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

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Published
Publication Date
03-Sep-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
04-Sep-2019
Completion Date
04-Sep-2019

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SLOVENSKI STANDARD
SIST EN 17212:2019
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
SIST EN 17212:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17212:2019
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SIST EN 17212:2019
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.
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SIST EN 17212:2019
EN 17212:2019 (E)
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

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EN 17212:2019 (E)

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

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SIST EN 17212:2019
EN 17212:2019 (E)
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.
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SIST EN 17212:2019
EN 17212:2019 (E)
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.
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EN 17212:2019 (E)
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 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.
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 6497, Animal feeding stuffs - Sampling (ISO 6497)

EN ISO 6498:2012, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498:2012)

EN ISO/IEC 17043:2010, Conformity assessment — General requirements for proficiency testing

(ISO/IEC 17043:2010)

ISO 3534-2:2006, Statistics — Vocabulary and symbols — Part 2: Applied statistics

ISO 16577:2016, Molecular biomarker analysis — Terms and definitions

ISO 24276:2006, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — General requirements and definitions

ISO/IEC GUIDE 99:2007, International vocabulary of metrology — Basic and general concepts and

associated terms (VIM)
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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
calibration

complete set of operations which estimates under specified conditions the calibration function from

observations of the response variable obtained on reference states
[SOURCE: ISO 3534-2:2006, 3.5.13]
3.2
calibration function

functional relationship between the expected value of the response variable and the value of the net state

variable
[SOURCE: ISO 3534-2:2006, 3.5.12]
3.3
canned pet food

feed product for pets which has been processed, packaged, sealed and sterilized for preservation in cans

or similar containers
[SOURCE: EN ISO 6498:2012, 2.3.15]
3.4
collaborative trial
interlaboratory comparisons

organization, performance and evaluation of measurements or tests on the same or similar items by two

or more laboratories in accordance with predetermined conditions

Note 1 to entry: Interlaboratory comparisons are widely used for a number of purposes and their use is increasing

internationally. Typical purposes for interlaboratory comparisons include the evaluation of the performance

characteristics of a method – often described as collaborative trials [SOURCE: EN ISO/IEC 17043:2010,

Introduction].
[SOURCE: EN ISO/IEC 17043:2010, 3.4]
3.5
complementary feed

compound feed which has a high content of certain substances but which, by reason of its composition,

is sufficient for a daily ration only if used in combination with other feed
[SOURCE: Regulation (EC) No 767/2009 (Article 3, 1j)] [1]
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EN 17212:2019 (E)
3.6
complete feed

compound feed which, by reason of its composition, is sufficient for a daily ration

[SOURCE: Regulation (EC) No 767/2009 (Article 3, 1i)] [1]
3.7
compound feed

mixture of at least two feed materials, whether or not containing feed additives, for oral animal feeding

in the form of complete or complementary feed
[SOURCE: Regulation (EC) No 767/2009 (Article 3, 1h)] [1]
3.8
contaminated materials

feed containing a level of undesirable substances in excess of that which is acceptable under Directive

2002/32/EC
[SOURCE: Regulation (EC) No 767/2009 (Article 3, 1p) [1]
3.9
detection limit
limit of detection

measured quantity value, obtained by a given measurement procedure, for which the probability of

falsely claiming the absence of a component in a material is β, given a probability α of falsely claiming its

presence
Note 1 to entry: IUPAC recommends default values for α and β equal to 0,05.
Note 2 to entry: The abbreviation LOD is sometimes used.
Note 3 to entry: The term 'sensitivity' is discouraged for ‘detection limit’.
[SOURCE: ISO/IEC Guide 99:2007, 4.18]
3.10
feed material

products of vegetable or animal origin, whose principal purpose is to meet animals’ nutritional needs, in

their natural state, fresh or preserved, and products derived from the industrial processing thereof, and

organic or inorganic substances, whether or not containing feed additives, which are intended for use in

oral animal-feeding either directly as such, or after processing, or in the preparation of compound feed,

or as carrier of premixtures
[SOURCE: Regulation (EC) No 767/2009 (Article 3, 1g)] [1]
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3.11
HorRat

ratio of the reproducibility relative standard deviation to that calculated from the Horwitz equation

–0,15
Note 1 to entry: Predicted relative standard deviation PRSDR = 2 C
HorRat = RSD / PRSD
RR R
(1)
HorRat = RSD / PRSD
r r R
(2)

Note 2 to entry: If applied to within-laboratory studies, the normal range of HorRat(r) is 0,30 – 1,30.

