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

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Published
Publication Date
13-Feb-2018
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
14-Feb-2018
Completion Date
14-Feb-2018

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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

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SIST EN 17049:2018
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

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SIST EN 17049:2018
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

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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.
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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,

“water” means demineralized water with a conductivity of at least 10 MΩ.cm . Guaranteed purity is

required for each lot of reference standard.
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SIST EN 17049:2018
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.
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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
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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,

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
...

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