1、November 2009DEUTSCHE NORM English price group 8No 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!$ZaF“1556235www
2、.din.deDDIN EN 15782Animal feeding stuffs Determination of nicarbazin High-performance liquid chromatographic methodEnglish version of DIN EN 15782:2009-11Futtermittel Bestimmung von Nicarbazin Hochleistungsflssigchromatographisches VerfahrenEnglische Fassung DIN EN 15782:2009-11www.beuth.deDocument
3、 comprises pages12DIN EN 15782:2009-11 National foreword This standard has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Lebensm
4、ittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-03-03 AA Futtermittel. The DIN Standards corresponding to the International Standards referred to in this document are as follows, whereby EN ISO Standards are only listed below if
5、 these have not been published as DIN EN ISO Standards with the same number: EN ISO 3696 DIN ISO 3696 EN ISO 6498 DIN EN ISO 6498 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods E DIN EN ISO 6498, Animal feeding stuffs Gui
6、delines for sample preparation 2 EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15782August 2009ICS 65.120English VersionAnimal feeding stuffs - Determination of nicarbazin - High-performance liquid chromatographic methodAliments des animaux - Dtermination de la nicarbazine -Mthode de chromatogra
7、phie liquide hautes performancesFuttermittel - Bestimmung von Nicarbazin -Hochleistungsflssigchromatographisches VerfahrenThis European Standard was approved by CEN on 3 July 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
8、 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 Standard exists in three official versions (Eng
9、lish, 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 standards bodies of Austria, Belgium, Bulgaria,
10、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 STANDARDIZATIONCO
11、MIT 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 15782:2009: EEN 15782:2009 (E) 2 Contents Page Foreword 3 1 Scope 4 2
12、 Principle 4 3 Reagents .4 4 Apparatus .5 5 Sampling .5 6 Preparation of test sample 5 7 Procedure .6 8 Calculation and expression of results .7 9 Precision .8 10 Test report 8 Annex A (informative) Results of an interlaboratory study 9 Bibliography . 10 DIN EN 15782:2009-11 EN 15782:2009 (E) 3 Fore
13、word This document (EN 15782:2009) 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 endorsement, at the lates
14、t by February 2010, and conflicting national standards shall be withdrawn at the latest by February 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 identifying any or all
15、such patent rights. 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, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Icela
16、nd, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15782:2009-11 EN 15782:2009 (E) 4 1 Scope This European Standard specifies a method for the determination of additiv
17、e use of nicarbazin in animal feeding stuffs and premixtures (maximum concentration 2,5% nicarbazin) using high performance liquid chromatography. Nicarbazin is a 1:1 equimolar mixture of 4,4-dinitrocarbanilide (DNC) and 4,6-dimethyl-2-pyriminol (HDP). Nicarbazin is generally determined by using DNC
18、 as the target compound. In this method the DNC moiety of nicarbazin is detected. The limit of quantitation is 20 mg/kg. The limit of detection is 0,5 mg/kg NOTE A lower limit of quantitation may be achievable but should be validated by the user. 2 Principle Samples are extracted using an acetonitri
19、le/methanol mixture. For feeding stuffs, water is added additionally. An aliquot of the extract is assayed using a reverse phase isocratic HPLC method which measures the 4,4dinitrocarbanilide moiety at a wavelength of 350 nm. 3 Reagents Use only reagents of recognized analytical grade, unless otherw
20、ise specified. 3.1 Water, resistance 10 MOhm.cm-1.3.2 Acetonitrile (CH3CN), HPLC grade. 3.3 Methanol (CH3OH), HPLC grade. 3.4 Extraction solvent Mix 500 ml of acetonitrile (3.2) with 500 ml of methanol (3.3). Mix well using a magnetic stir plate and stir bar. 3.5 Eluent for liquid chromatography Mix
21、 650 ml acetonitrile (3.2) with 350 ml of purified water (3.1). Mix well using a magnetic stir plate and stir bar and degas (e.g. with helium) before use. 3.6 Nicarbazin reference standard 3.7 Standard solutions 3.7.1 Nicarbazin stock standard solution, 100 g/ml Dissolve 10 mg, weighed to the neares
