1、BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06Animal feeding stuffs: Methods of sampling and analysis Identification of tylosin, spiramycin, virginiamycin, carbadox and olaquindox at sub-additive levels in compound feed Confirmatory analysis by LC-MSBS EN 17049:201
2、8National forewordThis British Standard is the UK implementation of EN 17049:2018.The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can be obtained on request to its secretary.This publicati
3、on does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2018 Published by BSI Standards Limited 2018ISBN 978 0 580 91795 0ICS 65.120Compliance with a British Standard cannot confer immunity from l
4、egal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 March 2018.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN 17049:2018EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 17049February 201
5、8ICS 65.120EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGCEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2018 CEN Ref. No. EN 17049:2018: EAll rights of exploitation in any form and by any means reserved worldwide for CEN national
6、MembersAnimal feeding stuffs: Methods of sampling and analysis - Identification of tylosin, spiramycin, virginiamycin, carbadox and olaquindox at sub-additive levels in compound feed - Confirmatory analysis by LC-MSAliments des animaux: Mthodes dchantillonnage et danalyse - Identification de la tylo
7、sine, spiramycine, virginiamycine, du carbadox et de lolaquindix dans les aliments composs pour animaux des concentrations infrieures celles des additifs - Analyse de confirmation par CL-SMFuttermittel: Probenahme- und Untersuchungsverfahren - Identifizierung von Tylosin, Spiramycin, Virginiamycin,
8、Carbadox und Olaquindox in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln - Besttigungsanalyse mittels LC-MSThis European Standard was approved by CEN on 8 January 2018.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giv
9、ing 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 offi
10、cial 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
11、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,
12、Spain, Sweden, Switzerland, Turkey and United Kingdom.English VersionEN 17049:2018 (E)European foreword 41 Scope . 52 Normative references 53 Principle 54 Reagents and materials . 54.1 General . 54.2 Reagents and materials . 54.2.1 Acetonitrile (LC-MS grade) . 54.2.2 Methanol (LC-MS grade) . 54.2.3
13、Formic acid (LC-MS grade) 64.2.4 Tylosin. 64.2.5 Spiramycin . 64.2.6 Virginiamycin 64.2.7 Carbadox . 64.2.8 Olaquindox 64.3 Solutions . 64.3.1 HPLC Mobile phase A: Formic acid 5mM 64.3.2 HPLC Mobile phase B: Formic acid 50 mM/ acetonitrile (10/90, v/v) . 64.4 Standard solutions . 64.4.1 Stock soluti
14、on tylosin (500 g/ml) 64.4.2 Stock solution spiramycin (500 g/ml) . 64.4.3 Stock solution virginiamycin (500 g/ml) 64.4.4 Stock solution carbadox (500 g/ml) 64.4.5 Stock solution olaquindox (500 g/ml) . 74.4.6 Mixed stock solution 1 74.4.7 Mixed stock solution 2 74.4.8 Calibration standard . 75 Appa
15、ratus . 76 Sampling 87 Sample preparation . 87.1 Sample pre-treatment . 87.2 Quality control samples . 87.3 Sample extraction . 87.4 Sample purification . 97.5 Sample preparation for LC-MS/MS . 97.6 Confirmation control . 98 LC-MS/MS analysis 98.1 General . 98.2 LC-MS/MS experimental conditions 98.3
16、 Initial test . 98.4 Analysis of samples . 99 Data processing and interpretation of results .109.1 Data processing . 109.2 Recording and calculation of identification parameters 1010 Criteria for acceptance of the analytical results .1010.1 General 1010.2 Run acceptance 1110.3 Identification of the
17、analyte 1110.3.1 General. 1110.3.2 Retention time criterion . 1110.3.3 Ion ratio criterion .112Contents PageBS EN 17049:2018EN 17049:2018 (E)11 Test report 11Annex A (informative) Results of the interlaboratory study 12Annex B (informative) Run and sample acceptance form .22Annex C (informative) Qua
18、ntitative analysis 23Annex D (informative) Run and sample acceptance form .30Bibliography .31 ISO ISO pub-date year All rights reserved 3BS EN 17049:2018EN 17049:2018 (E)European forewordThis document (EN 17049:2018) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs - Method
19、s 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 August 2018, and conflicting national standards shall be withdrawn at the lates
