Animal feeding stuffs: Methods of sampling and analysis - Detection of tylosin, spiramycin and virginiamycin - Thin Layer Chromatography and bioautography

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.
NOTE    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 may 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] [9]. For confirmatory purposes, LCMS is required.

Futtermittel - Probenahme- und Untersuchungsverfahren - Nachweis von Tylosin, Spiramycin und Virginiamycin - Dünnschichtchromatographie und Bioautographie

Das Verfahren ermöglicht es, Spiramycin, Tylosin und Virginiamycin in Futtermitteln (Futtermittel-Ausgangserzeugnisse hauptsächlich pflanzlichen Ursprungs und Mischfuttermittel) nachzuweisen und zu identifizieren, davon ausgeschlossen sind Mineralfuttermittel und Vormischungen. Die Nachweisgrenze (en: limit of detection, LOD) beträgt 2 mg/kg bei Spiramycin, 1 mg/kg bei Tylosin und 1 mg/kg bei Virginiamycin. In einigen Milchaustausch-Futtermitteln kann die Nachweisgrenze bei Virginiamycin leicht über 1 mg/kg liegen.
Die berichteten Nachweisgrenzen werden wahrscheinlich ein wenig überschätzt, sie wurden aber während des Ringversuchs vollständig validiert (siehe Anhang B). In jedem Laboratorium, an jedem Tag der Untersuchung werden aufgestockte Blindwertproben mit 1 mg/kg bei Spiramycin und Virginiamycin sowie mit 0,5 mg/kg bei Tylosin zur Prüfung der unteren Nachweisgrenzen (siehe 9.2 und 9.3) untersucht. Diese unteren Nachweisgrenzen sind erreichbar, sie sollten jedoch zuerst mit einer internen Validierung bestimmt werden.
Einige andere Antibiotika können den Nachweis dieser drei spezifischen Makrolid-Antibiotika stören. Die bekannten Störungen (Interferenzen) sind in Anhang A des Verfahrens angegeben.
Das Verfahren sollte als ein qualitatives Screeningverfahren und/oder Post-Screeningverfahren (beispiels-weise nach dem mikrobiologischen Plattentest) angewendet werden. Die Nachuntersuchung hinsichtlich des Vorhandenseins der Antibiotika kann durch andere Untersuchungstechniken (LC- und/oder LC-MS-Verfahren) [4] [9] erfolgen. Für Bestätigungszwecke ist die Flüssigchromatographie mit Massenspektrometrie (en: liquid chromatography/mass spectrometry, LC-MS) erforderlich.

Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Détection de tylosine, spiramycine et virginiamycine - Chromatographie sur couche mince et bioautographie

La méthode permet de détecter et d’identifier la présence de spiramycine, de tylosine et de virginiamycine dans les aliments pour animaux (matières premières alimentaires d’origine végétale principalement et aliments composés), à l’exclusion des aliments minéraux et des prémélanges. La limite de détection est d’environ 2 mg/kg pour la spiramycine, 1 mg/kg pour la tylosine et 1 mg/kg pour la virginiamycine. Dans certains lactoremplaceurs, elle peut être légèrement supérieure à 1 mg/kg pour la virginiamycine.
Les limites de détection publiées sont probablement légèrement surévaluées, mais ont été entièrement validées au cours de l’essai interlaboratoires (voir Annexe B). Dans chaque laboratoire, chaque jour d’analyse, des échantillons témoins enrichis de 1 mg/kg pour la spiramycine et la virginiamycine et de 0,5 mg/kg pour la tylosine sont analysés pour vérifier les limites de détection inférieures (voir 9.2 et 9.3). Ces limites de détection inférieures peuvent être atteintes, mais il convient de les établir préalablement par une validation interne.
D’autres antibiotiques sont susceptibles d’interférer dans la détection de ces 3 antibiotiques macrolides spécifiques. Les interférences connues sont spécifiées dans l’Annexe A de la méthode.
Il convient d’utiliser la présente méthode comme une méthode de dépistage qualitatif et/ou de dépistage ultérieur (après un essai sur plaque microbiologique, par exemple). Le suivi de la présence d’antibiotiques peut être assuré par d’autres techniques d’analyse (techniques SM et/ou CL-SM) [4] [9]. La technique CL-SM est requise à des fins de confirmation.

