Animal feeding stuffs: Methods of sampling and analysis - Screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compound feed by a microbiological plate test

This method describes the screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in complete feeding stuffs and milk replacers by a microbiological 3-plate test. The limit of detection of the method is 1 mg/kg for avoparcin, tylosin, spiramycin, virginiamycin and 5 mg/kg for zinc bacitracin. The presence of other (veterinary) antibiotics may interfere with the method. Furthermore, high concentrations of metals (Cu, Zn) may interfere. The method should be used as a qualitative screening method. Positive results can be analysed further by TLC, for confirmatory purposes LCMS is recommended [1].
A lower limit of detection for zinc bacitracin (3 mg/kg) is achievable (see Table 2), but should be established with an in house validation first.

Futtermittel - Probenahme- und Untersuchungsverfahren - Screening auf die Antibiotika Tylosin, Virginiamycin, Spiramycin, Bacitracin-Zink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln mittels mikrobiologischem Plattentest

Dieses Verfahren beschreibt das Screening auf die Antibiotika Tylosin, Virginiamycin, Spiramycin, Bacitracin-Zink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Alleinfuttermitteln und Milchaustausch-Futtermitteln mittels mikrobiologischem 3-Platten-Test (Allgemeiner Hemmstofftest). Die Nachweisgrenze des Verfahrens beträgt 1 mg/kg bei Avoparcin, Tylosin, Spiramycin und Virginiamycin sowie 5 mg/kg bei Bacitracin-Zink. Das Vorhandensein weiterer Antibiotika (aus Tierarzneimitteln) kann das Verfahren beeinträchtigen. Weiterhin können hohe Konzentrationen von Metallen (Cu, Zn) stören. Das Verfahren sollte als ein qualitatives Screeningverfahren angewendet werden. Positive Ergebnisse können mittels Dünnschichtchromatographie (en: thin layer chromatography, TLC) weiter analysiert werden, für Bestätigungszwecke ist die Flüssigchromatographie mit Massenspektrometrie (en: liquid chromatography/mass spectrometry, LC-MS) empfehlenswert [1].
Eine niedrigere Nachweisgrenze bei Bacitracin-Zink (3 mg/kg) ist erreichbar (siehe Tabelle 2), sie sollte jedoch erst durch eine laborinterne Validierung etabliert werden.

Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Dépistage des antibiotiques tylosine, virginiamycine, spiramycine, bacitracine-zinc et avoparcine à des niveaux sous-additifs dans les aliments composés par essai sur plaque microbiologique

La présente Norme européenne présente une méthode décrivant le dépistage des antibiotiques tylosine, virginiamycine, spiramycine, bacitracine-zinc et avoparcine à des niveaux sous-additifs dans les aliments complets pour animaux et les lactoremplaceurs par un essai microbiologique sur 3 plaques.
La limite de détection de la méthode est de 1 mg/kg pour l’avoparcine, la tylosine, la spiramycine, la virginiamycine et de 5 mg/kg pour la bacitracine-zinc. La présence d’autres antibiotiques (à usage vétérinaire) peut interférer avec la méthode.
En outre, des concentrations élevées en métaux (Cu, Zn) peuvent également causer des interférences. Il convient d’utiliser la méthode comme une méthode de dépistage qualitatif. Dans le cas de résultats positifs, l’analyse peut être approfondie par CCM (chromatographie sur couche mince). Il est nécessaire d’utiliser la CL-SM (chromatographie liquide couplée à la spectrométrie de masse) pour confirmer les résultats [1].
Il est possible d’atteindre une limite de détection inférieure pour la bacitracine-zinc (3 mg/kg) (voir le Tableau 2), mais il convient de l’établir préalablement par une validation en interne.

