1、Animal feeding stuffs - Methods of sampling and analysis - Detection of tylosin, spiramycin and virginiamycin - Thin Layer Chromatography and bioautographyBS EN 16939:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NOR
2、M EN 16939 August 2017 ICS 65.120 English Version Animal feeding stuffs: Methods of sampling and analysis - Detection of tylosin, spiramycin and virginiamycin - Thin Layer Chromatography and bioautography Aliments pour animaux : Mthodes dchantillonnage et danalyse - Dtection de tylosine, spiramycine
3、 et virginiamycine - Chromatographie sur couche mince et bioautographie Futtermittel - Probenahme- und Untersuchungsverfahren - Nachweis von Tylosin, Spiramycin und Virginiamycin - Dnnschichtchromatographie und Bioautographie This European Standard was approved by CEN on 24 April 2017. CEN members a
4、re 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
5、 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 C
6、entre 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithu
7、ania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marni
8、x 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16939:2017 ENational forewordThis British Standard is the UK implementation of EN 16939:2017.The UK participation in its preparation was entrusted to Techni
9、cal Committee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standar
10、ds Institution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 91797 4ICS 71.040.50; 65.120Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 Septembe
11、r 2017.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN 16939:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16939 August 2017 ICS 65.120 English Version Animal feeding stuffs: Methods of sampling and analysis - Detection of tylosin, spiramycin and virg
12、iniamycin - Thin Layer Chromatography and bioautography Aliments pour animaux : Mthodes dchantillonnage et danalyse - Dtection de tylosine, spiramycine et virginiamycine - Chromatographie sur couche mince et bioautographie Futtermittel - Probenahme- und Untersuchungsverfahren - Nachweis von Tylosin,
13、 Spiramycin und Virginiamycin - Dnnschichtchromatographie und Bioautographie This European Standard was approved by CEN on 24 April 2017. 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 nationa
14、l 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 versio
15、n 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, Cze
16、ch 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 United Ki
17、ngdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16939
18、:2017 EBS EN 16939:2017EN 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
19、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 ex
20、tracts 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
21、 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
22、) Preparation of bacterial suspensions . 24 C.1 General . 24 C.2 Classical/old fashion preparation 24 C.2.1 Maintenance of stock culture . 24 BS EN 16939:2017EN 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 2
23、5 C.4 Bacterial count . 25 C.5 Storage 25 Bibliography . 26 BS EN 16939:2017EN 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. Thi
24、s 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
25、 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 thi
26、s 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
27、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 Yugosl
28、av 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. BS EN 16939:2017EN 16939:2017 (E) 5 1 Scop
29、e 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
30、 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 s
31、amples 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
32、 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
33、 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 t
34、he 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 def
35、initions 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 proper
36、ty 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. Not
37、e 2 to entry The microbiological activity is expressed as International Unit/mg or g/mg with possibility to have microbiological activities higher or lower than 1 000 g/mg. 3.2 retardation factor Rf ratio of the distance which the product travelled by the distance which the solvent front travelled u
38、sing 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. BS EN 16939:2017EN 16939:2017 (E) 6 Figure 1 TLC plate with 2 inhibition zones 3.3 sensitivity of a method SE ability of the met
39、hod 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 equivale
40、nt 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 (m
41、acrocin) 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,
42、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 BS EN 16939:2017EN 16939:2017 (E) 7 4 Principle The sample is extracted with a mixture of methanol and water. The extracts are pu
43、rified 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 valu
44、es 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 rhizo
45、phila 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 med
46、ia 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
47、 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 g/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
48、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. BS EN 16939:2017EN 16939:2017 (E) 8 5.3.4 Antibiotic medium 11 supplemented with me
49、thanol, 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 g/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
