1、BS EN 15782:2009ICS 65.120,NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal feeding stuffs Determination ofnicarbazin High-performance liquidchromatographicmethodThis British Standardwas published under theauthority of the StandardsPolicy and StrategyCommi
2、ttee on 31 October2009 BSI 2009ISBN 978 0 580 61798 0Amendments/corrigenda issued since publicationDate CommentsBS EN 15782:2009National forewordThis British Standard is the UK implementation of EN 15782:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal f
3、eeding 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 its correct application.Compliance with a British Standard cannot confer im
4、munityfrom legal obligations.BS EN 15782:2009EUROPEAN 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
5、 chromatographie 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
6、 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 Standard exists in three official v
7、ersions (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 standards bodies of Austria, Belgium
8、, 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 Kingdom.EUROPEAN COMMITTEE FOR STAND
9、ARDIZATIONCOMIT 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: EBS EN 15782:2009EN 15782:2009 (E) 2 Contents
10、 Page Foreword 3 1 Scope 4 2 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 BS EN 15782:2009E
11、N 15782:2009 (E) 3 Foreword 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 en
12、dorsement, at the latest 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
13、identifying any or all 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,
14、 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 15782:2009EN 15782:2009 (E) 4 1 Scope This European Standard specifies a method for the determ
15、ination of additive 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 deter
16、mined by using DNC 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 u
17、sing an acetonitrile/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 gr
18、ade, unless otherwise 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
19、chromatography Mix 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, wei
20、ghed to the nearest 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 (se
21、e remark 3.7.1). NOTE 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
22、standard solution (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 BS EN 15782:2009EN 15782:2009 (E) 5 absorbance measured between 340 nm and 350 nm should be within a margin of 5% o
23、f the default 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-degr
24、adation is well 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
25、 and 10 g/ml 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
26、autosampler or 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
27、used provided 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.
28、4 Ultrasonic 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
29、in EN ISO 6497. 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 BS EN 15782:2009EN 15782:2009 (E) 6 mill equipped with a 1 mm screen. After milling, mix the entire sample thoroughly. Store the sample
30、at room 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 bef
31、ore weighing 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 uprig
32、ht position 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 t
33、horoughly 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 f
34、lask of 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
35、) and sonicate 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 standa
36、rd curve 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
37、of water. Take care that the whole sample becomes wet. BS EN 15782:2009EN 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
38、 min. 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 mi
39、x. 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.
40、 NOTE 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 m
41、ay 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,
42、0 g/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 co
43、rresponding 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 t
44、he mass fraction of nicarbazin in the test sample by the equation: BS EN 15782:2009EN 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 feeding
45、 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 9.1
46、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 single
47、 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
48、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 Recovery The recovery obtained for compound feeds should be in
49、 the range of 90% to 110% at spike levels between 20 mg/kg and 200 mg/kg. 10 Test report The test report shall specify: a) information necessary for the complete identification of the test sample; b) the sampling method used, if known; c) the test method used, with reference to this European Standard; d) operating details not specified in this European Standard, or regarded as optional, together with details of any incidents which may have influenced the test results(s); e) the test result(s) obtained, or, if the repeatability has been checked,
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