20、t by August 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 Commissi
21、on and the European Free Trade Association.WARNING The method described in this standard implies the use of reagents that pose a hazard to health. The standard does not claim to address all associated safety problems. It is the responsibility of the user of this standard to take appropriate measures
22、 for the health and safety protection of the personnel prior to use of the standard and to ensure that regulatory and legal requirements are complied with.According to the CEN-CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this Eu
23、ropean 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, Slo
24、vakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.4 BS EN 17049:2018EN 17049:2018 (E)1 ScopeThis European Standard specifies a high performance liquid chromatography mass spectrometry (LC-MS/MS) method for the identification of tylosin, spiramycin, virginiamycin, carbadox an
25、d olaquindox in animal feeds.The method is suitable for the identification of low concentrations of tylosin, spiramycin, virginiamycin, carbadox and olaquindox in compound animal feeds. A limit of identification of 1 mg/kg for tylosin, spiramycin and virginiamycin, 4 mg/kg for carbadox and 3 mg/kg f
26、or olaquindox should be obtained by using the described method. The method was fully validated during a collaborative study (see Annex A).Since tylosin, spiramycin and virginiamycin are fermentation products consisting of a mixture of several closely related compounds, the analysis is based on detec
27、tion and identification of the most abundant constituents. For tylosin the marker is tylosin A, for spiramycin the marker is spiramycin I and II and for virginiamycin the marker is virginiamycin M1 and S1. The other isomers and forms can be readily detected with the same method but adjustment of the
28、 MS parameters according to the molecular mass of precursor and product ions need to be made. Carbadox and olaquindox are analysed as such.2 Normative referencesThere are no normative references in this document.3 PrincipleThe compounds are extracted from the feed with a mixture of water and methano
29、l. An aliquot of the liquid phase is diluted and applied to a pre-conditioned SPE column. After washing of the SPE column, compounds of interest are eluted with methanol. The obtained extract is evaporated and re-dissolved in dilute formic acid. The resulting extract is analysed by LC-MS/MS. Separat
30、ion is carried out on a silica-based C18 bonded phase column and detection is performed by mass spectrometry in multiple reaction monitoring mode.The validation of this method was performed at concentration levels that were calculated on a weight (w/w) basis. Expression of working ranges in terms of
31、 w/w concentration is common practice in residue analysis of veterinary drugs, in fact Maximum Residue Limits (MRL) are exclusively expressed on a w/w basis. For feed additives however, tolerances have been expressed traditionally as microbiological activity. To translate the validation experiments
32、concerning the level at which they were performed, to units expressed as microbiological activity, the w/w concentrations should be corrected for the microbiological potency of the preparation used for spiking experiments.4 Reagents and materialsWARNING Use all solvents and solutions in a fume hood.
33、 Wear safety glasses, protective clothing and avoid skin contact.4.1 GeneralAll reagents are of Analytical reagent grade or better unless otherwise stated. Throughout this method, “water” means demineralized water with a conductivity of at least 10 M.cm1. Guaranteed purity is required for each lot o
34、f reference standard.4.2 Reagents and materials4.2.1 Acetonitrile (LC-MS grade)4.2.2 Methanol (LC-MS grade)5BS EN 17049:2018EN 17049:2018 (E)4.2.3 Formic acid (LC-MS grade)4.2.4 Tylosin4.2.5 Spiramycin4.2.6 Virginiamycin4.2.7 Carbadox4.2.8 Olaquindox4.3 Solutions4.3.1 HPLC Mobile phase A: Formic aci
35、d 5mMMeasure 200 l formic acid (4.2.3) and transfer to a volumetric flask of 1 000 ml, make up to the mark with water. Filter and degas before use.4.3.2 HPLC Mobile phase B: Formic acid 50 mM/ acetonitrile (10/90, v/v)Measure 200 l formic acid (4.2.3) and transfer to a volumetric flask of 1 000 ml,
