SIST EN 17049:2018

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 nah verwandter Wirkstoffe 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 CL-SM

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. Il convient 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 en utilisant la méthode décrite. 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

• Status: Published
• Public Enquiry End Date: 19-Dec-2016
• Publication Date: 13-Mar-2018
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 01-Mar-2018
• Due Date: 06-May-2018
• Completion Date: 14-Mar-2018
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/521 - Mandate for standardisation addressed to CEN for methods of analysis in the field of animal nutrition - part 1
• Ref Project: EN 17049:2018 - 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

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN 17049:2018
01-april-2018
.UPDPHWRGHY]RUþHQMDLQDQDOL]H8JRWDYOMDQMHWLOR]LQDVSLUDPLFLQD
YLUJLQLDPLFLQDNDUEDGRNVDLQRODNYLQGRNVDSULNRQFHQWUDFLMDKPDQMãLKRG
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
SIST EN 17049:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17049:2018

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SIST EN 17049:2018


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.

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SIST EN 17049:2018
EN 17049:2018 (E)
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
2

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EN 17049:2018 (E)
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
3

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EN 17049:2018 (E)
C.2.8.1 LC-MS/MS experimental conditions . 27
C.2.8.2 Initial test . 28
C.2.8.3 Analysis of samples . 28
C.3 Data processing and interpretation of results . 29
C.3.1 Data processing . 29
C.3.2 Recording and calculation of identification parameters . 29
C.3.3 Calculation of the amount of analyte in the sample . 29
C.3.4 Calculation of recovery percentage . 29
C.4 Criteria for the acceptance of the analytical result . 29
C.4.1 General . 29
C.4.2 Run acceptance . 30
C.4.3 Identification of the analyte . 30
C.4.3.1 General . 30
C.4.3.2 Retention time criterion . 30
C.4.3.3 Ion ratio criterion . 30
C.5 Notes on the procedure . 31
C.5.1 Influence of ionization suppression . 31
C.5.2 Comments on the quantitive accuracy . 31
C.5.3 Comments on the relevance of recovery percentage . 31
Annex D (informative) Run and sample acceptance form . 32
Bibliography . 33

4

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SIST EN 17049:2018
EN 17049:2018 (E)
European foreword
This document (EN 17049:2018) 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 August 2018, and conflicting national standards shall
be withdrawn at the latest 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 Commission 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 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 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.
5

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SIST EN 17049:2018
EN 17049:2018 (E)
1 Scope
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.
2 Normative references
There are no normative references in this document.
3 Principle
The compounds are extracted from the feed with a mixture of water and methanol. 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. Separation 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 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 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 materials
WARNING — Use all solvents and solutions in a fume hood. Wear safety glasses, protective clothing and
avoid skin contact.
4.1 General
All reagents are of 'Analytical reagent' grade or better unless otherwise stated. Throughout this method,
−1
“water” means demineralized water with a conductivity of at least 10 MΩ.cm . Guaranteed purity is
required for each lot of reference standard.
6

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EN 17049:2018 (E)
4.2 Reagents and materials
4.2.1 Acetonitrile (LC-MS grade)
4.2.2 Methanol (LC-MS grade)
4.2.3 Formic acid (LC-MS grade)
4.2.4 Tylosin
4.2.5 Spiramycin
4.2.6 Virginiamycin
4.2.7 Carbadox
4.2.8 Olaquindox
4.3 Solutions
4.3.1 HPLC Mobile phase A: Formic acid 5mM
Measure 200 μl 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 μl formic acid (4.2.3) and transfer to a volumetric flask of 1 000 ml, add 100 ml water and
make up to the mark with acetonitrile (4.2.1). Filter and degas before use.
4.4 Standard solutions
4.4.1 Stock solution tylosin (500 μg/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 methanol (4.2.2) and add that amount (on a weight basis) to obtain a
standard solution of 500 μg/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 μg/ml)
Weigh between 10 and 50 mg of spiramycin standard substance (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 μg/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 solution virginiamycin (500 μg/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 μg/ml. Store this stock solution in the dark at 4-8 °C. Under these
conditions it is stable for at least one month.
7

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SIST EN 17049:2018
EN 17049:2018 (E)
4.4.4 Stock solution carbadox (500 μg/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 a weight basis) to
obtain a standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 °C. Under these
conditions it is stable for at least one month.
4.4.5 Stock solution olaquindox (500 μg/ml)
Weigh between 10 and 50 mg of olaquindox standard substance (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 μg/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 stock solution 1
Measure 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 flask. 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.
4.4.7 Mixed stock solution 2
Mix 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 solution fresh daily.
4.4.8 Calibration standard
Measure 50 μl 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, 400 and 300 μg/l respectively. Prepare this calibration
standard fresh daily.
5 Apparatus
Usual 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 mg
5.2 Balance suitable to accurately weigh between 0 and 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
8

