SIST EN 16858:2017

Foodstuffs - Determination of melamine and cyanuric acid in foodstuffs by liquid chromatography and tandem mass spectrometry (LC-MS/MS)

This European Standard specifies a method for the determination of melamine and cyanuric acid in foodstuffs with liquid chromatography in combination with tandem mass spectrometry. The method has been validated in an interlaboratory study via the analysis of spiked samples of milk based infant formula, soy based infant formula, milk powder, whole milk, soy drink and milk chocolate ranging from 0,71 mg/kg to 1,43 mg/kg for melamine and 0,57 mg/kg to 1,45 mg/kg for cyanuric acid. The limits of quantification (LOQ) for melamine and cyanuric acid in food are 0,05 mg/kg and 0,25 mg/kg, respectively. The upper limit of the working range is up to 10 mg/kg for melamine and up to 25 mg/kg for cyanuric acid.

Lebensmittel - Bestimmung von Melamin und Cyanursäure in Lebensmitteln mit Flüssigchromatographie und Tandem-Massenspektrometrie (LC-MS/MS)

Diese Europäische Norm legt ein Verfahren zur Bestimmung von Melamin und Cyanursäure in Lebensmitteln mit Flüssigchromatographie und Tandem Massenspektrometrie (LC MS/MS) fest. Das Verfahren wurde in einem Ringversuch durch die Analyse von aufgestockten Proben von Babynahrung auf Milchbasis, Babynahrung auf Sojabasis, Milchpulver, Vollmilch, Sojagetränk und Milchschokolade im Bereich von 0,71 mg/kg bis 1,43 mg/kg für Melamin und von 0,57 mg/kg bis 1,45 mg/kg für Cyanursäure validiert. Die Bestimmungsgrenzen (en: limit of quantification, LOQ) für Melamin und Cyanursäure in Lebensmitteln liegen bei 0,05 mg/kg beziehungsweise 0,25 mg/kg. Die Obergrenze des Arbeitsbereichs für Melamin reicht bis 10 mg/kg und für Cyanursäure bis 25 mg/kg.

Produits alimentaires - Détermination de la teneur en mélamine et en acide cyanurique dans les produits alimentaires par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (CL-SM/SM)

La présente Norme européenne spécifie une méthode de détermination de la teneur en mélamine et acide cyanurique dans les produits alimentaires par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem. La méthode a été validée lors d’un essai interlaboratoires par l’analyse d’échantillons enrichis de formule infantile à base de lait, de formule infantile à base de soja, de lait en poudre, de lait entier, de jus de soja et de lait chocolaté, présentant des teneurs en mélamine comprises entre 0,71 mg/kg et 1,43 mg/kg et des teneurs en acide cyanurique comprises entre 0,57 mg/kg et 1,45 mg/kg. Les limites de quantification (LOQ) pour la mélamine et l’acide cyanurique dans les aliments sont respectivement de 0,05 mg/kg et 0,25 mg/kg. La limite supérieure du domaine de mesure peut atteindre 10 mg/kg pour la mélamine et 25 mg/kg pour l’acide cyanurique.

Živila - Določevanje melamina in cianurinske kisline v živilih s tekočinsko kromatografijo in tandemsko masno spektrometrijo (LC-MS/MS)

Ta evropski standard opredeljuje metodo za določevanje melamina in cianurinske kisline v živilih s tekočinsko kromatografijo in tandemsko masno spektrometrijo (LC-MS/MS). Ta metoda je bila potrjena v medlaboratorijski študiji z analizo primešanih vzorcev formule za dojenčke na osnovi mleka, formule za dojenčke na osnovi soje, mleka v prahu, polnomastnega mleka, sojinega mleka in mlečne čokolade v razponu 0,71–1,43 mg/kg za melamin in 0,57–1,45 mg/kg za cianurinsko kislino. Mejne vrednosti kvantifikacije (LOQ) za melamin in cianurinsko kislino v hrani so 0,05 mg/kg in 0,25 mg/kg. Zgornja meja delovnega razpona je do 10 mg/kg za melamin in do 25 mg/kg za cianurinsko kislino.

