SIST EN 15781:2009

Animal feeding stuffs - Determination of maduramicin-ammonium by reversed-phase HPLC using post-column derivatisation

This European Standard specifies a high performance liquid chromatography (HPLC) method for the
determination of the content of maduramicin in feeding stuffs and premixtures.
The usual concentration of maduramicin in feedstuffs is 5 mg/kg, in premixtures 500 mg/kg. The limit of
quantification is 2 mg/kg. The limit of detection is 0,5 mg/kg.
NOTE A lower limit of quantification may be achievable but shall be validated by the user.

Futtermittel - Bestimmung von Maduramicin-Ammonium durch Umkehrphasen HPLC-Verfahren mittels Nachsäulenderivatisierung

Diese Europäische Norm legt ein hochleistungsflüssigchromatographisches (HPLC-) Verfahren zur Bestimmung
des Gehaltes von Maduramicin in Futtermitteln und Vormischungen fest.
Die übliche Konzentration von Maduramicin in Futtermitteln beträgt 5 mg/kg, in Vormischungen 500 mg/kg.
Die Bestimmungsgrenze beträgt 2 mg/kg. Die Nachweisgrenze beträgt 0,5 mg/kg.
ANMERKUNG Eine niedrigere Bestimmungsgrenze lässt sich erreichen, jedoch muss sie durch den Anwender validiert
werden.

Aliments des animaux - Détermination de la maduramicine ammonium par HPLC en phase inverse à l'aide de la dérivation post-colonne

La présente Norme européenne spécifie une méthode de chromatographie liquide haute performance (HPLC)
pour déterminer la teneur en maduramicine dans les aliments et les prémélanges.
La teneur en maduramicine est de 5 mg/kg dans les aliments, de 500 mg/kg dans les prémélanges. La limite
de quantification est de 2 mg/kg. La limite de détection est de 0,5 mg/kg.
NOTE Une limite de quantification plus faible peut être atteignable mais elle doit être validée par l’utilisateur.

Krma - Določevanje amonijevega maduramicina s tekočinsko kromatografijo visoke ločljivosti s postkolonsko derivatizacijo

General Information

• Status: Published
• Publication Date: 16-Nov-2009
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 03-Nov-2009
• Due Date: 08-Jan-2010
• Completion Date: 17-Nov-2009
• 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: EN 15781:2009 - Animal feeding stuffs - Determination of maduramicin-ammonium by reversed-phase HPLC using post-column derivatisation

Overview - EN 15781:2009 (maduramicin in feed by HPLC)

EN 15781:2009 is a CEN European Standard that specifies a validated analytical method for the determination of maduramicin‑ammonium in animal feeding stuffs and premixtures. The method uses reversed‑phase high performance liquid chromatography (HPLC) with post‑column derivatisation (vanillin reagent) and UV detection at 520 nm. Typical concentration ranges covered are about 5 mg/kg in feedstuffs and 500 mg/kg in premixtures. The standard reports a limit of quantification (LOQ) of 2 mg/kg and a limit of detection (LOD) of 0.5 mg/kg (users may validate lower LOQs).

Key topics and technical requirements

• Scope: Determination of maduramicin in feeding stuffs and premixtures using reversed‑phase HPLC with post‑column derivatisation.
• Sample extraction: Methanol extraction of ground samples (feed: 10 g; premixes: 1 g) with ultrasonic bath and fat removal steps.
• Chromatography: C18 column (250 x 4.6 mm, 5 µm), mobile phase based on phosphate buffer:methanol, column oven 40°C, flow 0.4 ml/min, injection 50 µl, retention ~25 min.
• Post‑column derivatisation: Vanillin reagent (daily fresh, cooled) delivered by a reagent pump (~0.4 ml/min) and reacted at ~95°C; Annex B notes DMAB as an alternative reagent (requires user validation).
• Calibration & quantification: Multi‑point calibration solutions, replicate injections, calculation by reference to calibration graph; recovery check (90–110% acceptable).
• Performance & precision: Results from an interlaboratory study (10 labs) are summarized; repeatability and reproducibility limits defined in the standard.
• Safety & quality: Maduramicin is highly toxic - the standard emphasizes laboratory safety, reagent grades, and use of EN ISO 3696 water.

Applications - who uses EN 15781

• Feed analysis laboratories performing routine QC/assurance testing for ionophore residues.
• Feed manufacturers validating premixture and finished‑feed concentrations.
• Regulatory and compliance bodies monitoring maduramicin levels in commercial feeds.
• Contract testing labs offering accredited HPLC methods for veterinary drug residues.
• Method developers and instrument vendors integrating post‑column derivatisation systems.

Related standards and references

• EN ISO 3696 - Water for analytical laboratory use (cited for reagent quality)
• ISO 6497 / ISO 6498 - Recommended sampling and sample preparation methods (referenced for representative sampling and test portion preparation)
• Annex A (EN 15781) - Interlaboratory study results; Annex B - DMAB post‑column conditions

EN 15781:2009 is practical for ensuring accurate, traceable measurement of maduramicin in feeds, supporting food‑animal safety, regulatory compliance and quality control.