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SIST EN 17049:2018
EN 17049:2018 (E)
® 1
5.8 Oasis HLB cartridges polymer phase, 60 mg, 3 ml (Waters WAT094226 or equivalent)
5.9 Sample vials suitable for the auto-sampler system that is used (5.11.1)
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 Symmetry 300 C18 150 × 3 mm,
2
5 μm particle size (Waters WAT106154 or comparable)
NOTE During the method validation, this recommended LC column has proved to be fit for purpose.
5.11.3 LC-MS/MS equipment comprised of mass spectrometer suitable for tandem MS
measurement (triple quadrupole or ion trap) and equipped with an electrospray interface.
6 Sampling
The laboratory should receive a sample that is truly representative and has not been damaged or
changed during transport or storage.
NOTE 1 Sampling is not part of the method specified in this European Standard. Sampling is described in
Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis
for the official control of feed. [7]
NOTE 2 For quantification of the content multi-level standard addition is applied to account for the
considerable variability of feed composition. This procedure is described in Annex C.
7 Sample preparation
7.1 Sample pre-treatment
Weigh (5,0±0,1) g each test sample and transfer in a 50 ml centrifuge tube (5.6) and proceed with the
procedure at 7.3.
7.2 Quality control samples
A known negative sample, preferably of approximately the same composition, is included in each series
(code S0). Weigh (5,0±0,1) g of the known negative sample as indicated in 7.1 and proceed with the
procedure at 7.3.
Also in each series a confirmation control sample is prepared by spiking an aliquot of the extract of the
negative control sample S0 obtained after sample extraction (see 7.6).

1 ®
 Oasis HLB cartridges 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.
2 ®
 Symmetry 300 C18 150 × 3 mm, 5 μm particle size is
...

SLOVENSKI STANDARD
oSIST prEN 17049:2016
01-december-2016
Ugotavljanje tilozina, spiramicina, virginiamicina, karbadoksa in olakvindoksa pri
koncentracijah, manjših od vsebnosti dodatkov v krmnih mešanicah - Potrditvena
analiza z LCMS
Identification of tylosin, spiramycin, virginiamycin, carbadox and olaquindox at sub-
additive levels in compound feed - Confirmatory analysis by LCMS
Bestimmung von Tylosin, Spiramycin, Virginiamycin, Carbadox und Olaquindox in
Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln - Bestätigungsanalyse
mittels LC-MS
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: prEN 17049
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 17049:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 17049:2016


DRAFT
EUROPEAN STANDARD
prEN 17049
NORME EUROPÉENNE

EUROPÄISCHE NORM

October 2016
ICS 65.120
English Version

Identification of tylosin, spiramycin, virginiamycin,
carbadox and olaquindox at sub-additive levels in
compound feed - Confirmatory analysis by LCMS
Identification de la tylosine, spiramycine, Bestimmung von Tylosin, Spiramycin, Virginiamycin,
virginiamycine, du carbadox et de l'olaquindix dans les Carbadox und Olaquindox in Konzentrationen
aliments composés pour animaux à des concentrations unterhalb von Zusatzstoffen in Mischfuttermitteln -
inférieures à celles des additifs - Analyse de Bestätigungsanalyse mittels LC-MS
confirmation par CL-SM
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 327.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17049:2016 E
worldwide for CEN national Members.

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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) . 7
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
5.1 Analytical balance suitable to accurately weigh between 0 and 10 g with an accuracy
of 0,1 mg . 8
5.2 Balance suitable to accurately weigh between 0 and 1 500 g with an accuracy of 0,1 g . 8
5.3 Centrifuge . 8
5.4 Ultrasonic bath . 8
5.5 Evaporation unit . 8
5.6 Centrifuge tubes of different volumes, adapted to the centrifuge . 8
5.7 SPE Vacuum manifold . 8
®
5.8 Oasis HLB cartridges polymer phase, 60 mg, 3 ml (Waters WAT094226 or
equivalent) . 9
5.9 Sample vials suitable for the auto-sampler system that is used (5.11.1) . 9
5.10 Head-over-head shaker . 9
5.11 LC-MS/MS equipment . 9
5.11.1 LC-MS/MS equipment comprised of gradient HPLC system . 9
5.11.2 LC-MS/MS equipment comprised of analytical column Symmetry® 300 C18
150 × 3 mm, 5 μm particle size (Waters WAT106154 or comparable) . 9
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5.11.3 LC-MS/MS equipment comprised of mass spectrometer suitable for tandem MS
measurement (triple quadrupole or ion trap) and equipped with an electrospray
interface. . 9
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 Run acceptance . 12
10.2 Identification of the analyte . 12
10.2.1 Retention time criterion . 12
10.2.2 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 . 16
A.4.1 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 . 26
C.3 Data processing and interpretation of results . 28
C.3.1 Data processing . 28
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C.3.2 Recording and calculation of identification parameters . 29
C.3.3 Calculation of the amount of analyte in the sample. 29
C.3.4 Calculation of recovery percentage . 29
C.4 Criteria for the acceptance of the analytical result . 29
C.4.1 Run acceptance . 29
C.4.2 Identification of the analyte . 30
C.5 Notes on the procedure . 30
C.5.1 Influence of ionization suppression . 30
C.5.2 Comments on the quantitative accuracy . 30
C.5.3 Comments on the relevance of recovery percentage . 31
Annex D (informative) Run and sample acceptance form . 32
Bibliography . 33

