EN 17299:2019

Animal feeding stuffs: Methods of sampling and analysis - Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography - Tandem Mass Spectrometry detection (LC-MS/MS)

This document specifies requirements for light fire storage units providing protection against fire.
The method of test is specified to determine the ability of light fire storage units to protect paper media from the effects of fire. Two levels of fire exposure periods (LFS 30 and LFS 60) are specified using the maximum temperature increase permitted within the storage space of the light fire storage unit.
Protection after the fire exposure of 30 min (LFS 30) or 60 min (LFS 60) is not ensured by this document, but by European Standard EN 1047-1. Requirements are also specified for the test specimen, the technical documentation for the test specimen, correlation of the test specimen with the technical documentation, preparation for type testing and test procedures.
A scheme to classify the light fire storage units from the test results is also given (see Table 1).

Futtermittel - Probenahme‑ und Untersuchungsverfahren - Screening und Bestimmung von zugelassenen Kokzidiostatika in Konzentrationen von Zusatzstoffen und deren Verschleppungen im Bereich von 1 % und 3 % sowie von nicht registrierten Kokzidiostatika und von einem Antibiotikum in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln mittels Hochleistungs-Flüssigchromatographie mit Tandem-Massenspektrometrie-Nachweis (LC‑MS/MS)

Dieses Dokument legt ein Verfahren fest zum gleichzeitigen Screening und/oder zur Bestimmung des Ge-halts der elf zugelassenen Kokzidiostatika (Halofuginon, Hydrobromid, Robenidinhydrochlorid, Nicarbazin, Diclazuril, Decoquinat, Monensin Natrium, Salinomycin Natrium, Narasin, Lasalocid A Natrium, Semdura-micin Natrium und Maduramicin Ammonium Alpha) durch Hochleistungs Flüssigchromatographie in Kombination mit Tandem Massenspektrometrie (HPLC MS/MS) in Mischfuttermitteln und Einzelfuttermitteln pflanzlichen Ursprungs in Konzentrationen von Zusatzstoffen und deren Verschleppungen und von den fünf nicht registrierten Kokzidiostatika (Ethopabat, Clopidol, Ronidazol, Dimetridazol und Amprolium) in Konzentra¬tionen unterhalb von Zusatzstoffen und zum Screening auf das verbotene Antibiotikum Furazolidon in Konzentrationen unterhalb von Zusatzstoffen, in den gleichen Matrices. Das Verfahren ist nur für Geflügel , Rinder  und Schweinemischfuttermittel vollständig validiert worden.
Der Anwendungsbereich des Verfahrens ist geeignet für das Screening auf und die Bestimmung von allen elf registrierten Kokzidiostatika bei den in der europäischen Gesetzgebung festgelegten Werten, für die nicht registrierten Kokzidiostatika und für das Screening auf das verbotene Antibiotikum.

Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Recherche et dosage dans des aliments composés pour animaux des coccidiostatiques autorisés aux taux d’additif et de contamination croisée à 1 % et 3 %, de coccidiostatiques non enregistrés et d’un antibiotique aux taux sub-additifs, par chromatographie en phase liquide à haute performance couplée à une détection par spectrométrie de masse en tandem (CL-SM/SM)

Le présent document spécifie une méthode de chromatographie liquide à haute performance - spectrométrie de masse en tandem (CL-SM/SM) pour la recherche simultanée des onze coccidiostatiques autorisés (halofuginone, chlorhydrate de robénidine, nicarbazine, diclazuril, décoquinate, monensine sodium, salinomycine sodium, narasine, lasalocide sodium, semduramicine sodium et maduramicine ammonium alpha) et/ou leur dosage dans les aliments composés pour la volaille, le bétail et les porcs aux taux d’additif et de contamination croisée et de cinq coccidiostatiques non enregistrés (éthopabate, clopidol, ronidazole, dimétridazole et amprolium) à des taux sub-additifs, ainsi que pour la recherche de l’antibiotique interdit furazolidone au taux sub-additif, dans les mêmes matrices.
La plage d’application de la méthode est adéquate aux fins de recherche et de dosage de la totalité des onze coccidiostatiques aux valeurs définies par la législation européenne et des coccidiostatiques non enregistrés, ainsi que de recherche de l’antibiotique interdit.

