EN 16162:2012
(Main)Animal feeding stuffs - Determination of decoquinate by HPLC with fluorescence detection
Animal feeding stuffs - Determination of decoquinate by HPLC with fluorescence detection
This European Standard specifies a method for the determination of decoquinate. This high-performance liquid chromatographic (HPLC) method with a fluorescence detection is applicable to the quantification of decoquinate content in complete and complementary compound feeds, medicated feeds, semi-liquid feeds, premixtures and feed additives.
The method was fully validated from LOQ to 60 000 mg/kg on different matrices during an international collaborative study [11], especially on complete compound feeds for poultry, at trace contamination level of 3 mg/kg and at European authorized level of 20 mg/kg to 40 mg/kg [12].
The limit of detection is between 0,1 mg/kg and 0,3 mg/kg and the limit of quantification is around 0,5 mg/kg. These limits were validated during the collaborative study [11], from results on the blank feed. Lower limits of detection or quantification could be reached but a single laboratory validation is then requested.
Futtermittel - Bestimmung von Decoquinat mit Hochleistungs-Flüssigchromatographie (HPLC) und Fluoreszenzdetektion
Diese Europäische Norm legt ein Verfahren zur Bestimmung von Decoquinat fest. Dieses hochleistungsflüssigchromatographische (HPLC) Verfahren mit Fluoreszenzdetektion ist für die Quantifizierung des Decoquinatgehaltes in Allein- und Ergänzungsfuttermitteln, Medizinalfutter, semi-Flüssigfutter, Vormischungen und Futterzusatzstoffen geeignet.
Das Verfahren wurde vollständig für verschiedene Matrices für den Bereich von LOQ bis 60 000 mg/kg während eines international durchgeführten Ringversuches [11] validiert, besonders für Alleinfuttermittel für Geflügel, bei einem Spurenverunreinigungsgehalt von 3 mg/kg und bei einem europäisch zugelassenen Gehalt von 20 mg/kg bis 40 mg/kg [12].
Die Nachweisgrenze liegt zwischen 0,1 mg/kg und 0,3 mg/kg und die Bestimmungsgrenze ungefähr bei 0,5 mg/kg. Diese Grenzen wurden während des Ringversuchs [11] anhand der mit dem Kontrollfuttermittel ohne Zusatz erzielten Ergebnisse validiert. Niedrigere Nachweis und Bestimmungsgrenzen könnten erreicht werden, jedoch ist dann eine Validierung durch ein Einzellaboratorium erforderlich.
Aliments des animaux - Dosage du décoquinate par chromatographie liquide à haute performance (CLHP) avec détection par fluorescence
La présente Norme européenne spécifie une méthode de dosage du décoquinate. La présente méthode de chromatographie liquide à haute performance (CLHP) avec détection par fluorescence est applicable à la détermination de la teneur en décoquinate dans les aliments composés complets et complémentaires pour animaux, les aliments médicamenteux, les aliments semi-liquides, les prémélanges et additifs utilisés en alimentation animale.
La méthode a été validée dans sa totalité, de la limite de quantification à 60 000 mg/kg, selon différentes matrices, lors d’un essai interlaboratoires [11], en particulier pour les aliments complets pour volailles, au niveau de la contamination à l’état de traces de 3 mg/kg et à la teneur autorisée en Europe comprise entre 20 mg/kg et 40 mg/kg [12].
La limite de détection est comprise entre 0,1 mg/kg et 0,3 mg/kg et la limite de quantification est d’environ 0,5 mg/kg. Ces limites ont été validées lors d’un essai interlaboratoires [11], à partir des résultats obtenus sur l’aliment témoin. Des limites de détection ou de quantification inférieures pourraient être atteintes à condition d’être validées en interne (validation intralaboratoire).