Krma: metode vzorčenja in analize - Presejalna analiza antibiotikov tilozina, virginiamicina, spiramicina, bacitracin-cinka in avoparcina pri koncentracijah pod vsebnostmi dodatkov v krmnih mešanicah s preskusom z mikrobiološko ploščo

Ta metoda opisuje presejalno analizo antibiotikov tilozin, virginiamicin, spiramicin, bacitracin-cink in avoparcin pri koncentracijah pod vsebnostmi dodatkov v popolnih krmnih mešanicah in mlečnih nadomestkih s preskusom s tremi mikrobiološkimi ploščami. Meja zaznavanja te metode je 1 mg/kg za avoparcin, tilozin, spiramicin in virginiamicin ter 5 mg/kg za bacitracin-cink. Metoda lahko ovira prisotnost drugih (veterinarskih) antibiotikov. Metodo lahko ovirajo tudi visoke vsebnosti kovin (Cu, Zn). Metodo je treba uporabiti kot kvalitativno presejalno metodo. Pozitivne rezultate je mogoče dodatno analizirati s TLC, za namen potrditve pa se priporoča metoda LCMS [1].
Spodnjo mejo detekcije za bacitracin-cink (3 mg/kg) je mogoče doseči (glej preglednico 2), vendar jo je treba najprej določiti z internim preskusom.

General Information

Status
Published
Public Enquiry End Date
14-Feb-2016
Publication Date
10-Aug-2017
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
02-Aug-2017
Due Date
07-Oct-2017
Completion Date
11-Aug-2017

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Probenahme- und Untersuchungsverfahren - Screening auf die Antibiotika Tylosin, Virginiamycin, Spiramycin, Bacitracin-Zink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln mittels mikrobiologischem PlattentestAliments pour animaux : Méthodes d’échantillonnage et d’analyse - Dépistage des antibiotiques tylosine, virginiamycine, spiramycine, bacitracine-zinc et avoparcine à des niveaux sous-additifs dans les aliments composés par essai sur plaque microbiologiqueAnimal feeding stuffs: Methods of sampling and analysis - Screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compound feed by a microbiological plate test65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16936:2017SIST EN 16936:2017en,fr,de01-september-2017SIST EN 16936:2017SLOVENSKI

STANDARD
SIST EN 16936:2017
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16936
May
t r s y ICS
x wä s t r English Version

Animal feeding stuffsã Methods of sampling and analysis æ Screening on the antibiotics tylosiná virginiamyciná spiramyciná bacitracinæzinc and avoparcin at subæadditive levels in compound feed by a microbiological plate test Aliments pour animaux ã Méthodes d 5échantillonnage et d 5analyse æ Dépistage des antibiotiques tylosineá virginiamycineá spiramycineá bacitracineæzinc et avoparcine à des niveaux sousæadditifs dans les aliments composés par essai sur plaque microbiologique

Futtermittel æ Probenahmeæ und Untersuchungsverfahren æ Screening auf die Antibiotika Tylosiná Virginiamyciná Spiramyciná BacitracinæZink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln mittels mikrobiologischem Plattentest This European Standard was approved by CEN on

x February
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

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Avenue Marnix 17,
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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 xã t r s y ESIST EN 16936:2017

EN 16936:2017 (E) 2 Contents Page European foreword ....................................................................................................................................................... 3 1 Scope .................................................................................................................................................................... 4 2 Normative references .................................................................................................................................... 4 3 Principle ............................................................................................................................................................. 4 4 Reagents and materials ................................................................................................................................. 4 5 Apparatus ........................................................................................................................................................... 8 6 Sample preparation ........................................................................................................................................ 8 6.1 Preparation of the test plates ..................................................................................................................... 8 6.2 Sample extraction ........................................................................................................................................... 9 7 Measurements .................................................................................................................................................. 9 7.1 Procedure........................................................................................................................................................... 9 7.2 Interpretation ................................................................................................................................................... 9 7.3 Identification .................................................................................................................................................... 9 8 Determination of concentrations ........................................................................................................... 10 9 Precision .......................................................................................................................................................... 10 9.1 Interlaboratory study ................................................................................................................................. 10 10 Test report ...................................................................................................................................................... 11 Annex A (informative)

