1、Foodstuffs Determination of melamine and cyanuric acid in foodstuffs by liquid chromatography and tandem mass spectrometry (LC-MS/MS)BS EN 16858:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16858 May 2017 IC
2、S 67.050 English Version Foodstuffs - Determination of melamine and cyanuric acid in foodstuffs by liquid chromatography and tandem mass spectrometry (LC-MS/MS) Produits alimentaires - Dtermination de la teneur en mlamine et en acide cyanurique dans les produits alimentaires par chromatographie en p
3、hase liquide couple la spectromtrie de masse en tandem (CL-SM/SM) Lebensmittel - Bestimmung von Melamin und Cyanursure in Lebensmitteln mit Flssigchromatographie und Tandem-Massenspektrometrie (LC-MS/MS) This European Standard was approved by CEN on 10 April 2017. CEN members are bound to comply wit
4、h 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 Manag
5、ement 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 sta
6、tus 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, Malt
7、a, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
8、2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16858:2017 ENational forewordThis British Standard is the UK implementation of EN 16858:2017.The UK participation in its preparation was entrusted to Technical Committee AW/275,
9、Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Insti
10、tution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 80861 6ICS 67.050Compliance with a British Standard cannot confer immunity from legal obligations. This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2017.Amendments/corri
11、genda issued since publicationDate Text affectedBRITISH STANDARDBS EN 16858:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16858 May 2017 ICS 67.050 English Version Foodstuffs - Determination of melamine and cyanuric acid in foodstuffs by liquid chromatography and tandem mass spectrometry
12、(LC-MS/MS) Produits alimentaires - Dtermination de la teneur en mlamine et en acide cyanurique dans les produits alimentaires par chromatographie en phase liquide couple la spectromtrie de masse en tandem (CL-SM/SM) Lebensmittel - Bestimmung von Melamin und Cyanursure in Lebensmitteln mit Flssigchro
13、matographie und Tandem-Massenspektrometrie (LC-MS/MS) This European Standard was approved by CEN on 10 April 2017. 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
14、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
15、 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, E
16、stonia, 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 COMMITT
17、EE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16858:2017 EBS EN 16858:2017
18、EN 16858:2017 (E) 2 Contents Page European foreword . 3 1 Scope 4 2 Normative references 4 3 Principle . 4 4 Reagents . 4 5 Apparatus . 7 6 Procedure. 8 7 System settings. 9 8 Calculation and expression of results . 14 9 Precision data 17 10 Test report 18 Annex A (informative) Typical HPLC chromato
19、grams . 20 Annex B (informative) Precision data . 24 Bibliography . 26 BS EN 16858:2017EN 16858:2017 (E) 3 European foreword This document (EN 16858:2017) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European
20、Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by November 2017, and conflicting national standards shall be withdrawn at the latest by November 2017. Attention is drawn to the possibility that some of the elemen
21、ts of this document may be the subject of patent rights. CEN and/or CENELEC 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 use of
22、 this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to take appropriate measures for ensuring safety and health of the personnel prio
23、r to application of the standard and to fulfil statutory requirements for this purpose. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
24、 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
25、Kingdom. BS EN 16858:2017EN 16858:2017 (E) 4 1 Scope 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
26、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 aci
27、d 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. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for
28、its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principle A test por
29、tion of the homogenous food sample is fortified with 13C labelled internal standards (melamine and cyanuric acid). After incubation for at least one hour, water is added to the sample and after shaking, the slurry is dissolved in a mixture of acetonitrile and water. The sample is shaken and centrifu
30、ged. After separation of supernatant from sediments benzoguanamine is added as a recovery standard. An aliquot of the aqueous supernatant is injected into a LC-MS/MS system. The triple quadrupole mass spectrometer is coupled either to high performance liquid chromatography (HPLC) or to ultra perform
31、ance liquid chromatography (UHPLC). Chromatography is based on hydrophilic interaction liquid chromatography (HILIC). Ionization is achieved by electrospray ionization (ESI) in multiple reaction monitoring (MRM). 4 Reagents Use only reagents of recognized analytical grade and water complying with gr
32、ade 1 of EN ISO 3696:1995, unless otherwise specified. Use only reagents with purity suitable for melamine and cyanuric acid analysis. Check the purity of the reagents and reference materials (e.g. standard solutions) by performing a blank test under the same conditions as used in the method. The ch
33、romatogram shall not show any interfering impurity at the retention time of compounds of interest. BS EN 16858:2017EN 16858:2017 (E) 5 4.1 Formic acid, mass fraction is 98 % to 100 % (CAS 64-18-6). 4.2 Acetonitrile, HPLC gradient grade (CAS 75-05-8). 4.3 Ammonium acetate, mass fraction approximately
34、 98 % (CAS 631-61-8). 4.4 Methanol, Ultra LC-MS grade (CAS 67-56-1). 4.5 Melamine, mass fraction 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
35、 g/ml. 4.9 13C Cyanuric acid, 13C3 (99 %), 15N3 (98 %), solution with = 1 000 g/ml. 4.10 Preparation of stock solutions 4.10.1 Melamine stock solution, = 1 000 g/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
36、water to achieve a concentration of 1 000 g/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 g/ml Weigh, to the nearest 0,01 mg, approximately 1
37、00 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 g/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.
38、10.3 Benzoguanamine stock solution, = 1 000 g/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 g/ml. Store the solution in a refrigerator at 4 C (3 C). Th
39、e 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 g/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 g/ml. T
40、ransfer 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 g/ml Pipette 1,0 ml of 13C cyanuric acid (4.9) into a 50 ml volumetric flask (5.3). Make up to
41、 the mark with water and mix. The final concentration is 20 g/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. BS EN 16858:2017EN 16858:2017 (E) 6 4.11 Preparation of standard solution
42、s 4.11.1 Melamine standard solution I, = 20 g/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 g/ml. Transfer this solution into a 100 ml glass flask (5.2) and store it in
43、 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 g/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
44、mark with dilution solution (4.16) and mix. The concentration is 20 g/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 stan
45、dard solution II, = 0,2 g/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 g/ml. Transfer this solution into a 100 ml glass flask (5.2) and store it in a refrigerat
46、or 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 g/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
47、with dilution solution (4.16) and mix. The concentration is 0,2 g/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 s
48、tandard solution I, = 20 g/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 g/ml. Transfer this solution into a 100 ml glass flask (5.2) and store it in a refrigerat
49、or 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 g/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 g/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