Krma - Določevanje dekokvinata s HPLC s fluorescentno detekcijo
Ta evropski standard določa metodo za določevanje dekokvinata. Ta metoda tekočinske kromatografije visoke ločljivosti (HPLC) s fluorescenčnim zaznavanjem se uporablja za kvantifikacijo vsebnosti dekokvinata v polnovrednih in dopolnilnih krmnih mešanicah, medicirani krmi, poltekoči krmi, premiksih in krmnih dodatkih. Metoda je bila z mednarodno medlaboratorijsko študijo v celoti validirana za razpon od meje kvantifikacije do 60.000 mg/kg na različnih matricah, zlasti na polnovrednih krmnih mešanicah za perutnino pri vrednosti onesnaženja v sledovih 3 mg/kg in evropski dovoljeni vrednosti 20 mg/kg do 40 mg/kg. Meja detekcije je med 0,1 mg/kg in 0,3 mg/kg, meja kvantifikacije pa približno 0,5 mg/kg. Ti meji sta bili validirani z medlaboratorijsko študijo na podlagi rezultatov iz slepe krme. Doseči je mogoče nižjo mejo detekcije ali kvantifikacije, vendar se za to zahteva ena sama laboratorijska validacija.
General Information
Overview
EN 16162:2012 (CEN) defines a validated analytical method for the determination of decoquinate in animal feeding stuffs using HPLC with fluorescence detection. Published March 2012, this European Standard covers complete and complementary compound feeds, medicated and semi‑liquid feeds, premixtures and feed additives. The method was validated across multiple matrices from the limit of quantification (LOQ) up to 60 000 mg/kg, with demonstrated trace-level performance and suitability for regulatory control and quality assurance.
Key topics and technical requirements
- Analytical principle: Reversed‑phase HPLC with fluorescence detection; positive trace samples confirmed using an alternate excitation wavelength.
- Sample extraction: Decoquinate is extracted using a solution of 1% calcium chloride in methanol with mechanical shaking (≈60 min), followed by centrifugation or filtration.
- Chromatography & detection: Typical configuration uses a C18 column (5 µm, 4.6 × 250 mm) and fluorescence detector (excitation wavelengths ~330 nm and 260 nm; emission 390 nm). Mobile phase: methanol/water (825/175 v/v) containing calcium chloride.
- Calibration & standards: Stock and calibration solutions prepared from certified decoquinate reference standard; calibration range supports quantification down to target extract concentrations (≈0.3–0.6 µg/ml).
- Performance characteristics:
- Limit of detection (LOD): between 0.1 mg/kg and 0.3 mg/kg
- Limit of quantification (LOQ): around 0.5 mg/kg (validated in collaborative study)
- Validated at trace contamination level (3 mg/kg) and at common European authorized levels 20–40 mg/kg.
- Quality control: Use of blank and spiked feeds, duplicated analyses recommended, and procedural requirements for premixtures and high‑concentration samples.
Practical applications
- Regulatory monitoring of decoquinate residues in animal feed to ensure compliance with authorized levels.
- Feed mill quality control to detect cross‑contamination from medicated feeds.
- Routine laboratory testing of feed additives, premixtures and finished feeds (including poultry feeds, where validation focused).
- Confirmation of suspect trace positives using alternate excitation parameters for robust identification.
Who should use this standard
- Accredited feed testing laboratories and analytical chemists.
- National and regional regulatory authorities performing surveillance of animal feeding stuffs.
- Feed manufacturers, QA/QC teams and formulators handling medicated feeds and feed additives.
- Proficiency testing providers and method validators.
Related standards
- prEN ISO 6498 - Guidelines for sample preparation (normative reference in EN 16162).
- EN ISO 6497 - Recommended sampling procedures (referenced for representative sampling).
Keywords: EN 16162:2012, decoquinate, HPLC, fluorescence detection, animal feed analysis, limit of detection, limit of quantification, feed additives, premixtures, CEN.
