ISO/PRF 23970

Milk, milk products and infant formula -- Determination of melamine and cyanuric acid by liquid chromatography and tandem mass spectrometry (LC-MS/MS)

Lait, produits laitiers et formule infantile -- Détermination de la teneur en mélamine et en acide cyanurique par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (CL-SM/SM)

General Information

• Status: Published
• ICS: 67.100.10 - Milk and processed milk products
• Technical Committee: ISO/TC 34/SC 5 - Milk and milk products
• Drafting Committee: ISO/TC 34/SC 5 - Milk and milk products

Standards Content (sample)

INTERNATIONAL ISO
STANDARD 23970
IDF 252
First edition
Milk, milk products and infant
formula — Determination of
melamine and cyanuric acid by liquid
chromatography and tandem mass
spectrometry (LC-MS/MS)
Lait, produits laitiers et formule infantile — Détermination de la
teneur en mélamine et en acide cyanurique par chromatographie en
phase liquide couplée à la spectrométrie de masse en tandem (CL-SM/
SM)
PROOF/ÉPREUVE
Reference numbers
ISO 23970:2021(E)
IDF 252:2021(E)
ISO and IDF 2021
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ISO 23970:2021(E)
IDF 252:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO and IDF 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

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Published in Switzerland
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ISO 23970:2021(E)
IDF 252:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 1

5 Reagents ........................................................................................................................................................................................................................ 1

5.1 List of reagents ........................................................................................................................................................................................ 1

5.2 Preparation of stock solutions ................................................................................................................................................... 2

5.3 Preparation of standard solutions ......................................................................................................................................... 3

5.4 Preparation of calibration solutions ..................................................................................................................................... 4

6 Apparatus ..................................................................................................................................................................................................................... 5

7 Procedure..................................................................................................................................................................................................................... 6

7.1 Sample preparation ............................................................................................................................................................................ 6

7.2 Extraction .................................................................................................................................................................................................... 6

7.2.1 General...................................................................................................................................................................................... 6

7.2.2 Extraction procedure ................................................................................................................................................... 6

7.3 Determination ......................................................................................................................................................................................... 6

8 System settings ....................................................................................................................................................................................................... 7

8.1 HPLC parameters .................................................................................................................................................................................. 7

8.2 HPLC-MS/MS parameters .............................................................................................................................................................. 7

8.3 UHPLC parameters .............................................................................................................................................................................. 9

8.4 UHPLC-MS/MS parameters .......................................................................................................................................................... 9

8.5 Preparation of the LC-MS/MS system and samples .............................................................................................10

8.5.1 Tuning of the LC-MS/MS system .....................................................................................................................10

8.5.2 Checking of the instrument settings ............................................................................................................10

8.5.3 Checking of the sensitivity of the system .................................................................................................10

8.5.4 Sample list ..........................................................................................................................................................................11

9 Calculation and expression of results ..........................................................................................................................................11

9.1 General ........................................................................................................................................................................................................11

9.2 Calibration criteria ...........................................................................................................................................................................11

9.3 Identification criteria .....................................................................................................................................................................11

9.4 Recovery ....................................................................................................................................................................................................12

9.5 Calculation of results ......................................................................................................................................................................13

10 Precision data .......................................................................................................................................................................................................14

10.1 General ........................................................................................................................................................................................................14

10.2 Repeatability ..........................................................................................................................................................................................14

10.3 Reproducibility ....................................................................................................................................................................................14

11 Test report ................................................................................................................................................................................................................15

Annex A (informative) Precision data ..............................................................................................................................................................16

Bibliography .............................................................................................................................................................................................................................18

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ISO 23970:2021(E)
IDF 252:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national

standards bodies (ISO member bodies). The work of preparing International Standards is normally

carried out through ISO technical committees. Each member body interested in a subject for which

a technical committee has been established has the right to be represented on that committee.