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European foreword
This document (prEN 17049:2016) 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 document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission 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 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.
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1 Scope
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.
2 Normative references
No normative references apply to this document.
3 Principle
The compounds are extracted from the feed with a mixture of water and methanol. 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. Separation 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 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 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 materials
WARNING — Use all solvents and solutions in a fume hood. Wear safety glasses, protective clothing and
avoid skin contact.
4.1 General
All reagents are of 'Analytical reagent' grade or better unless otherwise stated. Throughout this method,
“water” means demineralised water with a conductivity of at least 10 Mohm.cm-1. Guaranteed purity is
required for each lot of reference standard.
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4.2 Reagents and materials
4.2.1 Acetonitrile (LC-MS grade)
4.2.2 Methanol (LC-MS grade)
4.2.3 Formic acid (LC-MS grade)
4.2.4 Tylosin
4.2.5 Spiramycin
4.2.6 Virginiamycin
4.2.7 Carbadox
4.2.8 Olaquindox
4.3 Solutions
4.3.1 HPLC Mobile phase A: Formic acid 5mM
Measure 200 μl 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 μl formic acid (4.2.3) and transfer to a volumetric flask of 1 000 ml, add 100 ml water and
make up to the mark with acetonitrile (4.2.1). Filter and degas before use.
4.4 Standard solutions
4.4.1 Stock solution tylosin (500 μg/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 methanol (4.2.2) and add that amount (on a weight basis) to obtain a
standard solution of 500 μg/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 μg/ml)
Weigh between 10 and 50 mg of spiramycin standard substance (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 μg/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 solution virginiamycin (500 μg/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 μg/ml. Store this stock solution in the dark at 4-8 °C. Under these
conditions it is stable for at least one month.
4.4.4 Stock solution carbadox (500 μg/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 a weight basis) to
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obtain a standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 °C. Under these
conditions it is stable for at least one month.
4.4.5 Stock solution olaquindox (500 μg/ml)
Weigh between 10 and 50 mg of olaquindox standard substance (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 μg/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 stock solution 1
Measure 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 flask. 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.
4.4.7 Mixed stock solution 2
Mix 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 solution fresh daily.
4.4.8 Calibration standard
Measure 50 μl 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, 400 and 300 μg/l respectively. Prepare this calibration
standard fresh daily.
5 Apparatus
Usual 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 mg
5.2 Balance suitable to accurately weigh between 0 and 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
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®
5.8 Oasis HLB cartridges polymer phase, 60 mg, 3 ml (Waters WAT094226 or
1
equivalent)
5.9 Sample vials suitable for the auto-sampler system that is used (5.11.1)
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 Symmetry® 300 C18 150 × 3 mm,
2
5 μm particle size (Waters WAT106154 or comparable)
NOTE During the method validation, this recommended LC column has proved to be fit for purpose.
5.11.3 LC-MS/MS equipment comprised of mass spectrometer suitable for tandem MS
measurement (triple quadrupole or ion trap) and equipped with an electrospray interface.
6 Sampling
The laboratory should receive a sample that is truly representative and has not been damaged or
changed during transport or storage.
NOTE 1 Sampling is not part of the method specified in this European standard. Sampling is described in
Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis
for the official control of feed. [7]
NOTE 2 For quantification of the content multi-level standard addition is applied to account for the
considerable variability of feed composition. This procedure is described in Annex C.
7 Sample preparation
7.1 Sample pre-treatment
Weigh 5,0 g ± 0,1 g each test sample and transfer in a 50 ml centrifuge tube (5.6) and proceed with the
procedure at 7.3.
7.2 Quality control samples
A known negative sample, preferably of approximately the same composition, is included in each series
(code S0). Weigh 5,0 g ± 0,1 g of the known negative sample as indicated in 7.1 and proceed with the
procedure at 7.3.
Also in each series a confirmation control sample is prepared by spiking an aliquot of the extract of the
negative control sample S0 obtained after sample extraction (see 7
...