General Information

• Status: Published
• Public Enquiry End Date: 29-Jul-2015
• Publication Date: 10-Aug-2017
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 02-Aug-2017
• Due Date: 07-Oct-2017
• Completion Date: 11-Aug-2017
• 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/463 - Mandate for Standardisation addressed to CEN for the methods of analysis for food contaminants
• Ref Project: EN 16858:2017 - Foodstuffs - Determination of melamine and cyanuric acid in foodstuffs by liquid chromatography and tandem mass spectrometry (LC-MS/MS)

Overview

EN 16858:2017 (CEN) defines a validated analytical method for the determination of melamine and cyanuric acid in foodstuffs using liquid chromatography coupled with tandem mass spectrometry (LC‑MS/MS). The method uses hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization in multiple reaction monitoring (MRM) on a triple‑quadrupole MS. It was validated in an interlaboratory study on a range of dairy and soy matrices and provides limits of quantification and working ranges suitable for food safety control.

Key topics and technical requirements

• Analytes: Melamine and cyanuric acid in foodstuffs.
• Technique: Liquid chromatography (HPLC or UHPLC) coupled to triple‑quadrupole MS/MS (LC‑MS/MS) with ESI and MRM.
• Sample preparation: Fortification with 13C‑labelled internal standards, incubation (≥1 h), extraction with acetonitrile/water, centrifugation, addition of benzoguanamine as recovery standard, and injection of aqueous supernatant. Typical sample mass for extraction is 1 g.
• Validated matrices and spike levels: Milk‑based infant formula, soy‑based infant formula, milk powder, whole milk, soy drink and milk chocolate. Spiked ranges used in validation: melamine 0.71–1.43 mg/kg, cyanuric acid 0.57–1.45 mg/kg.
• Performance limits: Limits of quantification (LOQ) - melamine 0.05 mg/kg, cyanuric acid 0.25 mg/kg. Upper working range - melamine up to 10 mg/kg, cyanuric acid up to 25 mg/kg.
• Standards and reagents: Use high‑purity reagents and water complying with EN ISO 3696 (grade 1). Method details include preparation of labelled internal standards, calibration solutions and mobile phases for HILIC separations.
• Instrumentation examples: HILIC columns (e.g. amide or BEH HILIC), LC pumps capable of gradients, injectors (e.g. 5 µL), centrifuges and sonication for extraction.

Applications

• Regulatory monitoring: Routine surveillance of commercial dairy and soy products for melamine and cyanuric acid contamination.
• Food safety investigations: Response testing following suspected adulteration or contamination incidents (e.g., infant formula safety events).
• Product quality control: Manufacturer and third‑party laboratory testing to verify raw materials and finished products.
• Accreditation and proficiency testing: Method suitable for use in accredited laboratories and interlaboratory comparison studies.

Who should use this standard

• Food testing laboratories (public and private) performing chemical contaminant analysis.
• National and regional food safety authorities and regulatory agencies.
• Manufacturers of dairy, soy and infant formula products for compliance testing.
• Accredited reference laboratories and proficiency test organizers.

Related standards (if applicable)

• EN ISO 3696:1995 - Water for analytical laboratory use (normative reference cited in EN 16858:2017).
• CEN/TC 275 - Technical committee responsible for horizontal food analysis methods.

Keywords: EN 16858:2017, melamine, cyanuric acid, LC‑MS/MS, HILIC, food safety, LOQ, infant formula, CEN.

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.WHNRþLQVNRLebensmittel - Bestimmung von Melamin und Cyanursäure in Lebensmitteln mit Flüssigchromatographie und Tandem-Massenspektrometrie (LC-MS/MS)Produits alimentaires - Détermination de la teneur en mélamine et en acide cyanurique dans les produits alimentaires par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (CL-SM/SM)Foodstuffs - Determination of melamine and cyanuric acid in foodstuffs by liquid chromatography and tandem mass spectrometry (LC-MS/MS)67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 16858:2017SIST EN 16858:2017en,fr,de01-september-2017SIST EN 16858:2017SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16858
May
t r s y ICS
x yä r w r English Version
Foodstuffs æ Determination of melamine and cyanuric acid in foodstuffs by liquid chromatography and tandem mass Produits alimentaires æ Détermination de la teneur en mélamine et en acide cyanurique dans les produits alimentaires par chromatographie en phase liquide
Lebensmittel æ Bestimmung von Melamin und Cyanursäure in Lebensmitteln mit Flüssigchromatographie und TandemæThis European Standard was approved by CEN on
s r April
t r s yä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä
EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels
t r s y CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s x z w zã t r s y ESIST EN 16858:2017

