EN 16939:2017
(Main)Animal feeding stuffs: Methods of sampling and analysis - Detection of tylosin, spiramycin and virginiamycin - Thin Layer Chromatography and bioautography
General Information
- Abstract
The method makes it possible to detect and identify spiramycin, tylosin and virginiamycin in animal feeding stuffs (feed raw materials of mainly plant origin and compound feeds) excluding mineral feeds and premixtures. The limit of detection is about 2 mg/kg for spiramycin, 1 mg/kg for tylosin and 1 mg/kg for virginiamycin. In some milk replacers, it can be slightly higher than 1 mg/kg for virginiamycin.
Reported limits of detection are probably little overestimated but were fully validated during the collaborative study (see Annex B). In each laboratory, each day of analysis, spiked blank samples at 1 mg/kg for spiramycin and virginiamycin and at 0,5 mg/kg for tylosin are analysed for checking lower detection limits (see 9.2 and 9.3). These lower limits of detection are achievable, but should be established with an in-house validation first.
Some other antibiotics can interfere in the detection of these 3 specific macrolide antibiotics. The known interferences are specified in Annex A of the method.
That method should be used as a qualitative screening and/or a post-screening method (after microbiological plate test, for example). The follow-up of the antibiotics presence may be done by other analytical technics (LC and/or LC-MS technics) ([4], [10]). For confirmatory purposes, LCMS is required.
- Status
- Published
- Publication Date
- 08-Aug-2017
- Withdrawal Date
- 27-Feb-2018
- 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
- 01-Jan-2024
- Completion Date
- 23-Sep-2026
Overview
EN 16939:2017 (CEN) specifies a qualitative screening method for the detection and identification of three macrolide antibiotics - tylosin, spiramycin and virginiamycin - in animal feeding stuffs. The procedure uses Thin Layer Chromatography (TLC) combined with bioautography (agar seeded with Micrococcus luteus / Kocuria rhizophila ATCC 9341) after extraction and purification of feed samples. The method applies to plant‑origin feed materials and compound feeds, excluding mineral feeds and premixtures.
Key topics and technical requirements
- Scope and purpose: Screening and post‑screening detection of spiramycin, tylosin and virginiamycin in feed; not a standalone confirmatory technique.
- Analytical principle: Extraction with methanol/water, liquid–liquid partition into chloroform, concentration, TLC on silica gel, and identification by Rf values and inhibition zones via bioautography.
- Biological detection: Uses Micrococcus luteus (Kocuria rhizophila) ATCC 9341; specific antibiotic media formulations and pH adjustments improve diffusion and detection.
- Limits of detection (LOD): approximately 2 mg/kg for spiramycin, 1 mg/kg for tylosin and 1 mg/kg for virginiamycin (virginiamycin LOD can be slightly higher in some milk replacers). Reported LODs were validated in a collaborative study (Annex B).
- In‑lab checks: Daily analysis of spiked blank samples - 1 mg/kg for spiramycin and virginiamycin, 0.5 mg/kg for tylosin - is recommended to verify lower detection capability. In‑house validation is required to demonstrate achievable LODs.
- Interferences: Other antibiotics can produce inhibition zones; known interferences are listed in Annex A.
- Follow-up and confirmation: Suitable for qualitative screening or as a post‑screening tool (for example after microbiological plate tests). Quantitative follow‑up and confirmatory analysis require LC or LC‑MS techniques; LC‑MS is required for confirmatory purposes.
Practical applications
- Routine feed monitoring for macrolide contamination in feed mills and testing laboratories.
- Regulatory screening by national control labs and food/feed safety agencies prior to confirmatory testing.
- Quality assurance in feed production to detect inadvertent antibiotic carry‑over.
- Investigative testing during suspected non‑compliance or contamination incidents.
Who should use this standard
- Accredited feed testing laboratories and contract analytical services
- National regulatory and control authorities for animal feed safety
- Feed manufacturers’ QA/QC teams and R&D laboratories
- Veterinary drug surveillance and public‑health laboratories
Related standards
- EN ISO 6498 (sample preparation guidelines) is normatively referenced for sample handling and preparation.