Krma: metode vzorčenja in analize - Določevanje tilozina, spiromicina in virginiamicina - Tenkoplastna kromatografija in bioavtografija

Metoda omogoča zaznavanje in prepoznavanje spiramicina, tilozina in virginiamicina v krmi (surovinah krme večinoma rastlinskega izvora in krmnih mešanicah), razen mineralnih krmah in premiksih. Meja zaznavanja je približno 2 mg/kg za spiramicin, 1 mg/kg za tilozin in 1 mg/kg za virginiamicin. Pri nekaterih mlečnih nadomestkih je lahko meja zaznavanja za virginiamicin rahlo višja od 1 mg/kg.
OPOMBA    Navedene meje zaznavanja so morda nekoliko precenjene, vendar so bile med medlaboratorijsko študijo v celoti potrjene (glej dodatek B). V vseh laboratorijih so vsak dan med potekom analize analizirani slepi vzorci z vrhom pri 1 mg/kg za spiramicin in virginiamicin ter 0,5 mg/kg za tilozin za namene preverjanja spodnjih meja zaznavanja (glej točki 9.2 in 9.3). Te spodnje meje zaznavanja je mogoče doseči, vendar jih je treba najprej določiti z internim preskusom.
Nekateri drugi antibiotiki lahko vplivajo na zaznavanje teh 3 specifičnih makrolidnih antibiotikov. Znani vplivi so navedeni v dodatku A metode.
Metodo je treba uporabiti kot kvalitativno presejalno metodo in/ali popresejalno metodo (po mikrobiološkem preskusu s ploščicami na primer). Poznejše spremljanje prisotnosti antibiotikov se lahko opravi z drugimi analitičnimi tehnikami (tehnika LC in/ali LC-MS) [4] [9]. Za namen potrditve se zahteva tehnika LCMS.

General Information

Status
Published
Public Enquiry End Date
09-Feb-2016
Publication Date
07-Sep-2017
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
24-Aug-2017
Due Date
29-Oct-2017
Completion Date
08-Sep-2017

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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
SIST EN 16939:2017
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

EN 16939:2017 (E) 2 Contents Page European foreword ....................................................................................................................................................... 4 1 Scope .................................................................................................................................................................... 5 2 Normative references .................................................................................................................................... 5 3 Terms and definitions ................................................................................................................................... 5 4 Principle ............................................................................................................................................................. 7 5 Reagents and culture media ........................................................................................................................ 7 5.1 General ................................................................................................................................................................ 7 6 Apparatus ........................................................................................................................................................ 10 7 Sampling .......................................................................................................................................................... 11 8 Preparation of test samples ..................................................................................................................... 11 9 Procedure........................................................................................................................................................ 11 9.1 Extraction ........................................................................................................................................................ 11 9.2 Blank feed sample spiked with 1 mg/kg of spiramycin and 0,5 mg/kg of tylosin (microbiological activity) .......................................................................................................................... 11 9.3 Blank feed sample spiked with 1 mg/kg of virginiamycin (microbiological activity) ........ 11 9.4 Purification and concentration ............................................................................................................... 11 9.5 Thin-layer chromatography ..................................................................................................................... 11 9.5.1 Spotting of feed extracts and of spiked feed extracts ..................................................................... 11 9.5.2 Chromatography ........................................................................................................................................... 12 9.6 Bioautography ............................................................................................................................................... 12 9.6.1 General ............................................................................................................................................................. 12 9.6.2 For spiramycin and tylosin ....................................................................................................................... 12 9.6.3 For virginiamycin ......................................................................................................................................... 12 10 Results .............................................................................................................................................................. 13 10.1 Observation of inhibition zones .............................................................................................................. 13 10.2 Interferences ................................................................................................................................................. 14 11 Precision .......................................................................................................................................................... 14 11.1 Interlaboratory study ................................................................................................................................. 14 11.2 Repeatability .................................................................................................................................................. 14 11.3 Reproducibility ............................................................................................................................................. 14 12 Test report ...................................................................................................................................................... 15 Annex A (informative)

