procedures 6.1 Sample preparation It

45、 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 prepared in accordance with European legislation where applicable 2. Samples should be finely ground and thoroughly mixed usi

46、ng 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 instances if the sample has been frozen allow it to thaw completely before sampling. Stir the sample thoroughly before removing

47、 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 solvent (4.10). Mix briefly by hand to obtain a homogeneous suspension then either shake for 1 h on a shaker (5.2), or sonica

48、te 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 filte

49、red extract into a 250 ml graduated cylinder with stopper (5.10). Dilute the extract in the cylinder with PBS (4.9) to the 150 ml mark. Mix and filtrate approximately 20 ml of this diluted extract through a glass microfiber filter (5.16) into a glass beaker by applying a slight vacuum (5.22). Do not apply too strong a vacuum in the beginning of the filtration process, as this can lead to turbid filtered extracts after filtration. Discard these 20 ml and filtrate another approximately 70 ml for analysis. Proceed