Keywords: EN 16939:2017, animal feeding stuffs, tylosin, spiramycin, virginiamycin, thin layer chromatography, bioautography, feed testing, LC‑MS, detection limits, macrolide antibiotics.
Relations
- Effective Date
- 28-Jan-2026
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Frequently Asked Questions
EN 16939:2017 is a standard published by the European Committee for Standardization (CEN). Its full title is "Animal feeding stuffs: Methods of sampling and analysis - Detection of tylosin, spiramycin and virginiamycin - Thin Layer Chromatography and bioautography". This standard covers: The method makes it possible to detect and identify spiramycin, tylosin and virginiamycin in animal feeding stuffs (feed raw materials of mainly plant origin and compound feeds) excluding mineral feeds and premixtures. The limit of detection is about 2 mg/kg for spiramycin, 1 mg/kg for tylosin and 1 mg/kg for virginiamycin. In some milk replacers, it can be slightly higher than 1 mg/kg for virginiamycin. Reported limits of detection are probably little overestimated but were fully validated during the collaborative study (see Annex B). In each laboratory, each day of analysis, spiked blank samples at 1 mg/kg for spiramycin and virginiamycin and at 0,5 mg/kg for tylosin are analysed for checking lower detection limits (see 9.2 and 9.3). These lower limits of detection are achievable, but should be established with an in-house validation first. Some other antibiotics can interfere in the detection of these 3 specific macrolide antibiotics. The known interferences are specified in Annex A of the method. That method should be used as a qualitative screening and/or a post-screening method (after microbiological plate test, for example). The follow-up of the antibiotics presence may be done by other analytical technics (LC and/or LC-MS technics) ([4], [10]). For confirmatory purposes, LCMS is required.
The method makes it possible to detect and identify spiramycin, tylosin and virginiamycin in animal feeding stuffs (feed raw materials of mainly plant origin and compound feeds) excluding mineral feeds and premixtures. The limit of detection is about 2 mg/kg for spiramycin, 1 mg/kg for tylosin and 1 mg/kg for virginiamycin. In some milk replacers, it can be slightly higher than 1 mg/kg for virginiamycin. Reported limits of detection are probably little overestimated but were fully validated during the collaborative study (see Annex B). In each laboratory, each day of analysis, spiked blank samples at 1 mg/kg for spiramycin and virginiamycin and at 0,5 mg/kg for tylosin are analysed for checking lower detection limits (see 9.2 and 9.3). These lower limits of detection are achievable, but should be established with an in-house validation first. Some other antibiotics can interfere in the detection of these 3 specific macrolide antibiotics. The known interferences are specified in Annex A of the method. That method should be used as a qualitative screening and/or a post-screening method (after microbiological plate test, for example). The follow-up of the antibiotics presence may be done by other analytical technics (LC and/or LC-MS technics) ([4], [10]). For confirmatory purposes, LCMS is required.
EN 16939:2017 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 16939:2017 has the following relationships with other standards: It is inter standard links to EN ISO 6498:2012. Understanding these relationships helps ensure you are using the most current and applicable version of the standard.
EN 16939:2017 is associated with the following European legislation: EU Directives/Regulations: 882/2004; Standardization Mandates: M/521. 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.
EN 16939:2017 is available in PDF format for immediate download after purchase. The document can be added to your cart and obtained through the secure checkout process. Digital delivery ensures instant access to the complete standard document.