22、t 0,1 mg, of nicarbazin reference standard (3.6) in 100 ml extraction solvent (3.4). To aid with dissolution, sonication for approximately 5 min is recommended. Mix well. This solution is stable for 24 h when stored in subdued light at ambient or refrigerated storage conditions (see remark 3.7.1). N
23、OTE 1 The solubility of the nicarbazin reference standard in extraction solvent is critical. The nicarbazin concentrations in the prepared stock solutions must be monitored by use of a cuvet spectrophotometer as follows. Prepare a solution of 10 g/ml by diluting the prepared stock standard solution
24、(3.7.1) with acetonitrile. Record a UV-Vis spectrum between 220 nm and 450 nm using a mixture of methanol/acetonitrile (5:95 v/v) as a reference solution. The maximum DIN EN 15782:2009-11 EN 15782:2009 (E) 5 absorbance measured between 340 nm and 350 nm should be within a margin of 5% of the default
25、 value. The default value should be established in your own laboratory by preparing a stock standard solution in duplicate and monitoring the UV-Vis spectra as described above. The default value is the mean result of the duplicates. NOTE 2 The susceptibility of nicarbazin to photon-degradation is we
26、ll known. To avoid degradation during sample preparation and analysis, protect the samples and extracts from daylight at all times. 3.7.2 Nicarbazin working standard solutions Prepare a range of calibration working standards containing 0 g/ml; 0,25 g/ml; 0,5 g/ml; 1 g/ml; 2 g/ml; 5 g/ml and 10 g/ml
27、nicarbazin by diluting the stock standard solution (3.7.1) with HPLC eluent (3.5). Working standards must be prepared daily. 4 Apparatus Usual laboratory apparatus and, in particular, the following. 4.1 High performance liquid chromatography system consisting of the following: 4.1.1 An autosampler o
28、r manual injector set to inject a volume of 20 l. 4.1.2 A pump set to deliver a constant eluent flow rate of 1,0 ml/min. 4.1.3 HPLC column, 250 mm x 4,6 mm packed with RP C18, 5 m material, or equivalent. NOTE A Nova-Pak or Bonda-Pak column is recommended, but also other columns can be used provided
29、 that a satisfactory separation of DNC is achieved. 4.1.4 A detector allowing the measurement of absorbance of UV light at a wavelength of 350 nm. 4.2 Mechanical shaker (e.g. Gyratory shaker, wrist action shaker or equivalent). 4.3 Micro filters for sample filtration, 0,2 m to 0,5 m. 4.4 Ultrasonic
30、bath 4.5 Waterbath, 50C 2C. 5 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this European Standard. A recommended sampling method is given in EN ISO 649
31、7. 6 Preparation of test sample 6.1 General The milling and mixing of compound feed samples prior to assay is obligatory. Grind feed samples through a DIN EN 15782:2009-11 EN 15782:2009 (E) 6 mill equipped with a 1 mm screen. After milling, mix the entire sample thoroughly. Store the sample at room
32、temperature in subdued light. Premix samples are not milled. NOTE The susceptibility of nicarbazin to photon-degradation is well known. To avoid degradation during sample preparation and analysis, protect the samples and extracts from daylight at all times. 6.2 Mixing of the test samples before weig
33、hing The container should be filled to a maximum of 50% of the total volume. Bring the container in a horizontal position and rotate bottom and top of the container in circles moving the container up and down along the virtual centre of the container for 30 sec. Put the container in an upright posit
34、ion and wait a few seconds for settlement of the generated dust. 6.3 Spiked feed samples; 100 mg/kg Transfer 2,5 ml of the stock standard solution (3.7.1) in the sample tube or flask. Evaporate to a small volume (less than 0,5 ml) with a gentle stream of nitrogen, add 2,5 g blank feed, mix thoroughl
35、y and wait 10 min before starting the extraction procedure by adding water for swelling (see 7.2.2). 7 Procedure 7.1 General Complete each assay within one working day. 7.2 Extraction 7.2.1 Premixtures Accurately weigh 1 g to the nearest 0,01 g of the test sample directly into a volumetric flask of