Note 3 to entry: To check proper calculation of PRSDR, a C of 10 should give a PRSDR of 16 %. C is concentration

expressed as a mass fraction (both numerator and denominator expressed in the same units). The HorRat is

indicative of method performance for a large majority of methods in chemistry. Normal values lie between 0,50 and

2,00.
[SOURCE: ISO 16577:2016, 3.75]
3.12
milk replacer

compound feed administered in dry form or after dilution in a given quantity of liquid for feeding young

animals as a complement to, or substitute for, post-colostral milk or for feeding young animals such as

calves, lambs or kids intended for slaughter
[SOURCE: Regulation (EC) No 767/2009 (Article 3, 1l] [1]]
3.13
precision

closeness of agreement between indications or measured quantity values obtained by replicate

measurements on the same or similar objects under specified conditions

Note 1 to entry: Measurement precision is usually expressed numerically by measures of imprecision, such as

standard deviation, variance, or coefficient of variation under the specified conditions of measurement.

Note 2 to entry: The ‘specified conditions’ can be, for example, repeatability conditions of measurement,

intermediate precision conditions of measurement, or reproducibility conditions of measurement (see ISO 5725-1)

[2].

Note 3 to entry: Measurement precision is used to define measurement repeatability, intermediate measurement

precision, and measurement reproducibility.

Note 4 to entry: Sometimes 'measurement precision' is erroneously used to mean 'measurement accuracy'.

[SOURCE: ISO-IEC Guide 99:2007, 2.15]
3.14
repeatability
precision under repeatability conditions

Note 1 to entry: Repeatability can be expressed quantitatively in terms of the dispersion characteristics of the

results.
[SOURCE: ISO 3534-2:2006, 3.3.5]
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3.15
repeatability conditions

observation conditions where independent test/measurement results are obtained with the same

method on identical test/measurement items in the same test or measuring facility by the same operator

using the same equipment within short intervals of time
Note 1 to entry: Repeatability conditions include:
— the same measurement procedure or test procedure;
— the same operator;
— the same measuring or test equipment used under the same conditions;
— the same location;
— repetition over a short period of time.
[SOURCE: ISO 3534-2:2006, 3.3.6]
3.16
repeatability limit
repeatability critical difference for a specified probability of 95 %
[SOURCE: ISO 3534-2:2006, 3.3.9]
3.17
repeatability standard deviation

standard deviation of test results or measurement results obtained under repeatability conditions

Note 1 to entry: It is a measure of the dispersion of the distribution of test or measurement results under

repeatability conditions.

Note 2 to entry: Similarly, “repeatability variance” and “repeatability coefficient of variation” can be defined and

used as measures of the dispersion of test or measurement results under repeatability conditions.

[SOURCE: ISO 3534-2:2006, 3.3.7]
3.18
reproducibility
precision under reproducibility conditions

Note 1 to entry: Reproducibility can be expressed quantitatively in terms of the dispersion characteristics (0) of the

results.
Note 2 to entry: Results are usually understood to be corrected results.
[SOURCE: ISO 3534-2:2006, 3.3.10]
3.19
reproducibility conditions

observation conditions where independent test/measurement results are obtained with the same

method on identical test/measurement items in different test or measurement facilities with different

operators using different equipment
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[SOURCE: ISO 3534-2:2006, 3.3.11]
3.20
reproducibility limit
reproducibility critical difference for a specified probability of 95 %
[SOURCE: ISO 3534-2:2006, 3.3.14]
3.21
reproducibility standard deviation

standard deviation of test results or measurement results obtained under reproducibility conditions

Note 1 to entry: It is a measure of the dispersion of the distribution of test or measurement results under

reproducibility conditions.

Note 2 to entry: Similarly, “reproducibility variance” and “reproducibility coefficient of variation” can be defined

and used as measures of the dispersion of test or measurement results under reproducibility conditions.

[SOURCE: ISO 3534-2:2006, 3.3.12]
3.22
screening method

method that will rapidly and reliably eliminate (screen) a large number of negative (or positive) test

samples and restrict the number of test samples requiring the application of a rigorous method

[SOURCE: ISO 24276:2006, 3.1.24]
4 Principle

In order to check for the presence of melamine and cyanuric acid in a screening method, samples are

extracted by a mixture of trichloroacetic acid, acetonitrile and water.

The extract is further diluted with acetonitrile, centrifuged and injected into the LC-MS/MS system. For

samples tested positive in the screening method, a confirmatory method shall be performed.

