EN 15782:2009

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
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 3 - Feed additives and drugs
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 04-Mar-2024
• Completion Date: 14-Apr-2025
• Directive: 70/373/EC - Council Directive 70/373/EEC of 20 July 1970 on the introduction of Community methods of sampling and analysis for the official control of feeding-stuffs
• Mandate: M/382 - Mandate for standardisation addressed to CEN in the field of Methods of analysis for animal Feedingstuffs
• Ref Project: SIST EN 15782:2009 - Animal feeding stuffs - Determination of nicarbazin - High-performance liquid chromatographic method

Overview

EN 15782:2009 (CEN) defines a validated analytical procedure for the determination of nicarbazin additive use in animal feedingstuffs and premixtures (maximum 2.5% nicarbazin) using high-performance liquid chromatography (HPLC). Nicarbazin is an equimolar mixture of 4,4'-dinitrocarbanilide (DNC) and 4,6-dimethyl-2-pyriminol (HDP); this method targets the DNC moiety. The method provides a declared limit of quantitation (LOQ) of 20 mg/kg and a limit of detection (LOD) of 0.5 mg/kg, with a note that a lower LOQ may be achievable but must be validated by the user.

Key practical controls in the method include protecting samples from light to prevent photon-degradation of nicarbazin and preparing calibration standards daily.

Key Topics

• Analyte: Nicarbazin (DNC as target compound)
• Sample types: Animal feedingstuffs and premixtures (premix concentration up to 2.5%)
• Extraction solvent: 50:50 v/v acetonitrile : methanol
• HPLC eluent: 65:35 v/v acetonitrile : purified water (degassed)
• HPLC conditions (guidance):
  • Reversed-phase C18 column (250 × 4.6 mm, 5 µm)
  • Isocratic elution at 1.0 ml/min
  • UV detection at 350 nm
  • Injection volume 20 µl
• Sample preparation:
  • Premixtures: 1.00 g extracted to 200 ml
  • Feedingstuffs: 2.50 g extracted to 100 ml after wetting with water
  • Mechanical shaking, sonication and filtration (0.2–0.5 µm) prior to injection
• Calibration and quantification: Daily working standards from a 100 µg/ml stock; calibration range provided in the standard. Calculation converts measured concentration in extract to mg/kg in the original sample.
• Quality considerations: Protect extracts from daylight; verify stock solubility and UV response; perform dilutions to fit calibration range.

Applications

• Routine quality control of feed production and premix manufacturing.
• Regulatory compliance testing where monitoring of coccidiostat additives is required.
• Method implementation in official or contract testing laboratories for surveillance of additive levels, verifying LOQ/LOD and recovery in specific matrices.
• Useful baseline for method validation, interlaboratory studies and proficiency testing (the standard includes precision data from a multi-lab study).

Related Standards

• EN ISO 6497 - Animal feeding stuffs - Sampling
• ISO 6498 - Animal feeding stuffs - Preparation of test samples
• EN ISO 3696 - Water for analytical laboratory use
• Relevant CANFAS reports and interlaboratory study literature cited in the standard for method development and validation

For laboratories adopting EN 15782:2009, follow the method steps, verify performance in your matrix, and record operating details in the test report to ensure traceability for QC and compliance purposes.

Standards Content (Sample)

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
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

Results of an interlaboratory study .9 Bibliography . 10
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

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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