Krma: metode vzorčenja in analize - Pregled in določevanje dovoljenih kokcidiostatikov v koncentracijah dodatkov in njihovih nosilcih v območju od 1 do 3 % v krmnih mešanicah s tekočinsko kromatografijo visoke ločljivosti - Tandemska masna spektrometrija (LC-MS/MS)

Ta dokument določa metodo tekočinske kromatografije visoke ločljivosti z masno spektrometrijo (LC-MS/MS) za hkratni pregled in/ali določevanje vsebnosti enajstih dovoljenih kokcidiostatikov (halofuginon, robenidin hidroklorid, nikarbazin, diklazuril, dekokvinat, natrijev monenzin, natrijev salinomicin, narazin, lasalocid natrija, natrijev semduramicin in alfa amonijev maduramicin) v krmi za perutnino, govedo in prašiče pri koncentracijah dodatkov in navzkrižnega onesnaženja ter petih neregistriranih kokcidiostatikov (etopabat, klopidol, ronidazol, dimetridazol in amprolium) pri koncentracijah, manjših od vsebnosti dodatkov, in za pregled prepovedanega antibiotika furazolidon pri koncentracijah, manjših od vsebnosti dodatkov, pri istih matrikah.
Območje uporabe metode je primerno za namen pregleda in določevanja vseh enajstih kokcidiostatikov pri vrednostih, ki so določene z evropsko zakonodajo, neregistriranih kokcidiostatikov in pregleda prepovedanega antibiotika.

General Information

• Status: Published
• Publication Date: 06-Aug-2019
• Withdrawal Date: 28-Feb-2020
• ICS: 65.120 - Animal feeding stuffs
• ICS: 71.040.50 - Physicochemical methods of analysis
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 3 - Feed additives and drugs
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 07-Aug-2019
• Due Date: 07-Jan-2019
• Completion Date: 07-Aug-2019
• 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/522 - Mandate for standardisation addressed to CEN for methods of analysis in the field of animal nutrition - part II
• Ref Project: SIST EN 17299:2019 - Animal feeding stuffs: Methods of sampling and analysis - Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination levels, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography - Tandem Mass Spectrometry detection (LC-MS/MS)

Standards Content (Sample)