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Bestimmung von Decoquinat mit Hochleistungs-Flüssigchromatographie (HPLC) und FluoreszenzdetektionAliments des animaux - Dosage du décoquinate par CLHP avec détection par fluorescenceAnimal feeding stuffs - Determination of Decoquinate by HPLC with fluorescence detection65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16162:2012SIST EN 16162:2012en,fr,de01-september-2012SIST EN 16162:2012SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16162
March 2012 ICS 65.120 English Version
Animal feeding stuffs - Determination of decoquinate by HPLC with fluorescence detection
Aliments des animaux - Détermination du décoquinate par Chromatographie Liquide Haute Performance avec détection fluorimétrique
Futtermittel - Bestimmung von Decoquinat mit Hochleistungs-Flüssigchromatographie (HPLC) und Fluoreszenzdetektion This European Standard was approved by CEN on 4 February 2012.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC 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 translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, 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, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16162:2012: ESIST EN 16162:2012
Results inter-laboratory study . 13Annex B (informative)
Additional recovery results . 17B.1Recoveries from the familiarization phase . 17B.2Pre-trial; recovery test on a milk-replacer . 18B.3Recoveries from the single-laboratory validation . 18Annex C (informative)
Additional results for robustness purposes . 19C.1Robustness in terms of excitation wavelength, during the final collaborative study . 19C.2Comparison between ground and unground samples during the final collaborative study . 19C.3Robustness from one laboratory during the final collaborative study . 20Annex D (informative)
HPLC parameters and chromatogram examples . 22D.1General . 22D.2Chromatogram example: Calibration point at 0,6 µg/ml . 22D.3Chromatogram example: Commercial Lamb feed containing around 50 mg/kg of decoquinate22D.4Chromatogram example: same commercial Lamb feed, as in D.3, containing around 50 mg/kg of decoquinate . 23SIST EN 16162:2012
The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. prEN ISO 6498, Animal feeding stuffs — Guidelines for sample preparation (ISO/DIS 6498) 3 Principle Decoquinate is extracted from samples with a solution of 1 % calcium chloride in methanol using mechanical shaking or stirring for 60 min. After centrifugation or filtration, an aliquot is, if necessary, diluted with the extraction solvent and analysed by reversed phase HPLC with fluorescence detection. Positive trace level samples should be confirmed by HPLC analysis using an alternate excitation wavelength. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralised water or water of equivalent purity. WARNING — This method requires the handling of hazardous substances. It is recommended to use various regulations for potentially hazardous chemicals. Organisational, technical and personal safety has to be observed. 4.1 Methanol, HPLC grade. 4.2 Methanol, technical grade. 4.3 Calcium chloride anhydrous or Calcium chloride dihydrate, each > 99 % purity. 4.4 HPLC dilution solution. Dissolve a mass of calcium salt (4.3) equivalent to 10 g of calcium chloride anhydrous in methanol (4.1). Mix well and make up to 1 000 ml. 4.5 Extraction solvent. SIST EN 16162:2012
Evaluate precisely each exact concentration by using the exact concentration of the stock solution (4.6.2). All solutions described here (standards A/B/C/D) are prepared fresh daily. 4.7 HPLC Mobile Phase. Dissolve a mass of calcium salt (4.3) equivalent to 10 g calcium chloride anhydrous into 1 l of a solvent mixture of methanol (4.1) / water in proportion by volume of 825/175. Filter under vacuum (5.2) before use. NOTE HPLC shutdown solution. Prepare a methanol (4.1) / water solution in proportion by volume of 85/15 without calcium salt to flush the HPLC column and equipment at the end of each day of analysis. SIST EN 16162:2012