36、add 100 ml water and make up to the mark with acetonitrile (4.2.1). Filter and degas before use.4.4 Standard solutions4.4.1 Stock solution tylosin (500 g/ml)Weigh between 10 and 50 mg of tylosin standard substance (4.2.4) and transfer to a brown glass bottle. Calculate the required amount of methano
37、l (4.2.2) and add that amount (on a weight basis) to obtain a standard solution of 500 g/ml. Store this stock solution in the dark at 4-8 C. Under these conditions it is stable for at least one month.4.4.2 Stock solution spiramycin (500 g/ml)Weigh between 10 and 50 mg of spiramycin standard substanc
38、e (4.2.5) and transfer to a brown glass bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to obtain a standard solution of 500 g/ml. Store this stock solution in the dark at 4-8 C. Under these conditions it is stable for at least one month.4.4.3 Stock
39、solution virginiamycin (500 g/ml)Weigh between 10 and 50 mg of virginiamycin standard substance (4.2.6) and transfer to a brown glass bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to obtain a standard solution of 500 g/ml. Store this stock solution
40、 in the dark at 4-8 C. Under these conditions it is stable for at least one month.4.4.4 Stock solution carbadox (500 g/ml)Weigh between 10 and 50 mg of carbadox standard substance (4.2.7) and transfer to a brown glass bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on
41、a weight basis) to obtain a standard solution of 500 g/ml. Store this stock solution in the dark at 4-8 C. Under these conditions it is stable for at least one month.6 BS EN 17049:2018EN 17049:2018 (E)4.4.5 Stock solution olaquindox (500 g/ml)Weigh between 10 and 50 mg of olaquindox standard substan
42、ce (4.2.8) and transfer to a brown glass bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to obtain a standard solution of 500 g/ml. Store this stock solution in the dark at 4-8 C. Under these conditions it is stable for at least one month.4.4.6 Mixed
43、 stock solution 1Measure 1,0 ml of stock solutions 4.4.1, 4.4.2 and 4.4.3 and transfer into a 25 ml volumetric flask. Accurately measure 4,0 ml of stock solution 4.4.4 and transfer to the same volumetric flask. Accurately measure 3,0 ml of stock solution 4.4.5 and transfer to the same volumetric fla
44、sk. Make up to the mark with water and mix. The concentration of tylosin, spiramycin, virginiamycin, carbadox and olaquindox in this stock solution is 20, 20, 20, 80 and 60 mg/l respectively. Store the stock solution in the dark at 4-8 C. Under these conditions it is stable for at least one week.4.4
45、.7 Mixed stock solution 2Mix equal volumes of mixed stock solution 1 (4.4.6) and water. Store the stock solution in the dark at 4-8 C. The concentration of tylosin, spiramycin, virginiamycin, carbadox and olaquindox in this stock solution is 10, 10, 10, 40 and 30mg/l respectively. Prepare this stock
46、 solution fresh daily.4.4.8 Calibration standardMeasure 50 l of mixed stock solution 1 (4.4.6) and transfer to a volumetric flask of 10 ml. Make up to the mark with water. The concentration of tylosin, spiramycin, virginiamycin, carbadox and olaquindox in this calibration standard is 100, 100, 100,
47、400 and 300 g/l respectively. Prepare this calibration standard fresh daily.5 ApparatusUsual laboratory equipment and, in particular, the following:5.1 Analytical balance suitable to accurately weigh between 0 and 10 g with an accuracy of 0,1 mg5.2 Balance suitable to accurately weigh between 0 and
48、1 500 g with an accuracy of 0,1 g 5.3 Centrifuge 5.4 Ultrasonic bath 5.5 Evaporation unit 5.6 Centrifuge tubes of different volumes, adapted to the centrifuge 5.7 SPE Vacuum manifold 5.8 Oasis HLBcartridges polymer phase, 60 mg, 3 ml (Waters WAT094226 or equivalent) 1)5.9 Sample vials suitable for t
49、he auto-sampler system that is used ( 5.11.1 ) 1) Oasis HLBcartridges polymer phase, 60 mg, 3 ml is an example of a suitable product available commercially. This information is given for the convenience of users of this European standard and does not constitute an endorsement by CEN of this product.7BS EN 17049:2018EN 17049:2018 (E)5.10 Head-over-head shaker 5.11 LC-MS/MS equipment 5.11.1 LC-MS/MS equipment comprised of gradient HPLC system 5.11.2 LC-MS/MS equipment comprised of analytical column Symmetry300 C18 150 3 mm, 5
