EN 17212:2019

Animal Feeding stuffs - Methods of sampling and analysis - Determination of melamine and cyanuric acid content by liquid chromatographic method with mass spectrometric detection (LC-MS/MS)

This document specifies a high-performance liquid chromatographic (HPLC) mass spectrometric (MS) method for screening and quantification of melamine and cyanuric acid in the concentration range between 1 mg/kg and 100 mg/kg feed.
The method is validated in an international collaborative trial for melamine in complete feed, complementary feed, feed material, milk replacer and pet food including canned pet food in the range between 1 mg/kg and 80 mg/kg with particular regard to the maximum level of 2,5 mg/kg as established by the European Commission.
Laboratory experiences have shown that the method is also applicable for cyanuric acid in the same concentration range in complete feed (n = 7), complementary feed (n = 6), feed material (n = 7, resp. 9), milk replacer (n = 7) and pet food (n = 7) including canned pet food.
Since the LC-MS/MS sensitivity for cyanuric acid is lower than for melamine, it has to be ensured that the LC-MS/MS system is in excellent working order. The method is applicable to feeding stuffs but not tested for pre-mixtures and feed additives.
Quantification of concentrations above 100 mg/kg is possible, but the method has to be validated by the operator.

Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung des Gehaltes von Melamin und Cyanursäure mittels flüssigkeitschromatographischem Verfahren mit massenspektrometrischem Nachweis (LC-MS/MS)

Das vorliegende Dokument legt ein hochleistungsflüssigkeitschromatographisches Verfahren (HPLC) mit
massenspektrometrischem Nachweis (MS) für das Screening und die quantitative Bestimmung von Melamin
und Cyanursäure in dem Konzentrationsbereich zwischen 1 mg/kg und 100 mg/kg Futtermittel fest.
Das Verfahren wurde in einem internationalen Ringversuch für Melamin in Alleinfuttermittel,
Ergänzungsfuttermittel, Einzelfuttermittel, Milchaustausch-Futtermittel und Heimtierfuttermittel,
einschließlich Dosenfutter für Heimtiere, im Bereich zwischen 1 mg/kg und 80 mg/kg unter besonderer
Berücksichtigung des durch die Europäische Kommission festgelegten Höchstgehaltes von 2,5 mg/kg
validiert.
Laborerfahrungen haben gezeigt, dass das Verfahren auch für Cyanursäure im gleichen
Konzentrationsbereich in Alleinfuttermittel (n = 7), Ergänzungsfuttermittel (n = 6), Einzelfuttermittel (n = 7
bzw. 9), Milchaustausch-Futtermittel (n = 7) und Heimtierfuttermittel (n = 7), einschließlich Dosenfutter für
Heimtiere, anwendbar ist.
Da die Empfindlichkeit der LC-MS/MS für Cyanursäure geringer ist als für Melamin, ist sicherzustellen, dass
sich das LC-MS/MS-System in ausgezeichnetem Betriebszustand befindet. Das Verfahren gilt für
Futtermittel, wurde jedoch nicht für Vormischungen und Futtermittelzusatzstoffe geprüft.
Die quantitative Bestimmung von Konzentrationen über 100 mg/kg ist möglich, aber das Verfahren muss
durch den Bearbeiter validiert werden.

Aliments des animaux - Méthodes d'échantillonnage et d'analyse - Détermination des teneurs en mélanine et en acide cyanurique par chromatographie liquide couplée à la spectrométrie de masse (LC-MS/MS)

