EN 17049:2018
(Main)Animal 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-MS
Animal 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-MS
This European Standard specifies a high performance liquid chromatography - mass spectrometry (LC-MS/MS) method for the identification of tylosin, spiramycin, virginiamycin, carbadox and 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 for 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 detection 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 MS parameters according to the molecular mass of precursor and product ions need to be made. Carbadox and olaquindox are analysed as such.
Futtermittel: Probenahme- und Untersuchungsverfahren - Identifizierung von Tylosin, Spiramycin, Virginiamycin, Carbadox und Olaquindox in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln - Bestätigungsanalyse mittels LC-MS
Diese Europäische Norm legt ein Verfahren für die Hochleistungsflüssigkeitschromatographie mit Massenspektrometrie-Kopplung (LC-MS/MS) zur Identifizierung von Tylosin, Spiramycin, Virginiamycin, Carbadox und Olaquindox in Futtermitteln fest.
Dieses Verfahren eignet sich für die Identifizierung geringer Konzentrationen von Tylosin, Spiramycin, Virginiamycin, Carbadox und Olaquindox in Futtermitteln. Eine Nachweisgrenze von 1 mg/kg für Tylosin, Spiramycin und Virginiamycin, 4 mg/kg für Carbadox und 3 mg/kg für Olaquindox sollte bei Einhaltung des beschriebenen Verfahrens erreicht werden. Das Verfahren wurde in einem Ringversuch vollständig validiert (siehe Anhang A).
Da es sich bei Tylosin, Spiramycin und Virginiamycin um Fermentationsprodukte handelt, die aus einer Mischung von einigen nah verwandten Wirkstoffen bestehen, beruht die Analyse auf dem Nachweis und der Identifizierung der am häufigsten vorhandenen Bestandteile. Der Marker für Tylosin ist Tylosin A, der Marker für Spiramycin ist Spiramycin I und II und der Marker für Virginiamycin ist Virginiamycin M1 und S1. Die anderen Isomere und Formen können ohne weiteres mithilfe des gleichen Verfahrens nachgewiesen werden, jedoch müssen die MS-Parameter entsprechend der Molekülmasse der Eltern und Tochterionen angepasst werden. Carbadox und Olaquindox werden als solche analysiert.
Aliments des animaux : Méthodes d’échantillonnage et d’analyse - Identification de la tylosine, spiramycine, virginiamycine, du carbadox et de l’olaquindix dans les aliments composés pour animaux à des concentrations inférieures à celles des additifs - Analyse de confirmation par LC‐MS
La présente Norme européenne décrit une méthode de chromatographie liquide – spectrométrie de masse (LC-MS/MS) de haute performance pour l’identification de la tylosine, de la spiramycine, de la virginiamycine, du carbadox et de l’olaquindox dans les aliments pour animaux.
La méthode est adaptée à l’identification des faibles concentrations de tylosine, de spiramycine, de virginiamycine, de carbadox et d’olaquindox dans les aliments composés pour animaux. La méthode décrite permet d’obtenir une limite d’identification de 1 mg/kg pour la tylosine, la spiramycine et la virginiamycine, de 4 mg/kg pour le carbadox et de 3 mg/kg pour l’olaquindox. La méthode a été totalement validée lors d’une étude collaborative (voir Annexe A).
La tylosine, la spiramycine et la virginiamycine étant des produits de fermentation consistant en un mélange de plusieurs composés étroitement liés, l’analyse repose sur la détection et l’identification des constituants les plus abondants. Pour la tylosine le marqueur est la tylosine A, pour la spiramycine le marqueur est la spiramycine I et II et pour la virginiamycine le marqueur est la virginiamycine M1 et S1. Les autres isomères et formes peuvent être facilement détectés par la même méthode, mais l’ajustement des paramètres de MS en fonction de la masse moléculaire des ions du précurseur et du produit est nécessaire. Le carbadox et l’olaquindox sont analysés comme tels.
Krma: metode vzorčenja in analize - Ugotavljanje tilozina, spiramicina, virginiamicina, karbadoksa in olakvindoksa pri koncentracijah, manjših od vsebnosti dodatkov v krmnih mešanicah - Potrditvena analiza z LC-MS
Ta evropski standard določa metodo z uporabo tekočinske kromatografije visoke ločljivosti z masno spektrometrijo (LC-MS/MS) za ugotavljanje tilozina, spiramicina, virginiamicina, karbadoksa in olakvindoksa v krmi.
Metoda je primerna za ugotavljanje nizkih koncentracij tilozina, spiramicina, virginiamicina, karbadoksa in olakvindoksa v krmnih mešanicah. Z uporabo opisane metode naj bi bilo mogoče pridobiti ugotovljeno mejno vrednost 1 mg/kg za tilozin, spiramicin in virginiamicin, 4 mg/kg za karbadoks ter 3 mg/kg za olakvindoks. Metoda je bila v celoti potrjena med medlaboratorijsko študijo (glej dodatek A).