Blank feed to spike at 9 mg/kg Accurately weigh, to the nearest 0,1 g, 10,0 g of quality control blank feed in a 250 ml amber conical flask. Add 15 ml of intermediate solution (4.6.3.1). Wait for 15 min. Then add 85 ml of the extraction solution (4.5). Proceed as described in 8.4. The recovery shall be from 80 % to 120 %. 8.6 HPLC parameters These HPLC parameters are given for guidance. Other parameters should be applicable (column, flow rate adapted to HPLC columns and optimum response of the fluorescence detector…). Analytical column: C18 column; 5 µm; 4.6 mm x 250 mm as described in 5.4.3; Column Temperature: ambient or 30 °C; Mobile phase (4.7): MeOH/water in proportion by volume of 825/
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Frequently Asked Questions
EN 16162:2012 is a standard published by the European Committee for Standardization (CEN). Its full title is "Animal feeding stuffs - Determination of decoquinate by HPLC with fluorescence detection". This standard covers: This European Standard specifies a method for the determination of decoquinate. This high-performance liquid chromatographic (HPLC) method with a fluorescence detection is applicable to the quantification of decoquinate content in complete and complementary compound feeds, medicated feeds, semi-liquid feeds, premixtures and feed additives. The method was fully validated from LOQ to 60 000 mg/kg on different matrices during an international collaborative study [11], especially on complete compound feeds for poultry, at trace contamination level of 3 mg/kg and at European authorized level of 20 mg/kg to 40 mg/kg [12]. The limit of detection is between 0,1 mg/kg and 0,3 mg/kg and the limit of quantification is around 0,5 mg/kg. These limits were validated during the collaborative study [11], from results on the blank feed. Lower limits of detection or quantification could be reached but a single laboratory validation is then requested.
This European Standard specifies a method for the determination of decoquinate. This high-performance liquid chromatographic (HPLC) method with a fluorescence detection is applicable to the quantification of decoquinate content in complete and complementary compound feeds, medicated feeds, semi-liquid feeds, premixtures and feed additives. The method was fully validated from LOQ to 60 000 mg/kg on different matrices during an international collaborative study [11], especially on complete compound feeds for poultry, at trace contamination level of 3 mg/kg and at European authorized level of 20 mg/kg to 40 mg/kg [12]. The limit of detection is between 0,1 mg/kg and 0,3 mg/kg and the limit of quantification is around 0,5 mg/kg. These limits were validated during the collaborative study [11], from results on the blank feed. Lower limits of detection or quantification could be reached but a single laboratory validation is then requested.
EN 16162:2012 is classified under the following ICS (International Classification for Standards) categories: 65.120 - Animal feeding stuffs. The ICS classification helps identify the subject area and facilitates finding related standards.
EN 16162:2012 is associated with the following European legislation: EU Directives/Regulations: 70/373/EC; Standardization Mandates: M/382. When a standard is cited in the Official Journal of the European Union, products manufactured in conformity with it benefit from a presumption of conformity with the essential requirements of the corresponding EU directive or regulation.