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

EN 16939:2017 (E) 3 C.2.2 Preparation of the bacterial suspension .............................................................................................. 24 C.2.3 Reagents ........................................................................................................................................................... 24 C.3 Alternative preparation of bacterial suspension .............................................................................. 25 C.4 Bacterial count ............................................................................................................................................... 25 C.5 Storage .............................................................................................................................................................. 25 Bibliography ................................................................................................................................................................. 26

SIST EN 16939:2017

EN 16939:2017 (E) 4 European foreword This document (EN 16939:2017) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs - Methods of sampling and analysis”, 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 2018, and conflicting national standards shall be withdrawn at the latest by February 2018. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN shall not be held responsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. WARNING — The use of this protocol involves hazardous materials, operations and equipment. This protocol does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this protocol to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. According to the CEN-CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 16939:2017

EN 16939:2017 (E) 5 1 Scope 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. 2 Normative references 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. EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498) 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 microbiological activity of antibiotics ratio of the dose that inhibits the growth of a suitable susceptible microorganism to the dose of an International Chemical Reference Substance/Antibiotic that produces similar inhibition Note 1 to entry Microbiological activity is a property measured by a microbiological assay. The activity (potency) of an antibiotic product is expressed as the ratio of the dose that inhibits the growth of a suitable susceptible microorganism to the dose of an International Chemical Reference Substance/Antibiotic that produces similar inhibition. Note 2 to entry The microbiological activity is expressed as International Unit/mg or µg/mg with possibility to have microbiological activities higher or lower than 1 000 µg/mg. 3.2 retardation factor Rf ratio of the distance which the product travelled by the distance which the solvent front travelled using the initial spotting site as reference Note 1 to entry These values depend on the solvent used and the type of TLC plate and are not physical constants, see Figure 1. SIST EN 16939:2017

EN 16939:2017 (E) 6

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

EN 16939:2017 (E) 7 4 Principle The sample is extracted with a mixture of methanol and water. The extracts are purified by a liquid-liquid partition with chloroform. The chloroformic phase is concentrated. The concentrated feed extracts and reference spiked blank feeds extracts are subjected to thin-layer chromatography (TLC) on silica gel. The antibiotics are detected and identified by comparing their Rf values with those of the standard substances by bioautography with agar media inoculated with Micrococcus luteus. 5 Reagents and culture media 5.1 General All the reagents shall be of analytical grade. 5.2 Microorganism: Bacterial suspension of Micrococcus luteus ATCC 9341 (reclassified to Kocuria rhizophila ATCC 9341). See Annex C for preparation of bacterial suspensions. Other techniques for the preparation and the storage of the bacterial suspension may be used if the detection limits specified can be reached. 5.3 Culture media: 5.3.1 Antibiotic medium 1. Use the water bath (6.6) to liquefy media just before inoculating Micrococcus Luteus. Beef extract: 1,5 g Yeast extract: 3,0 g Pancreatic digest of casein: 4,0 g Meat peptone: 6,0 g Glucose: 1,0 g Agar: 10 g to 20 g Water: 1 000 ml Autoclave at 121 °C for 15 min

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

EN 16939:2017 (E) 8 5.3.4 Antibiotic medium 11 supplemented with methanol, pH 8 buffer and tylosin. Just before inoculating the medium with Micrococcus luteus, add to the antibiotic medium 11 (5.3.3): — 4 % (v/v) of methanol (5.4); — 10 % (v/v) of pH 8 buffer (5.15); — 0,2 % (v/v) of the tylosin solution at 4 µg/ml (5.21.10). Adjust the pH to 7,6 ± 0,1. NOTE The addition of methanol allows a better diffusion of spiramycin in the agar. The addition of tylosin allows a better identification and exacerbate detection in bioautography (9.6). 5.4 Methanol. 5.5 Mixture of methanol (5.4) and water 1/1 (v/v). 5.6 Chloroform. 5.7 Ethyl acetate. 5.8 Dichloromethane. 5.9 Acetone. 5.10 Glycerol. 5.11