Ker so tilozin, spiramicin in virginiamicin produkti fermentacije, sestavljeni iz mešanice več tesno povezanih spojin, analiza temelji na zaznavanju in ugotavljanju najpogostejših sestavin. Označevalec tilozina je tilozin A, označevalca spiramicina sta spiramicin I in II, označevalca virginiamicina pa sta virginiamicin M1 in S1. Druge izomere in oblike je mogoče zlahka zaznati z isto metodo, vendar je treba parametre masne spektrometrije prilagoditi glede na molekulsko maso prekurzorja in produktnih ionov. Karbadoks in olakvindoks sta analizirana kot taka.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-april-2018
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YVHEQRVWLGRGDWNRYYNUPQLKPHãDQLFDK3RWUGLWYHQDDQDOL]D]/&06
Animal 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-MS
Futtermittel: Probenahme- und Untersuchungsverfahren - Identifizierung von Tylosin,
Spiramycin, Virginiamycin, Carbadox und Olaquindox in Konzentrationen unterhalb von
Zusatzstoffen in Mischfuttermitteln - Bestätigungsanalyse mittels LC-MS
Aliments des animaux: Méthodes d'échantillonnage et d'analyse - Identification de la
tylosine, spiramycine, virginiamycine, du carbadox et de l’olaquindix dans les aliments
composés pour animaux à des concentrations inférieures à celles des additifs - Analyse
de confirmation par CL-SM
Ta slovenski standard je istoveten z: EN 17049:2018
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17049
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2018
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal 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-MS
Aliments des animaux: Méthodes d'échantillonnage et Futtermittel: Probenahme- und
d'analyse - Identification de la tylosine, spiramycine, Untersuchungsverfahren - Identifizierung von Tylosin,
virginiamycine, du carbadox et de l'olaquindix dans les Spiramycin, Virginiamycin, Carbadox und Olaquindox
aliments composés pour animaux à des concentrations in Konzentrationen unterhalb von Zusatzstoffen in
inférieures à celles des additifs - Analyse de Mischfuttermitteln - Bestätigungsanalyse mittels LC-
confirmation par CL-SM MS
This 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 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 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
Centre 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, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17049:2018 E
worldwide for CEN national Members.
Contents Page
European foreword . 5
1 Scope . 6
2 Normative references . 6
3 Principle . 6
4 Reagents and materials . 6
4.1 General . 6
4.2 Reagents and materials . 7
4.2.1 Acetonitrile (LC-MS grade) . 7
4.2.2 Methanol (LC-MS grade) . 7
4.2.3 Formic acid (LC-MS grade) . 7
4.2.4 Tylosin . 7
4.2.5 Spiramycin . 7
4.2.6 Virginiamycin . 7
4.2.7 Carbadox . 7
4.2.8 Olaquindox . 7
4.3 Solutions . 7
4.3.1 HPLC Mobile phase A: Formic acid 5mM . 7
4.3.2 HPLC Mobile phase B: Formic acid 50 mM/ acetonitrile (10/90, v/v) . 7
4.4 Standard solutions . 7
4.4.1 Stock solution tylosin (500 μg/ml) . 7
4.4.2 Stock solution spiramycin (500 μg/ml) . 7
4.4.3 Stock solution virginiamycin (500 μg/ml) . 7
4.4.4 Stock solution carbadox (500 μg/ml) . 8
4.4.5 Stock solution olaquindox (500 μg/ml) . 8
4.4.6 Mixed stock solution 1 . 8
4.4.7 Mixed stock solution 2 . 8
4.4.8 Calibration standard . 8
5 Apparatus . 8
6 Sampling . 9
7 Sample preparation . 9
7.1 Sample pre-treatment . 9
7.2 Quality control samples . 9
7.3 Sample extraction . 10
7.4 Sample purification . 10
7.5 Sample preparation for LC-MS/MS . 10
7.6 Confirmation control . 10
8 LC-MS/MS analysis . 10
8.1 General . 10
8.2 LC-MS/MS experimental conditions . 10
8.3 Initial test . 11
8.4 Analysis of samples . 11
9 Data processing and interpretation of results . 11
9.1 Data processing . 11
9.2 Recording and calculation of identification parameters . 11
10 Criteria for acceptance of the analytical results . 12
10.1 General . 12
10.2 Run acceptance . 12
10.3 Identification of the analyte . 12
10.3.1 General . 12
10.3.2 Retention time criterion . 12
10.3.3 Ion ratio criterion . 12
11 Test report . 13
Annex A (informative) Results of the interlaboratory study . 14
A.1 Procedure . 14
A.2 Materials. 14
A.3 Statistical analysis of results . 15
A.4 Results and interpretation - Precision . 16
Annex B (informative) Run and sample acceptance form . 24
Annex C (informative) Quantitative analysis . 25
C.1 General . 25
C.2 Procedure quantitative analysis. 25
C.2.1 Sample pre-treatment quantitative analysis . 25
C.2.2 Quality control samples . 25
C.2.3 Sample extraction . 26
C.2.4 Sample purification . 26
C.2.5 Sample preparation for LC-MS/MS . 26
C.2.6 Recovery control . 26
C.2.7 Confirmation control . 26
C.2.8 LC-MS/MS analysis . 27
C.2.8.1 LC-MS/MS experimental conditions .
...
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