Le présent document spécifie une méthode par chromatographie liquide haute performance (HPLC) couplée à la spectrométrie de masse (MS) pour détecter et quantifier la mélamine dans la gamme de concentration comprise entre 1 mg/kg et 100 mg/kg d’aliments pour animaux.
La méthode a été validée lors d’un essai interlaboratoires international portant sur la mélamine présente dans les aliments complets pour animaux, les aliments complémentaires pour animaux, les matières premières pour aliments des animaux, les aliments d’allaitement et les aliments pour animaux domestiques, y compris les aliments en conserve pour animaux domestiques dans la gamme comprise entre 1 mg/k et 80 mg/kg, avec une attention particulière portée au niveau maximal de 2,5 mg/kg établi par la Commission européenne.
Les expériences en laboratoire ont montré que la méthode est également applicable à l’acide cyanurique dans la même gamme de concentration dans les aliments complets pour animaux (n = 7), les aliments complémentaires pour animaux (n = 6), les matières premières pour aliments des animaux (n = 7, resp. 9), les aliments d’allaitement (n = 7) et les aliments pour animaux domestiques (n = 7), y compris les aliments en conserve pour animaux domestiques.
Étant donné que la sensibilité LC-MS/MS pour l’acide cyanurique est nettement moins élevée que pour la mélamine, il faut s’assurer que le système LC-MS/MS est en excellent état de marche. La méthode est applicable aux aliments pour animaux mais n’a pas été soumise à essai dans les prémélanges et les additifs pour l’alimentation animale.
Il est possible de quantifier des concentrations supérieures à 100 mg/kg. Cependant, la méthode doit être validée par l’opérateur.

Krma: metode vzorčenja in analize - Določevanje melamina in cianurne kisline z metodo tekočinske kromatografije z masno spektrometrično detekcijo (LC-MS/MS)

Ta dokument določa metodo tekočinske kromatografije visoke ločljivosti (HPLC) z masno spektrometrijo (MS) za presejanje in kvantifikacijo melamina v koncentracijah od < 1 mg/kg do 100 mg/kg krme.
Metodo potrjuje mednarodni primerjalni preskus melamina v popolni krmni mešanici, dopolnilni krmni mešanici, posamičnih krmilih, mlečnih nadomestkih in hrani za hišne živali, vključno s konzervirano hrano za hišne ljubljenčke, v območju med 1 mg/kg in 80 mg/kg, s posebnim poudarkom na največji stopnji 2,5 mg/kg, ki jo je določila Evropska komisija.
Laboratorijske izkušnje kažejo, da se lahko metoda uporablja tudi za cianurinsko kislino v enakem koncentracijskem območju v popolni krmni mešanici (n=7), dopolnilni krmni mešanici (n=6), posamičnih krmilih (n=7 oz. 9), mlečnih nadomestkih (n=7) in hrani za hišne živali (n=7), vključno s konzervirano hrano za hišne živali.
Ker je občutljivost metode LC-MS/MS na cianurinsko kislino bistveno nižja kot na melamin, je treba zagotoviti, da sistem LC-MS/MS brezhibno deluje. Metoda se uporablja za krmo, vendar ni preskušena za premikse in krmne dodatke.
Kvantifikacija koncentracij nad 100 mg/kg je mogoča, vendar mora metodo potrditi upravljavec.

General Information

• Status: Published
• Publication Date: 03-Sep-2019
• Withdrawal Date: 30-Mar-2020
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 1 - Minerals and Contaminants
• Current Stage: 9020 - Submission to 2 Year Review Enquiry - Review Enquiry
• Start Date: 15-Oct-2024
• Completion Date: 15-Oct-2024
• 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 17212:2019 - Animal Feeding stuffs: Methods of sampling and analysis - Determination of melamine and cyanuric acid content by liquid chromatographic method with mass spectrometric detection (LC-MS/MS)