Preparation of bacterial suspensions .................................................................. 12 A.1 General ............................................................................................................................................................. 12 A.2 Storage ............................................................................................................................................................. 12 Annex B (informative)

Procedure for the additional identification of interfering antibiotics ..... 13 B.1 General ............................................................................................................................................................. 13 B.2 Tetracyclines ................................................................................................................................................. 13 B.3 Quinolones/colistin ..................................................................................................................................... 13 B.4 Aminoglycosides ........................................................................................................................................... 13 B.5 Avilamycin ...................................................................................................................................................... 14 B.6 Macrolides / ß-lactams .............................................................................................................................. 14 B.7 Mixture of antibiotics ................................................................................................................................. 14 Bibliography ................................................................................................................................................................. 16

SIST EN 16936:2017

EN 16936:2017 (E) 3 European foreword This document (EN 16936:2017) 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 November 2017, and conflicting national standards shall be withdrawn at the latest by November 2017. 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. 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 16936:2017

EN 16936:2017 (E) 4 1 Scope This European Standard presents a method describing the screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in complete feeding stuffs and milk replacers by a microbiological 3-plate test. The limit of detection of the method is 1 mg/kg for avoparcin, tylosin, spiramycin and virginiamycin, and 5 mg/kg for zinc bacitracin. The presence of other (veterinary) antibiotics may interfere with the method. Furthermore, high concentrations of metals (Cu, Zn) may interfere. The method should be used as a qualitative screening method. Positive results can be analysed further by TLC; for confirmatory purposes LC-MS is required [1]. A lower limit of detection for zinc bacitracin (3 mg/kg) is achievable (see Table 2), but should be established with an in house validation first. 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 13969, Milk and milk products - Guidelines for a standardized description of microbial inhibitor tests (ISO 13969) 3 Principle The feed sample is extracted with a mixture of acetone, hydrochloric acid (HCl) and water. Neutralized extract is dispensed into wells in three different test plates. Each of these test plates holds a different composition with respect to culture medium, indicator bacterium and/or pH. After a 16-18 h incubation period, the presence of antibiotic residues is indicated by the appearance of a zone of growth inhibition around the sample. Comparison of the inhibition pattern with a reference set (Table 1) may yield a presumptive identification of the antibiotic. 4 Reagents and materials 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. 4.1 Test organisms: Kocuria rhizophila ATCC 9341 (formerly: Micrococcus luteus) Micrococcus luteus ATCC 10240 Bacillus megaterium ATCC 10778 See Annex A for the preparation of the bacterial suspensions. 4.2 Culture media: In order to improve the reproducibility of the method, it is recommended to use dehydrated basic components or dehydrated complete media for the preparation of culture media. Follow the manufacturers’ instructions. SIST EN 16936:2017

EN 16936:2017 (E) 5 4.2.1 Culture medium for Kocuria rhizophila ATCC 9341. Meat peptone 6 g Tryptone 4 g Yeast extract 3 g Meat extract 1,5 g Glucose 1 g Agar 15 g Dipotassium hydrogen phosphate (K2HPO4) 20 g (to be added after sterilization) Demineralized water 1000 ml The basic dehydrated medium is commercially available as Antibiotic medium No. 1. Dissolve the components in the water by heating. Autoclave the medium at 121 °C ± 1 °C for 15 min. Adjust the pH to 8,0 ± 0,1. 4.2.2 Culture medium for Micrococcus luteus ATCC 10240. Meat peptone 6 g Tryptone 4 g Yeast extract 3 g Meat extract 1,5 g Glucose 1 g Agar 15 g Demineralized water 1000 ml The basic dehydrated medium is commercially available as Antibiotic medium No. 1. Dissolve the components in the water by heating. Autoclave the medium at 121 °C ± 1 °C for 15 min. Adjust the pH to 6,5 ± 0,1. 4.2.3 Culture medium for Bacillus megaterium ATCC 10778. Pancreatic digest of casein 5 g Yeast extract 2,5 g Glucose 1 g Agar 15 g Demineralized water 1000 ml The basic dehydrated medium is commercially available as Plate Count Agar. Dissolve the components in the water by heating. Autoclave the medium at 121 °C ± 1 °C for 15 min. Adjust the pH to 6,0 ± 0,1. 4.3 Water, demineralized or deionized or at least equivalent. 4.4 Sodium chloride (NaCl). SIST EN 16936:2017