36、200 ml. Add 80 ml of extraction solvent (3.4), close the flask and mix manually by swirling. Put the flasks in a waterbath of 50C (4.5) for at least 15 min with intermediate swirling at 8 min. Mix thoroughly for 15 min using a mechanical means (4.2). Put the flasks in an ultrasonic bath (4.4) and so
37、nicate for 15 min. Cool down to room-temperature, adjust to volume with HPLC eluent (3.5) and mix. Allow sample solids to settle (minimum 30 min). If additional dilutions are required, dilute the samples with HPLC eluent (3.5) to a final nicarbazin concentration which falls within the standard curve
38、 levels (3.7.2). Filter an aliquot of the final dilution through a micro filter (4.3) for analysis by HPLC. 7.2.2 Animal feeding stuffs Accurately weigh 2,5 g to the nearest 0,01 g of the test sample in a 50 ml disposable centrifuge tube or directly in a volumetric flask of 100 ml. Add 5 ml of water
39、. Take care that the whole sample becomes wet. DIN EN 15782:2009-11 EN 15782:2009 (E) 7 Wait at least 10 min. Add 35 ml of extraction solvent (3.4), close the tube or flask and mix manually by swirling. Put the tubes or flasks in a waterbath of 50C (4.5) for 15 min with intermediate swirling at 8 mi
40、n. Mix thoroughly for 15 min using a mechanical means (4.2). Put the tubes or flasks in an ultrasonic bath (4.4) and sonicate for 15 min. If using disposable centrifuge tubes, transfer the sample extract quantitatively into a 100 ml volumetric flask with HPLC eluent (3.5), adjust to volume and mix.
41、If additional dilutions are required, allow samples to settle (minimum 30 min) and dilute the samples with HPLC eluent (3.5) to a final nicarbazin concentration which falls within the standard curve levels. Filter an aliquot of the final dilution through a micro filter (4.3) for analysis by HPLC. NO
42、TE For relatively inhomogeneous compound feed samples, the weighed sample amount should be increased to 10 gram with simultaneous up-scaling of the volume of extraction solvent used. 7.3 HPLC determination 7.3.1 HPLC conditions The following conditions are offered for guidance, other conditions may
43、be used provided that they give equivalent results. analytical column as in 4.1.3 mobile phase as in 3.5 flow rate 1,0 ml/min detection wavelength 350 nm injection volume 20 l Check the stability of the chromatographic system, injecting several times the calibration solution (3.7.2) containing 1,0 g
44、/ml, until constant peak areas and retention times are achieved. 7.3.2 Calibration graph Inject each calibration solution (3.7.2) to determine the peak area/height for each concentration. Plot a calibration graph using the peak areas/heights of the calibration solutions as the ordinate and the corre
45、sponding concentrations in g/ml as the abscissae. 7.3.3 Sample solution Inject the sample extract (7.2.1 or 7.2.2) at least 2 times using the same volume as taken for the calibration solutions and determine the peak area/height of the DNC peaks. 8 Calculation and expression of results Calculate the
46、mass fraction of nicarbazin in the test sample by the equation: DIN EN 15782:2009-11 EN 15782:2009 (E) 8 dneEfmVw =(1) where wEis the numerical value for the mass fraction of nicarbazin in the test sample in mg/kg; Veis the extraction volume, in ml, viz. 200 for premixtures (7.2.1) and 100 for feedi
47、ng stuffs (7.2.2); nis the numerical value of the nicarbazin mass in concentration in the sample extract in g/ml; m is the numerical value of the mass of the test sample, in g; fdis the dilution factor introduced to prepare final sample extracts fitting with the standard curve levels. 9 Precision 9.
48、1 Collaborative study Details of the collaborative study of the method are summarized in Annex A. The values derived from this collaborative study may not be applicable to concentration ranges and matrices other than those given. 9.2 Repeatability The absolute difference between two independent sing
49、le test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5% of the cases exceed the repeatability limit r . 9.3 Reproducibility The absolute difference between two single test results, obtained using the same method on identical test material in different laboratories with different operators using different equipment will in not more than 5% of the cases exceed the reproducibility limit R. 9.4