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-2019
Krma: metode vzorčenja in analize - Določevanje melamina in cianurne kisline z
metodo tekočinske kromatografije z masno spektrometrično detekcijo (LC-MS/MS)
Animal Feeding stuffs: Methods of sampling and analysis - Determination of melamine
and cyanuric acid content by liquid chromatographic method with mass spectrometric
detection (LC-MS/MS)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung des Gehaltes
von Melamin und Cyanursäure mittels flüssigkeitschromatographischen Verfahrens mit
massenspektrometrischem Nachweis (LC-MS/MS)
Aliments des animaux : Méthodes d'échantillonnage et d'analyse - Détermination des
teneurs en mélamine et en acide cyanurique par chromatographie liquide couplée à la
spectrométrie de masse (LC‐MS/MS)
Ta slovenski standard je istoveten z: EN 17212:2019
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17212
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2019
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal Feeding stuffs - Methods of sampling and analysis -
Determination of melamine and cyanuric acid content by
liquid chromatographic method with mass spectrometric
detection (LC-MS/MS)
Aliments des animaux - Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Détermination des teneurs en mélanine et Untersuchungsverfahren - Bestimmung des Gehaltes
en acide cyanurique par chromatographie liquide von Melamin und Cyanursäure mittels
couplée à la spectrométrie de masse (LC-MS/MS) flüssigkeitschromatographischem Verfahren mit
massenspektrometrischem Nachweis (LC-MS/MS)
This European Standard was approved by CEN on 7 July 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 17212:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 7
4 Principle .11
5 Reagents .11
5.1 General .11
5.2 Chemicals .12
5.3 Standard solutions .13
5.3.6 Cyanuric acid stock solution 1, c = 1 mg/ml .14
6 Apparatus .15
7 Preparation of sample .16
7.1 General .16
7.2 Laboratory sample .16
7.3 Test material .16
7.4 Sample material .16
8 Procedure.17
8.1 General .17
8.2 Screening method .17
8.2.1 Sample extraction .17
8.2.2 Sample dilution .17
8.2.3 Injection solutions for LC-MS/MS .17
8.3 Confirmatory method .17
8.3.1 Addition of internal standard .17
8.3.2 Sample extraction .18
8.3.3 Sample dilution and standard addition .18
8.3.4 Injection solution for LC-MS/MS .19
8.4 Quality control measures for screening method .20
8.4.1 Blank extract .20
8.4.2 Control standards .20
8.5 Quality control measures for confirmatory method .20
8.5.1 Blank extract .20
8.5.2 Control matrix sample .20
8.5.3 Control standards .21
9 HPLC-MS/MS analysis .21
9.1 General .21
9.2 HPLC operating conditions .21
9.3 Determination of melamine and cyanuric acid in sample test solutions .21
9.3.1 Example of an analysis sequence for screening method.21
9.3.2 Sample tested positive in the screening method .22
9.3.3 Example of an analysis sequence for confirmatory method . 22
10 Calculation . 22
10.1 Peak identification . 22
10.2 Quantification (confirmatory method) . 22
11 Precision . 24
11.1 Collaborative trial . 24
11.2 Repeatability limit . 24
11.3 Reproducibility limit . 24
12 Test report . 25
Annex A (informative) Examples for suitable HPLC-MS/MS conditions . 26
A.1 Example of suitable HPLC conditions . 26
A.2 Example suitable for SCIEX API 4000 Q-Trap or API 4000 . 27
Annex B (informative) Results of the international collaborative trial . 29
Annex C (informative) Results of the in-house validation of the method . 32
Annex D (informative) Typical chromatograms . 35
Bibliography . 37

European foreword
This document (EN 17212:2019) 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 March 2020, and conflicting national standards shall be
withdrawn at the latest by March 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 mandate 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.
Introduction
This document was developed in response to the demand for a reliable method to detect and quantify
melamine and cyanuric acid in feeding stuffs. Both substances were illegally mixed into feed.
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.
1 Scope
This document specifies a high-performance liquid chromatographic (HPLC) mass spectrometric (MS)
method for screening and quantification of melamine and cyanuric acid in the concentration range
between 1 mg/kg and 100 mg/kg feed.
The method is validated in an international collaborative trial for melamine in complete feed,
complementary feed, feed material, milk replacer and pet food including canned pet food in the range
between 1 mg/kg and 80 mg/kg with particular regard to the maximum level of 2,5 mg/kg as established
by the European Commission.
Laboratory experiences have shown that the method is also applicable for cyanuric acid in the same
concentration range in complete feed (n = 7), complementary feed (n = 6), feed material (n = 7, resp. 9),
milk r
...