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Probenahme- und Untersuchungsverfahren - Nachweis von Tylosin, Spiramycin und Virginiamycin - Dünnschichtchromatographie und BioautographieAliments pour animaux : Méthodes d’échantillonnage et d’analyse - Détection de tylosine, spiramycine et virginiamycine - Chromatographie sur couche mince et bioautographieAnimal feeding stuffs: Methods of sampling and analysis - Detection of tylosin, spiramycin and virginiamycin - Thin Layer Chromatography and bioautography65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16939:2017SIST EN 16939:2017en,fr,de01-oktober-2017SIST EN 16939:2017SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16939
August
t r s y ICS
x wä s t r English Version
Animal feeding stuffsã Methods of sampling and analysis æ Detection of tylosiná spiramycin and virginiamycin æ Thin Layer Chromatography and bioautography Aliments pour animaux ã Méthodes d 5échantillonnage et d 5analyse æ Détection de tylosineá spiramycine et virginiamycine æ Chromatographie sur couche mince et bioautographie
Futtermittel æ Probenahmeæ und Untersuchungsverfahren æ Nachweis von Tylosiná Spiramycin und Virginiamycin æ Dünnschichtchromatographie und Bioautographie This European Standard was approved by CEN on
t v April
t r s yä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä
EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels
t r s y CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s x { u {ã t r s y ESIST EN 16939:2017
Substances giving inhibition zones . 16 Annex B (informative)
Results of the interlaboratory study . 18 B.1 General . 18 B.2 Materials . 18 B.3 Statistics . 19 B.4 Result and interpretation . 20 Annex C (informative)
Preparation of bacterial suspensions . 24 C.1 General . 24 C.2 Classical/old fashion preparation . 24 C.2.1 Maintenance of stock culture . 24 SIST EN 16939:2017
Figure 1 — TLC plate with 2 inhibition zones 3.3 sensitivity of a method SE ability of the method to classify a positive sample as positive 3.4 specificity of a method SP ability to classify a negative sample as negative 3.5 spiramycin macrolide antibiotic and often a mixture of different cofactors (spiramycin I, II, III.) Note 1 to entry One mg of spiramycin base is considered to be equivalent to 3200 International Unit. 3.6 tylosin macrolide antibiotic and mixture of four macrolide antibiotics produced by a strain of Streptomyces fradiae and depending on the manufacturing source Note 1 to entry The main component of the mixture (>80 %) is tylosin A. Tylosin B (desmycosin), tylosin C (macrocin) and tylosin D (relomycin) may also be present. All four components contribute to the potency of tylosin, which is not less than 900 IU/mg, calculated with reference to the dried substance (European Pharmacopoeia). Relative antimicrobial activities of tylosin derivatives are: tylosin A – 1,0, tylosin B – 0,83, tylosin C – 0,75 and tylosin D – 0,35. 3.7 virginiamycin macrolide antibiotic and mixture of 2 major synergistic cofactors: virginiamycin components M1 and S1 SIST EN 16939:2017
Final pH: 6,5 ± 0,2 Antibiotic medium 1 may be conserved at least 6 months at 5 °C ± 3 °C. 5.3.2 Antibiotic medium 1 supplemented with tylosin. Just before inoculating with Micrococcus luteus, add to the antibiotic medium 1 (5.3.1), 0,2 % (v/v) of the solution of tylosin at 4 µg/ml (5.20.10). Adjust the pH to 6,5 ± 0,1. 5.3.3 Antibiotic medium 11. Use the water bath (6.6) to liquefy media just before inoculating Micrococcus Luteus. The composition is the same than that of antibiotic medium 1 (5.3.1) but the final pH is: 8,0 ± 0,2. Antibiotic medium 11 may be conserved at least 6 months at 5 °C ± 3 °C. SIST EN 16939:2017
2, 3, 5 triphenyltetrazolium chloride (TTC). 5.12 Silicon anti-foaming agent. Silicon anti-foaming agent type SE2 ® or equivalent. 5.13 Mixture of methanol and phosphate buffer solution pH 8,0 for the stock solution of spiramycin. Phosphate buffer solution: Dipotassium hydrogen phosphate K2HPO4: 16,7 g Potassium dihydrogen phosphate KH2PO4: 0,5 g Sodium hydrogen carbonate NaHCO3: 20,0 g Water to: 1 000 ml pH: 8,0 Mix one volume of methanol with one volume of phosphate buffer solution. 5.14 Phosphate buffer solution pH 7,0 for the stock solution of tylosine. Potassium dihydrogen phosphate KH2PO4: 5,5 g Dipotassium hydrogen phosphate K2HPO4: 13,6 g Water to: 1 000 ml pH: 7,0 SIST EN 16939:2017
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