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

EN 16939:2017 (E) 9 5.15 Phosphate buffer solution pH 8,0 for supplementing the antibiotic medium 11. Dipotassium hydrogen phosphate K2HPO4: 1,41 g Disodium hydrogen phosphate Na2HPO4, 2 H2O: 57,5 g Water to: 1 000 ml Sterilize for 15 min at 121 °C. pH: 8,0 5.16 Eluent 1 for spiramycin and tylosin detection. Methanol (5.4). 5.17 Eluent 2 for spiramycin and tylosin detection. Dichloromethane (5.8)/methanol (5.4)/acetone (5.9)/glycerol (5.10): 49/30/20/1 (v/v/v/v). Prepare freshly. 5.18 Eluent 3 for virginiamycin detection. Ethyl acetate saturated with water. In a separating funnel, shake ethyl acetate (5.7) with an excess of water. Discard the lower phase. Prepare freshly. 5.19 Contrast solution. Dissolve 0,1 g of TTC = 2, 3, 5 triphenyltetrazolium chloride (5.11) in 100 ml of water. 5.20 Blank feed sample (Commercial feed or internal manufactured feed), free of antibiotic. 5.21 Reference standard substances and standard stock solutions. 5.21.1 Standard substance of spiramycin. Spiramycin of known microbiological activity (in µg/mg or equivalent). To convert UI/mg to µg/mg: 1 mg of spiramycin is considered to be equivalent to 3200 International Unit. 5.21.2 Standard substance of tylosin. Tylosin of known microbiological activity (in µg/mg or equivalent). 5.21.3 Standard substance of virginiamycin. Virginiamycin of known microbiological activity (in µg/mg or equivalent). 5.21.4 Stock solution of spiramycin (1 000 µg/ml). Dissolve an accurately weighed quantity of the standard substance (5.21.1) in the mixture (5.13) and dilute with the same mixture to give a solution of 1 000 µg/ml of microbiological activity (equivalent to 3 200 IU/ml). 5.21.5 Stock solution of tylosin (500 µg/ml). Dry the standard substance (5.21.2) for 3 h at 60 °C in a vacuum oven. SIST EN 16939:2017

EN 16939:2017 (E) 10 Dissolve an accurately weighed quantity of the dried substance in 5 ml of methanol (5.4) and dilute with phosphate buffer solution, pH 7 (5.14) to obtain a tylosin concentration of 500 µg/ml of microbiological activity. 5.21.6 Stock solution of virginiamycin (1 000 µg/ml). Dissolve an accurately weighed quantity of the standard substance (5.21.3) in methanol and dilute with methanol (5.4) to obtain a virginiamycin concentration of 1 000 µg/ml of microbiological activity. 5.21.7 Solution of spiramycin for spiking the blank feed sample (equivalent to 100 µg/ml of microbiological activity). Dilute the stock solution (5.21.4) 1:10 with methanol (5.4). 5.21.8 Solution food tylosin for spiking the blank feed sample (equivalent to 50 µg/ml of microbiological activity). Dilute the stock solution (5.21.5) 1:10 with methanol (5.4). 5.21.9 Solution of virginiamycin for spiking the blank feed sample (equivalent to 100 µg/ml of microbiological activity). Dilute the stock solution (5.21.6) 1:10 with methanol (5.4). 5.21.10 Solution of tylosin (4 µg/ml) for supplementing the media 1 and 11. Dilute the stock solution (5.21.5) 1:125 with methanol (5.4). 6 Apparatus Usual laboratory equipment and, in particular, the following: 6.1 Incubator set at 35 °C ± 2 °C. 6.2 Incubator or oven set between 55 °C and 60 °C for drying TLC plates. 6.3 Magnetic stirrer. 6.4 Vortex mixer or equivalent. 6.5 Centrifuge. 6.6 Water bath set between 44 °C and 47 °C. 6.7 Nitrogen stream evaporator with vapor collector/condenser or vacuum vortex evaporator or equivalent. 6.8 TLC chambers for 10 cm x 10 cm plates. 6.9 TLC plates. HPTLC Glass plates coated with silica gel 60 Å, 10 cm x 10 cm, thickness 200 µm or equivalent. 6.10 Square Petri dishes 12,5 cm x 12,5 cm. SIST EN 16939:2017