Typical HPLC chromatograms . 20 Annex B (informative)
Precision data . 24 Bibliography . 26
99 %, solid, (CAS 108-78-1). 4.6 Cyanuric acid, solid, (CAS 108-80-5). 4.7 Benzoguanamine, solid, (CAS 91-76-9). 4.8
13C Melamine, 13C3 (99 %), Amino-15N3 (98 %), solution with mass concentration
= 1 000 µg/ml. 4.9
13C Cyanuric acid, 13C3 (99 %), 15N3 (98 %), solution with
= 1 000 µg/ml. 4.10 Preparation of stock solutions 4.10.1 Melamine stock solution,
= 1 000 µg/ml Weigh, to the nearest 0,01 mg, approximately 100 mg of melamine (4.5) into a 100 ml glass flask (5.2) and add by weighing an amount of water to achieve a concentration of 1 000 µg/ml. Store the solution in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 year if the mass is carefully monitored. 4.10.2 Cyanuric acid stock solution,
= 1 000 µg/ml Weigh, to the nearest 0,01 mg, approximately 100 mg of cyanuric acid (4.6) into a 100 ml glass flask (5.2) and add by weighing an amount of water to achieve a concentration of 1 000 µg/ml. Store the solution in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 year if the mass is carefully monitored. 4.10.3 Benzoguanamine stock solution,
= 1 000 µg/ml Weigh, to the nearest 0,01 mg, approximately 100 mg of benzoguanamine (4.7) into a 100 ml glass flask (5.2) and add by weighing an amount of methanol (4.4) to achieve a concentration of 1 000 µg/ml. Store the solution in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 year if the mass is carefully monitored. 4.10.4
13C Melamine stock solution,
= 20 µg/ml Pipette 1,0 ml of 13C melamine (4.8) into a 50 ml volumetric flask (5.3). Make up to the mark with water and mix. The final concentration is 20 µg/ml. Transfer this solution into a 100 ml glass flask (5.2). The solution is stable under these conditions during at least 1 year if the mass is carefully monitored. 4.10.5
13C Cyanuric acid stock solution,
= 20 µg/ml Pipette 1,0 ml of 13C cyanuric acid (4.9) into a 50 ml volumetric flask (5.3). Make up to the mark with water and mix. The final concentration is 20 µg/ml. Transfer this solution into a 100 ml glass flask (5.2). The solution is stable under these conditions during at least 1 year if the mass is carefully monitored. SIST EN 16858:2017