EN 16936:2017 (E) 6 4.5 Acetone, analytical grade. 4.6 Hydrochloric acid (HCI). 4.7 Dipotassium hydrogen phosphate (K2HPO4). 4.8 Potassium dihydrogen phosphate (KH2PO4). 4.9 Sodium hydroxide (NaOH). 4.10 Methanol, analytical grade. 4.11 Extraction solvent: acetone/ hydrochloric acid/water mixture (475/25/500 v/v/v): Transfer 500 ml of water (4.3) into a 1000 ml volumetric flask, ad 25 ml of hydrochloric acid (4.6) and mix. Make up to 1000 ml with acetone (4.5). 4.12 Phosphate buffer pH 6,5: Dissolve 18,6 g potassium dihydrogen phosphate (4.8) and 14,8 g dipotassium hydrogen phosphate (4.7) in water (4.3). Adjust the pH to 6,5 ± 0,1 and fill up to 1000 ml with water (4.3). 4.13 Hydrochloric acid solution: 0,1 M: Transfer 500 ml of water (4.3) into a 1000 ml volumetric flask. Slowly add 8,212 ml hydrochloric acid (4.6). Make up to a final volume of 1000 ml with water (4.3). 4.14 Standard solvent: Transfer 400 ml of 0,1 M hydrochloric acid (4.13) into a 1000 ml volumetric flask, ad 600 ml of acetone (4.5) and mix. 4.15 Standard solutions and control samples: Correct all weighing for purity and salt contents in accordance with EN ISO 13969. 4.15.1 Neomycin standard stock solution. Dissolve 50,0 mg ± 0,1 mg of neomycin in 100 ml water (4.3) and mix. The thus-prepared neomycin standard stock solution contains 500 µg/ml of neomycin. The neomycin standard stock solution may be kept for one month if stored at 0 °C to + 5 °C. 4.15.2 Tylosin solutions and control samples: 4.15.2.1 Tylosin standard stock solution: Dissolve 50,0 mg ± 0,1 mg of tylosin in 100 ml water (4.3) and mix. The thus-prepared tylosin standard stock solution contains 500 µg/ml of tylosin. The tylosin standard stock solution may be kept for one month if stored at 0 °C to + 5 °C. 4.15.2.2 Tylosin working solution: Dilute 2 ml of the tylosin standard stock solution (4.15.2.1) with water (4.3) to 100 ml and mix. The thus-prepared tylosin working solution contains 1 µg/ml of tylosin. SIST EN 16936:2017

EN 16936:2017 (E) 7 4.15.2.3 Tylosin control sample: Weigh 10,0 g of feed sample and spike with 0,2 ml of the tylosin standard stock solution (4.15.2.1). Mix and leave at room temperature for 30 min, before starting the analysis. This positive control sample contains 1 mg/kg of tylosin. 4.15.3 Avoparcin standard stock solution and control samples: 4.15.3.1 Avoparcin standard stock solution: Dissolve 5,0 mg ± 0,1 mg of avoparcin in standard solvent (4.14), and fill up to 100 ml. The thus-prepared avoparcin standard stock solution contains 50 µg/ml of avoparcin. The avoparcin standard stock solution may be kept for one month if stored at 0 °C to + 5 °C. 4.15.3.2 Avoparcin control sample: Weigh 10,0 g of

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