SLOVENSKI STANDARD
01-oktober-2019
Krma: metode vzorčenja in analize - Pregled in določevanje dovoljenih
kokcidiostatikov v koncentracijah dodatkov in njihovih nosilcih v območju od 1 do
3 % v krmnih mešanicah s tekočinsko kromatografijo visoke ločljivosti -
Tandemska masna spektrometrija (LC-MS/MS)
Animal feeding stuffs: Methods of sampling and analysis - Screening and determination
of authorized coccidiostats at additive and 1 % and 3 % cross-contamination levels, and
of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound
feed with High Performance Liquid Chromatography - Tandem Mass Spectrometry
detection (LC-MS/MS)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung zugelassener
Kokzidiostatika in Konzentrationen von Zusatzstoffen und deren Verschleppungen im
Bereich von 1 % bis 3 % in Mischfuttermitteln mittels Hochleistungs-LC-MS
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Recherche et dosage
dans des aliments composés pour animaux des coccidiostatiques autorisés au taux
d’additif et de contamination croisée à 1 % et 3 %, de coccidiostatiques non enregistrés
et d’un antibiotique aux taux sub-additifs, par chromatographie en phase liquide à haute
performance couplée à une détection par spectrométrie de masse en tandem (CL-
SM/SM)
Ta slovenski standard je istoveten z: EN 17299:2019
ICS:
65.120 Krmila Animal feeding stuffs
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17299
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2019
EUROPÄISCHE NORM
ICS 65.120; 71.040.50
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Screening and determination of authorized coccidiostats at
additive and 1 % and 3 % cross-contamination level, and
of non-registered coccidiostats and of one antibiotic at
sub-additive levels, in compound feed with High
Performance Liquid Chromatography - Tandem Mass
Spectrometry detection (LC-MS/MS)
Aliments des animaux: Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Recherche et dosage dans des aliments Untersuchungsverfahren - Bestimmung zugelassener
composés pour animaux des coccidiostatiques Kokzidiostatika in Konzentrationen von Zusatzstoffen
autorisés au taux d'additif et de contamination croisée und deren Verschleppungen im Bereich von 1 % bis 3
à 1 % et 3 %, de coccidiostatiques non enregistrés et % in Mischfuttermitteln mittels Hochleistungs-LC-MS
d'un antibiotique aux taux sub-additifs, par
chromatographie en phase liquide à haute
performance couplée à une détection par
spectrométrie de masse en tandem (CL-SM/SM)
This European Standard was approved by CEN on 17 June 2019.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, 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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17299:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Terms and definitions . 4
4 Principle . 5
5 Reagents and materials . 5
6 Apparatus . 21
7 Sampling . 23
8 Preparation of test sample . 23
8.1 General . 23
8.2 Laboratory sample . 23
8.3 Test sample . 23
8.4 Test portion . 23
9 Procedure. 23
9.1 General . 23
9.2 Positive and negative control samples . 23
9.2.1 General . 23
9.2.2 Positive control sample PCS . 24
9.2.3 Negative control sample NCS . 24
9.3 Sample preparation . 24
9.3.1 Extraction . 24
9.3.2 Filtration . 24
9.3.3 Dilution . 24
9.4 Quantification through standard addition . 25
9.5 HPLC analysis . 25
9.5.1 Analytical conditions . 25
9.5.2 HPLC determination . 27
9.5.3 Criteria for acceptance of the analytical result . 28
10 Calculation . 28
11 Test report . 30
Annex A (informative) Complementary instrumental information. 31
European foreword
This document (EN 17299:2019) 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 February 2020, and conflicting national standards shall
be withdrawn at the latest by February 2020.
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 standardization request given to CEN by the European
Commission and the European Free Trade Association.
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, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
1 Scope
This document specifies a high performance liquid chromatographic – tandem mass spectrometry (HPLC-
MS/MS) method for the simultaneous screening and/or determination of the eleven authorized
coccidiostats (halofuginone hydrobromide, robenidine hydrochloride, nicarbazin, diclazuril,
decoquinate, monensin sodium, salinomycin sodium, narasin, lasalocid A sodium, semduramicin sodium
and maduramicin ammonium alpha) contents in compound feeds and feed materials of plant origin at
additive and cross-contamination levels and of the five non-registered coccidiostats (ethopabate,
clopidol, ronidazole, dimetridazole and amprolium) at sub-additive levels and for the screening of the
prohibited furazolidone antibiotic at sub-additive level, in the same matrices. It has been fully validated
only for poultry, cattle and pig compound feeds.
The range of application of the method is fit for the purpose of the screening and determination of all
eleven registered coccidiostats at the values set by European legislation, of the non-registered
coccidiostats and of the screening of the banned antibiotic.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 6498, Animal feeding stuffs — Guidelines for sample preparation (ISO 6498)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
Internal Standard
I.S.
compound of known concentration added to a sample to facilitate the qualitative identification and/or
quantitative determination of the sample components
[SOURCE: ISO 20752:2014, 3.2]
3.2
target feed
feed for target animal species or categories for which the additives are intended
[SOURCE: Commission Regulation (EC) No 124/2009]
3.3
non-target feed
feed for which the use of coccidiostats or histomonostats are not authorised, such as feed intended for
animal species or categories not provided for in the additive authorisation
[SOURCE: Commission Regulation (EC) No 124/2009]
4 Principle
All 11 authorized coccidiostats, non-registered coccidiostats and the banned antibiotic are extracted
using solid-liquid extraction. The extracts are centrifuged and supernatants filtered. After a first
screening analysis, the analytes are determined by reverse phase HPLC using electrospray ionization with
further tandem mass spectrometry detection. The quantitation of the detected target analytes is
performed using a multi-level standard additions approach.
5 Reagents and materials
WARNING — Avoid inhalation of and exposure to the toxic standard materials and solutions thereof.
Work in a fume-hood when handling the solvents and solutions.
Use only reagents recognized as analytical grade at least unless otherwise stated.
5.1 Water, HPLC grade or equivalent (e.g. milli-Q purified water).
5.2 Acetonitrile, HPLC gradient grade or hypergrade LC-MS, minimum 99,9 % purity.
5.3 Methanol, HPLC grade or hypergrade LC-MS.
5.4 Formic acid, HPLC grade or hypergrade LC-MS, minimum purity 98 %.
5.5 Ethanol, absolute, minimum purity 99,9 %.
5.6 N, N-Dimethylformamide, minimum purity 99 %.
5.7 Dimethylsulfoxide, minimum purity 99,5 %.
5.8 Calcium chloride, anhydrous, minimum purity 93 %.
5.9 Acetonitrile solution in wate
...