EN 16939:2017 (E) 11 7 Sampling It is important that laboratory receives a sample that is truly representative and hasn't been damaged or changed during transport and storage. Sampling is not part of the method specified in this European Standard. A recommended sampling method is given in EN ISO 6497 [2] or in Regulation n°152/2009 Annex I [1]. 8 Preparation of test samples EN ISO 6498 are appropriate for the preparation of test samples. NOTE See also Regulation n°152/2009 Annex II point A [1]. Raw feed material and feed samples are grinded and sieved (at least 1 mm) before analysis. 9 Procedure 9.1 Extraction Weigh 10,0 g of sample. Add 50 ml of the mixture methanol / water (5.5). Stir for at least 30 min with the magnetic stirrer (6.3). Let decant or centrifuge at 2 000 g for 15 min (6.5), if necessary. 9.2 Blank feed sample spiked with 1 mg/kg of spiramycin and 0,5 mg/kg of tylosin (microbiological activity) Weigh 10,0 g of the feed sample (5.20). Add 0,1 ml of each spiramycin and tylosin spiking solutions (5.21.7 and 5.21.8 respectively); wait for about 10 min and extract as in 9.1. 9.3 Blank feed sample spiked with 1 mg/kg of virginiamycin (microbiological activity) Weigh 10,0 g of the feed sample (5.20); add 0,1 ml of the spiking solution (5.21.9); wait for about 10 min and extract as in 9.1. 9.4 Purification and concentration Pipette one volume of each extracts 9.1, 9.2 and 9.3 (for example 4 ml) in a separate test tube. Add the same volume of chloroform (5.6). Mix for some seconds with the vortex mixer (6.4). Pipette 2 ml of the chloroformic phase (lower phase) in a small test tube. Evaporate to dryness under a nitrogen stream (6.7) at a vapour temperature below 50 °C. Dissolve the residue in 0,2 ml of chloroform (5.6). 9.5 Thin-layer chromatography 9.5.1 Spotting of feed extracts and of spiked feed extracts Draw 1 cm line from the lower edge of the plates and spot extracts at intervals of 1,5 cm from each other: — For the detection of spiramycin and tylosin: on one TLC plate (6.9), spot 20 µl of the concentrated feed extracts and 20 µl of concentrated blank feed extract spiked with spiramycin and tylosin. — For the detection of virginiamycin: on another TLC plate (6.9), spot 20 µl of the concentrated feed extracts and 20 µl of the concentrated blank feed extract spiked with virginiamycin. SIST EN 16939:2017

