Animal feeding stuffs - Determination of nicarbazin - High-performance liquid chromatographic method

This European Standard specifies a method for the determination 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 determined 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.

Futtermittel - Bestimmung von Nicarbazin - Hochleistungsflüssigchromatographisches Verfahren

Diese Europäische Norm legt ein Verfahren zur Bestimmung von zugesetzten (eingemischten) Nicarbazin in
Futtermitteln und Vormischungen (maximale Konzentration 2,5 % Nicarbazin) mittels Hochleistungsflüssigchromatographie
fest. Nicarbazin ist ein 1 : 1 äquimolares Gemisch aus 4,4’-Dinitrocarbanilid (DNC) und
4,6-Dimethyl-2-pyriminol (HDP). Im Allgemeinen wird bei der Nicarbazin-Bestimmung DNC als Zielsubstanz
erfasst. In diesem Verfahren wird der DNC-Anteil von Nicarbazin bestimmt.
Die Bestimmungsgrenze beträgt 20 mg/kg. Die Nachweisgrenze beträgt 0,5 mg/kg.
ANMERKUNG Eine niedrigere Bestimmungsgrenze lässt sich erreichen, sie sollte jedoch durch den Anwender validiert
werden.

Aliments des animaux - Détermination de la nicarbazine - Méthode de chromatographie liquide haute performance

La présente Norme européenne spécifie une méthode permettant de déterminer l’utilisation d’un additif de
nicarbazine dans les aliments pour animaux et les pré-mélanges (concentration maximale en nicarbazine
2,5 %) à l’aide de la chromatographie liquide haute performance. La nicarbazine est un mélange
équimolaire 1:1 de 4,4’-dinitrocarbanilide (DNC) et de 4,6-diméthyl-2-pyrimidinol (HDP). La nicarbazine est
généralement déterminée par l’utilisation du DNC comme composé cible. La présente méthode permet de
détecter la fraction de DNC dans la nicarbazine.
La limite de quantification est de 20 mg/kg. La limite de détection est de 0,5 mg/kg.
NOTE Une limite de quantification plus faible est acceptable mais il convient que celle-ci soit validée par l’utilisateur.

Krma - Določevanje nikarbazina - Metoda tekočinske kromatografije visoke ločljivosti

General Information

Status
Published
Publication Date
04-Aug-2009
Current Stage
9060 - Closure of 2 Year Review Enquiry - Review Enquiry
Due Date
02-Sep-2018
Completion Date
02-Sep-2018

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.ORþOMLYRVWLFuttermittel - Bestimmung von Nicarbazin - Hochleistungsflüssigchromatographisches VerfahrenAliments des animaux - Détermination de la nicarbazine - Méthode de chromatographie liquide hautes performancesAnimal feeding stuffs - Determination of nicarbazin - High-performance liquid chromatographic method65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 15782:2009SIST EN 15782:2009en,fr,de01-december-2009SIST EN 15782:2009SLOVENSKI

STANDARD
SIST EN 15782:2009

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15782August 2009ICS 65.120 English VersionAnimal feeding stuffs - Determination of nicarbazin - High-performance liquid chromatographic methodAliments des animaux - Détermination de la nicarbazine -Méthode de chromatographie liquide hautes performancesFuttermittel - Bestimmung von Nicarbazin -Hochleistungsflüssigchromatographisches 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 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 (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, 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 STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:

Avenue Marnix 17,

B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15782:2009: ESIST EN 15782:2009

EN 15782:2009 (E) 2 Contents 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

SIST EN 15782:2009

EN 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 endorsement, 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 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, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. SIST EN 15782:2009

EN 15782:2009 (E) 4

1 Scope This European Standard specifies a method for the determination 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 determined 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 using 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 4,4’dinitrocarbanilide moiety at a wavelength of 350 nm. 3 Reagents Use only reagents of recognized analytical grade, 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 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 µµµµg/ml Dissolve 10 mg, weighed 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 (see 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 µg/ml by diluting the prepared stock 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 SIST EN 15782:2009

EN 15782:2009 (E) 5 absorbance measured between 340 nm and 350 nm should be within a margin of ± 5% of 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-degradation 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 µg/ml; 0,25 µg/ml; 0,5 µg/ml; 1 µg/ml; 2 µg/ml; 5 µg/ml and 10 µg/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 autosampler or manual injector set to inject a volume of 20 µl. 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 µm material, or equivalent.

NOTE A Nova-Pak or Bonda-Pak column is recommended, but also other columns can be 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

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