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SIST EN 16162:2012は、デコクアナイトの定量に関する方法を規定したヨーロッパ標準であり、動物飼料におけるこの化合物の分析において重要な役割を果たします。この標準は、高性能液体クロマトグラフィー（HPLC）を使用した蛍光検出法に基づいており、完全かつ補完的な飼料、医療飼料、半液体飼料、プレミックス、飼料添加物内のデコクアナイト含量を正確に定量することが可能です。 この標準の強みの一つは、国際的な共同研究を通じて、異なるマトリックスにおいて、定量限界（LOQ）から60,000 mg/kgまでの範囲で完全なバリデーションが行われている点です。特に、鶏用の完全飼料に対して、3 mg/kgという微量の汚染レベルや、ヨーロッパで許可されている20 mg/kgから40 mg/kgの範囲を対象として効力を発揮しています。このように、実際の飼料に基づいたデータを持ち、適切な評価がなされていることは、規格の信頼性を高める要素となっています。 また、検出限界が0.1 mg/kgから0.3 mg/kg、定量限界が約0.5 mg/kgと厳密に設定されているため、デコクアナイトの微量分析にも適用可能です。この限界は共同研究から得られた結果に基づいて検証されており、ひとつのラボによるバリデーションが必要であるものの、さらなる低い検出限界や定量限界の達成も可能である点が注目されます。 このように、SIST EN 16162:2012は、デコクアナイトの正確な測定が求められる飼料業界において、その手法の明確さと有効性から、非常に重要な標準であると言えます。この標準に従うことで、品質管理や安全性の向上に貢献し、作業の効率化にも繋がることでしょう。
SIST EN 16162:2012 표준은 동물 사료에서 데코쿠인산(decoquinate)의 정량을 위한 HPLC(고성능 액체 크로마토그래피) 및 형광 검출 방법을 규정하고 있습니다. 이 표준의 범위는 완전 및 보완 혼합 사료, 의약품 사료, 반액체 사료, 프리믹스 및 사료 첨가물의 데코쿠인산 함량을 정량화하는 데 적용 가능합니다. 이 방법은 다양한 매트릭스에서 LOQ(정량 한계)부터 60,000 mg/kg까지 완전히 검증되었으며, 특히 가금류를 위한 완전 혼합 사료에서 3 mg/kg의 추적 오염 수준과 유럽에서 승인된 20 mg/kg에서 40 mg/kg의 수준에서 적용되었습니다. 이러한 점에서 이 표준은 산업에서의 실용성과 신뢰성을 보장합니다. 검출 한계는 0.1 mg/kg에서 0.3 mg/kg 사이이며, 정량 한계는 약 0.5 mg/kg입니다. 이러한 한계는 협동 연구에서 검증되었으며, 불순물이 없는 사료에 대한 결과를 바탕으로 설정되었습니다. 더 낮은 검출 또는 정량 한계를 달성할 수 있지만, 이를 위해서는 단일 실험실 검증이 필요합니다. SIST EN 16162:2012 표준은 동물 사료의 안전성과 품질 관리를 위한 중요한 지침을 제공하며, 데코쿠인산의 정량화 방법에 대한 명확하고 신뢰할 수 있는 기준을 설정합니다. 이는 사료 산업에서 데코쿠인산의 규제 준수를 보장하기 위한 필수 자료로서, 관련 법규 쟁점 및 시장 수요에 대한 반응으로써 높은 중요성을 지니고 있습니다.
The EN 16162:2012 standard provides a comprehensive framework for the determination of decoquinate in various animal feeding stuffs. Its primary focus is on the use of high-performance liquid chromatography (HPLC) coupled with fluorescence detection, a method that ensures precise quantification of decoquinate content in a diverse range of feed categories, including complete and complementary compound feeds, medicated feeds, semi-liquid feeds, premixtures, and feed additives. One of the strengths of this standard lies in its extensive validation process. The HPLC method has been rigorously tested from the limit of quantification (LOQ) levels up to 60,000 mg/kg across various matrices. This demonstrates the method's reliability and versatility, particularly for complex matrices such as complete compound feeds for poultry. The validation achieved in international collaborative studies is a testament to its robustness, especially at critical contamination levels, thus enhancing the trustworthiness of results in regulatory contexts. Another notable aspect is the established limits of detection (LOD) and quantification (LOQ). The LOD ranges between 0.1 mg/kg and 0.3 mg/kg, while the LOQ hovers around 0.5 mg/kg. These figures are crucial for ensuring that even trace amounts of decoquinate can be detected and quantified effectively, adhering to European standards that demand safety and compliance in animal feed production. The ability to reach lower detection limits through single laboratory validation also offers flexibility and adaptability for specific testing requirements. The EN 16162:2012 standard is highly relevant in the context of animal feed safety and quality assurance. By providing a reliable methodology for quantifying decoquinate, it supports compliance with regulatory requirements and best practices in the industry. Its adoption is essential for manufacturers, quality assurance personnel, and regulatory bodies involved in the production and monitoring of animal feeding stuffs. Overall, the standard not only enhances the accuracy of decoquinate determination but also aids in safeguarding animal health and welfare through stringent feed control measures.
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