= 20 µg/ml Pipette 1,0 ml of the melamine stock solution (4.10.1) into a 50 ml volumetric flask (5.3). Make up to the mark with dilution solution (4.16) and mix. The concentration is 20 µg/ml. Transfer this solution into a 100 ml glass flask (5.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 month if the mass is carefully monitored. 4.11.2 Cyanuric acid standard solution I,
= 20 µg/ml Pipette 1,0 ml of the cyanuric acid stock solution (4.10.2) into a 50 ml volumetric flask (5.3). Make up to the mark with dilution solution (4.16) and mix. The concentration is 20 µg/ml. Transfer this solution into a 100 ml glass flask (5.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 month if the mass is carefully monitored. 4.11.3 Melamine standard solution II,
= 0,2 µg/ml Pipette 1,0 ml of the melamine standard solution I (4.11.1) into a 100 ml volumetric flask (5.3). Make up to the mark with dilution solution (4.16) and mix. The concentration is 0,2 µg/ml. Transfer this solution into a 100 ml glass flask (5.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 month if the mass is carefully monitored. 4.11.4 Cyanuric acid standard solution II,
= 0,2 µg/ml Pipette 1,0 ml of the cyanuric acid standard solution I (4.11.2) into a 100 ml volumetric flask (5.3). Make up to the mark with dilution solution (4.16) and mix. The concentration is 0,2 µg/ml. Transfer this solution into a 100 ml glass flask (5.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 month if the mass is carefully controlled. 4.11.5 Benzoguanamine standard solution I,
= 20 µg/ml Pipette 1,0 ml of the benzoguanamine stock solution (4.10.3) into a 50 ml volumetric flask (5.3). Make up to the mark with dilution solution (4.16) and mix. The concentration is 20 µg/ml. Transfer this solution into a 100 ml glass flask (5.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 month if the mass is carefully monitored. 4.11.6 Benzoguanamine standard solution II,
= 2 µg/ml Pipette 1,0 ml of the benzoguanamine standard solution I (4.11.5) into a 10 ml volumetric flask (5.3). Make up to the mark with dilution solution (4.16) and mix. The concentration is 2 µg/ml. Transfer this solution into a 20 ml glass flask (5.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 month if the mass is carefully controlled. 4.11.7
13C Melamine and 13C cyanuric acid standard solution,
= 2 µg/ml Pipette 0,5 ml of the 13C melamine stock solution (4.10.4) and 0,5 ml of the 13C cyanuric acid stock solution (4.10.5) into a 5 ml volumetric flask (5.3). Make up to the mark with water and mix. The concentrations for both compounds is 2 µg/ml. Transfer this solution into a 20 ml glass flask (5.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable under these conditions during at least 1 month if the mass is carefully monitored. 4.11.8 Preparation of calibration solutions Pipette the volumes as indicated in Table 1 into a 10 ml volumetric flask (5.3) and make up to the mark with the dilution solution (4.16). SIST EN 16858:2017

Calibration solution number
1 2 3 4 5 6 7 Melamine standard solution I (4.11.1), ml 0 0 0 0 0 0,05 0,25 Cyanuric acid standard solution I (4.11.2), ml 0 0 0 0 0 0,05 0,25 Melamine standard solution II (4.11.3), ml 0 0,05 0,25 1,25 2,5 0 0 Cyanuric acid standard solution II (4.11.4), ml 0 0,05 0,25 1,25 2,5 0 0 Benzoguanamine standard solution II (4.11.6), ml 0,5 0,5 0,5 0,5 0,5 0,5 0,5 13C Melamine – 13C cyanuric acid standard solution (4.11.7), ml 0,5 0,5 0,5 0,5 0,5 0,5 0,5 Concentration 13C melamine – 13C cyanuric acid, µg/ml 0,1 0,1 0,1 0,1 0,1 0,1 0,1 Concentration native melamine – cyanuric acid, µg/ml 0 0,001 0,005 0,025 0,05 0,1 0,5 4.12 Mobile phase A for HPLC, substance concentration c(ammonium acetate) = 10 mmol/l Dissolve 0,77 g of ammonium acetate (4.3) in 1 000 ml of water. 4.13 Mobile phase B for HPLC Acetonitrile (4.2). 4.14 Mobile phase A for UHPLC Mix 3 parts per volume of formic acid (4.1) with 97 parts per volume of water. 4.15 Mobile phase B for UHPLC, c(ammonium acetate) = 20 mmol/l in a mixture of 3 parts per volume of water and 97 parts per volume of acetonitrile. Dissolve 1,54 g of ammonium acetate (4.3) in 30 ml of water. Add 970 ml of acetonitrile (4.2) to the mixture and shake firmly. The turbid mixture will clear overnight. NOTE The optimal choice for the mobile phases A and B may depend on the instrument configuration (see 5.10 to 5.12), in more particular the type of column used. Equivalent products may be used if they can be shown to lead to the same results. 4.16 Dilution solution Transfer, using measuring cylinders, 70 ml of acetonitrile (4.2) and 30 ml of water into a 100 ml volumetric flask (5.3) and mix. Store at room temperature for no longer than one month. 5 Apparatus All technical descriptions are examples of possible system setups and parameters and shall be scaled or adopted to the user's equipment. Usual laboratory glassware and equipment and, in particular, the following. 5.1 Disposable polypropylene carbonate tube, of approximately 50 ml. SIST EN 16858:2017

1 cm. 6.2 Extraction 6.2.1 General The following samples shall be included in each series: — calibration solutions;
1) This 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. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16858:2017
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