EN 16939:2017 (E) 12 9.5.2 Chromatography 9.5.2.1 Chromatography for spiramycin and tylosin Pre-develop the plate on 9 cm with ethyl acetate (5.7). Dry the plate in the incubator set at 55 °C to 60 °C (6.2) for about 30 min. Pour eluent 1 (5.16) in a chromatographic chamber (6.8) to obtain a height of about 5 mm and let it saturate for at least 30 min. Develop the plate on 8 cm. If an interference is suspected after bioautography, use eluent 2 (5.17) in the same conditions. 9.5.2.2 Chromatography for virginiamycin Pour eluent 3 (5.18) in a chromatographic chamber (6.8) to obtain a height of about 5 mm and let it saturate for at least 30 min. Develop the plate on 8 cm. 9.6 Bioautography 9.6.1 General Place each plate in a Petri dish (6.10). Dry the plates by putting the Petri dishes in the incubator/oven set at 55 °C to 60 °C (6.2) for about 30 min. 9.6.2 For spiramycin and tylosin Inoculate the supplemented antibiotic medium 11 (5.3.4) with the Micrococcus Luteus bacterial suspension (5.2) at a rate of 1 % (v/v). Take the Petri dish containing the TLC plate from the incubator/oven and pour immediately in it 25 ml of the inoculated medium. NOTE 1 If bubbles form when pouring the medium on the plates, this can be avoided by: — pouring the agar not directly on the silica gel, but in a corner of the Petri dish and letting it spread on the plate; — spraying sterile water on the silica gel until the gel is wet (about 1 ml); or — adding about 0,1 % (w/v) of silicon anti-foaming agent (5.12) in the medium just before inoculation. The thickness of the medium above the TLC plate is 1,5 mm. NOTE 2 The sensitivity of antibiotics' detection is influenced by the thickness of the agar layer: a low thickness enhances it but can lead to less growth or no growth of the test microorganism. Incubate the plate for one night at 35 °C (6.1) Pour about 5 ml of the TTC solution (5.19) on the medium and re-incubate for some minutes. 9.6.3 For virginiamycin Inoculate the tylosine supplemented antibiotic medium 1 (5.3.2) with the Micrococcus Luteus bacterial suspension (5.2) at a rate of 1 % (v/v). Take the Petri dish containing the TLC plate from the incubator/oven and pour immediately in it 25 ml of the inoculated medium. SIST EN 16939:2017

EN 16939:2017 (E) 13 NOTE 1 If bubbles form when pouring the medium on the plates, this can be avoided by: — pouring the agar not directly on the silica gel, but in a corner of the Petri dish and letting it spread on the plate; — spraying sterile water on the silica gel until the gel is wet (about 1 ml); or — adding about 0,1 % (w/v) of silicon anti-foaming agent (5.12) in the medium just before inoculation. The thickness of the medium above the TLC plate is 1,5 mm. NOTE 2 The sensitivity of antibiotics' detection is influenced by the thickness of the agar layer: a low thickness enhances it but can lead to less growth or no growth of the test microorganism. Incubate the plate for one night at 35 °C (6.1). Pour about 5 ml of the TTC solution (5.19) on the medium and re-incubate for some minutes. 10 Results 10.1 Observation of inhibition zones Observe the inhibition zones for spiked blank feed extracts (coming from 9.2, 9.3 and 9.4) and the eventual inhibition zones for unknown feed extracts. If inhibition zones from unknown feed extracts are too large, the spotting volume shall be reduced. If inhibition zones are observed for unknown feed extracts, compare their retardation factor (Rf) with those of spiramycin, tylosin, and virginiamycin in spiked blank feed extracts. For bulky/distorted inhibition zone, a “centre” or equivalent needs to be estimated (see 3.2 and Figure 1). The detection is considered positive if the Rf is in accordance (±10 %) with that of one standard (spiked blank feeds 9.2 and 9.3). Reference values of Rf are given in Table 1. These values vary slightly from day to day according to non-controlled factors such as temperature and relative humidity. But if Rf of controls are too different (±20 %) from expected Rf in Table 1, analysis shall be restarted. Eluents 1 and 2 are used for the detection and identification of spiramycin and tylosin. Eluent 3 is used for the detection of virginiamycin. In the case of tylosin detection, the absence of virginiamycin shall be checked with eluent 3; if virginiamycin is present, and if there is also an inhibition zone at Rf 0 with eluent 3, the presence of tylosin cannot be excluded. Report results as “Positive” OR “Negative” (or equivalent wording). In case of doubts (potential interferences, mix of several antibiotics…), use another European Standard (confirmatory/identification methods, for example using LC-MS technics) to remove or confirm suspicions. SIST EN 16939:2017

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