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.UDILMRFuttermittel - Bestimmung von Maduramicin-Ammonium durch Umkehrphasen HPLC-Verfahren mittels NachsäulenderivatisierungAliments des animaux - Détermination de la maduramicine ammonium par HPLC en phase inverse à l'aide de la dérivation post-colonneAnimal feeding stuffs - Determination of maduramicin-ammonium by reversed-phase HPLC using post-column derivatisation65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 15781:2009SIST EN 15781:2009en,fr,de01-december-2009SIST EN 15781:2009SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15781August 2009ICS 65.120 English VersionAnimal feeding stuffs - Determination of maduramicin-ammonium by reversed-phase HPLC using post-columnderivatisationAliments des animaux - Détermination de la maduramicineammonium par HPLC en phase inverse à l'aide de ladérivation post-colonneFuttermittel - Bestimmung von Maduramicin-Ammoniumdurch Umkehrphasen HPLC-Verfahren mittelsNachsäulenderivatisierungThis 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 15781:2009: ESIST EN 15781:2009

Results of the interlaboratory study . 10 Annex B (informative)
Conditions for post-column derivatisation with DMAB . 12 Bibliography . 13
1 Scope This European Standard specifies a high performance liquid chromatography (HPLC) method for the determination of the content of maduramicin in feeding stuffs and premixtures.
The usual concentration of maduramicin in feedstuffs is 5 mg/kg, in premixtures 500 mg/kg. The limit of quantification is 2 mg/kg. The limit of detection is 0,5 mg/kg. NOTE A lower limit of quantification may be achievable but shall be validated by the user. 2 Normative reference(s) The following referenced documents are indispensable for the application of this document. 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 The sample is extracted with methanol. Maduramicin is determined by reversed-phase HPLC using post-column derivatization with vanillin and detection at 520 nm.
4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. WARNING — Use all solvents and solutions in a fume hood. Wear safety glasses, protective clothing and avoid skin contact. 4.1 Water, complying with EN-ISO 3696, grade 1 4.2 Methanol (CH3OH), HPLC grade 4.3 1,5-Dimethylhexylamine (CH3(CH2)4CH2N(CH3)2) 4.4 Sulfuric acid (H2SO4), purity 95% to 97% by volume 4.5 Ortho-phosphoric acid, (H3PO4), purity approximately 85% by volume 4.5.1 Diluted o-phosphoric acid Dissolve 10 ml of ortho-phosphoric acid (4.5) to 100 ml with water (4.1). 4.6 Potassium dihydrogen phosphate (KH2PO4) SIST EN 15781:2009

Vanillin, 4-hydroxy-3-methoxybenzaldehyde, minimum 98% purity by volume (HPLC grade) 4.9.1 Vanillin reagent Dissolve 10 g of vanillin (4.9) in a mixture of 250 ml of methanol (4.2) and 5,0 ml of sulphuric acid (4.4). Mix well and sonicate for some minutes under vacuum at room temperature. This solution has to be prepared daily prior to use and has to be cooled with ice water during use. NOTE Dimethylaminobenzaldehyde (DMAB) is also suitable as a reagent for post-column derivatisation (details are given in Annex B) although a full validation of this reagent has not been performed.
4.10 Maduramicin WARNING — Maduramicin is very toxic. Avoid inhalation and exposure to the toxic standard material and solutions thereof. 4.11 Standard solutions 4.11.1 Stock-standard-solution, 100 µµµµg/ml Accurately weigh 10 mg to the nearest 0,1 mg maduramicin (4.10) into a 100 ml volumetric flask. Dissolve in methanol (4.2) and dilute to volume. Store below 4°C. Prepare fresh every month. 4.11.2 Standard solution, 10 µµµµg/ml Dilute 10 ml of the stock-standard-solution (4.11.1) to 100 ml with methanol (4.2) in a 100 ml volumetric flask. Store below 4°C. Prepare fresh every week. 4.11.3 Calibration solutions The interlaboratory study was performed with 8 calibration solutions. Into a series of 50 ml volumetric flasks transfer 1,0 ml, 2,0 ml, 3,0 ml, 4,0 ml, 5,0 ml, 6,0 ml, 8,0 ml and 10,0 ml of the intermediate standard solution (4.11.2). Dilute to volume with methanol (4.2) and mix. These solutions correspond to 0,2 µg, 0,4 µg, 0,6 µg, 0,8 µg, 1,0 µg, 1,2 µg, 1,6 µg, and 2,0 µg of maduramicin per ml respectively. Alternatively, you may use 5 calibration solutions with maduramicin concentrations of 0,4 µg, 0,7 µg, 1,0 µg, 1,5 µg and 2,0 µg per ml respectively. Calibration solutions should be prepared on the day of analysis. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Centrifuge SIST EN 15781:2009
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