For the confirmatory method an internal standard is added. Extraction is performed thrice. The first time,

it is performed with a mixture of trichloroacetic acid, acetonitrile and water. The second time, it is

performed twice by a mixture of acetonitrile and water.

After dilution and centrifugation, the supernatant is injected into the LC-MS/MS system.

The quantification of melamine and/or cyanuric acid is carried out by means of standard addition.

5 Reagents
5.1 General

Use only reagents of recognized analytical grade or higher unless stated otherwise.

WARNING — Avoid inhalation of and exposure to the toxic materials and solutions thereof. Work in a

fume-hood when handling the solvents and solutions. Wear safety glasses, protective clothing and avoid

skin contact.
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5.2 Chemicals
5.2.1 Diethylamine (DEA)
5.2.2 Trichloroacetic acid (TCA)

5.2.3 Water, purity grade for HPLC or comparable quality (e.g. with Milli-Q purified water).

5.2.4 Acetonitrile, hypergrade for LCMS.
5.2.5 Methanol, hypergrade for LCMS.

5.2.6 Extraction solvent A: Weigh 80 g TCA (5.2.2) and add 600 ml extraction solvent B (solvent B

5.2.7).

5.2.7 Extraction solvent B: Mix acetonitrile (5.2.4) and water (5.2.3) at a rate of 10 + 30 (v/v).

5.2.8 Ammonium acetate
5.2.9 Ammonium acetate solution, c = 10 mmol/l.

Weigh 0,77 g ammonium acetate (5.2.8) to the nearest 0,01 g into a 1 000 ml volumetric flask (6.11) and

dissolve in water (5.2.3). Fill up to 1 000 ml with water (5.2.3). The solution is stable for one week.

5.2.10 Mobile Phase A for HPLC

Mix acetonitrile (5.2.4) and ammonium acetate solution (5.2.9) at the rate of 95 + 5 (v/v).

5.2.11 Mobile Phase B for HPLC

Mix ammonium acetate solution (5.2.9) and acetonitrile (5.2.4) at the rate of 95 + 5 (v/v).

5.2.12 Melamine (2,4,6-Triamino-1,3,5-triazine, sym-Triaminotriazine), MEL

Specification of the purity of each batch of the reference standards is necessary.

5.2.12.1 Melamine standard substance purity at least 99 %.
5.2.12.2 Alternatively melamine standard solution c = 1 mg/ml or c = 0,1 mg/ml.

5.2.13 Cyanuric acid (1,3,5-Triazine-2,4,6-triol, 2,4,6-Trihydroxy-1,3,5-triazine), CYA

Specification of the purity of each batch of the reference standards is necessary.

5.2.13.1 Cyanuric acid standard substance purity at least 98 %.
5.2.13.2 Alternatively cyanuric standard solution c = 1 mg/ml or c = 0,1 mg/ml.
5.2.14 C -Melamine

The standard substances or standard solutions can be purchased from Sigma-Aldrich, Restek or Witega. These are

examples for suitable products available commercially. This information is given for the convenience of users of this

standard and does not constitute an endorsement by CEN of these products. Other products could be used, if the

results are comparable.
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SIST EN 17212:2019
EN 17212:2019 (E)

NOTE If other isotopically labelled standards e.g. N -melamine are used as internal standard, the suitability

can be verified.
5.2.14.1 C -Melamine standard substance purity at least 95 %.
5.2.14.2 Alternatively, C -Melamine standard solution c = 0,1 mg/ml.
5.2.15 C -Cyanuric acid
5.2.15.1 C -Cyanuric acid standard substance purity at least 97 %.
5.2.15.2 Alternatively C -Cyanuric acid standard solution c = 0,1 mg/ml.
5.3 Standard solutions

The preparation of mixture solutions of melamine and cyanuric acid is not advisable.

5.3.1 Melamine stock solution 1, c = 1 mg/ ml

Weigh to the nearest 0,1 mg, 10 mg of melamine reference standard (5.2.12.1) into a 10 ml volumetric

flask (6.11). The true weight has to be recorded. Dissolve the standard in 2,5 ml DEA (5.2.1) and about

5 ml water (5.2.3), place in an ultrasonic bath (6.8) for 15 min to 30 min and dilute with water (5.2.3) up

to 10 ml. The stock solution is stored at −20 °C and is stable for six months.
Alternatively, use commercially available standard solutions (5.2.12.2).
5.3.2 Melamine stock solution 2, c = 0,1 mg/ml
Alter
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