International organizations, governmental and non-governmental, in liaison with ISO, also take part

in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all

matters of electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,

Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO

and IDF.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv PROOF/ÉPREUVE © ISO and IDF 2021 – All rights reserved
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ISO 23970:2021(E)
IDF 252:2021(E)

IDF (the International Dairy Federation) is a non-profit private sector organization representing the

interests of various stakeholders in dairying at the global level. IDF members are organized in National

Committees, which are national associations composed of representatives of dairy-related national

interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and

governments/food control authorities.

ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis

and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International

Standards using the logos and reference numbers of both organizations.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. IDF shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

This document was prepared by the IDF Standing Committee on Analytical Methods for Additives and

Contaminants and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk

products. It is being published jointly by ISO and IDF.

The work was carried out by the ISO/IDF Action Team A05 of the Standing Committee on Analytical

Methods for Additives and Contaminants under the aegis of its project leader, Dr S. Holroyd.

© ISO and IDF 2021 – All rights reserved PROOF/ÉPREUVE v
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ISO 23970:2021(E)
INTERNATIONAL STANDARD
IDF 252:2021(E)
Milk, milk products and infant formula — Determination of
melamine and cyanuric acid by liquid chromatography and
tandem mass spectrometry (LC-MS/MS)
1 Scope

This document specifies a method for the determination of melamine and cyanuric acid with liquid

chromatography in combination with tandem mass spectrometry (LC-MS/MS). 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.
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.

ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
4 Principle

A test portion of the homogenous food sample is fortified with C labelled internal standards (melamine

and cyanuric acid). After incubation for at least 1 h, 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 centrifuged. After

the 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 high

performance liquid chromatography (UHPLC). Chromatography is based on hydrophilic interaction

liquid chromatography (HILIC). Ionization is achieved by electrospray ionization (ESI) in multiple

reaction monitoring (MRM).
5 Reagents
5.1 List of reagents

Use only reagents of recognized analytical grade and water conforming to grade 1 of ISO 3696, unless

otherwise specified. Use only reagents with purity suitable for melamine and cyanuric acid analysis.

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ISO 23970:2021(E)
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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 chromatogram shall not show any

interfering impurity at the retention time of compounds of interest.
5.1.1 Formic acid, mass fraction of 98 % to 100 % (CAS RN® 64-18-6).
5.1.2 Acetonitrile, HPLC gradient grade (CAS RN® 75-05-8).
5.1.3 Ammonium acetate, mass fraction of approximately 98 % (CAS RN® 631-61-8).
5.1.4 Methanol, Ultra LC-MS grade (CAS RN® 67-56-1).
5.1.5 Melamine, mass fraction ≥99 %, solid (CAS RN® 108-78-1).
5.1.6 Cyanuric acid, solid (CAS RN® 108-80-5).
5.1.7 Benzoguanamine, solid (CAS RN® 91-76-9).
13 13 15

5.1.8 C melamine, C (99 %), Amino- N (98 %), solution with mass concentration ρ = 1 000 µg/

3 3

ml. Other isotope marked melamine/cyanuric acid solutions can be used if they lead to similar results;

this would need to be appropriately validated.

EXAMPLE 1,3,5-Triazine-2,4,6-triamine-13C3 (melamine), 1,3,5-Triazine-2,4,6-triol-13C3, 2,4,6-Trihydroxy-

1,3,5-triazine-13C (cyanuric acid).
13 13 15
5.1.9 C cyanuric acid, C (99 %), N (98 %), solution with ρ = 1 000 µg/ml.
3 3
NOTE Alternatives can be used as per 5.1.8.
5.2 Preparation of stock solutions
5.2.1 Melamine stock solution, ρ = 1 000 µg/ml.

Weigh, to the nearest 0,1 mg, approximately 100 mg of melamine (5.1.5) into a 100 ml glass flask (6.2)

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.

5.2.2 Cyanuric acid stock solution, ρ = 1 000 µg/ml.

Weigh, to the nearest 0,1 mg, approximately 100 mg of cyanuric acid (5.1.6) into a 100 ml glass flask

(6.2) 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.
5.2.3 Benzoguanamine stock solution, ρ = 1 000 µg/ml.

Weigh, to the nearest 0,1 mg, approximately 100 mg of benzoguanamine (5.1.7) into a 100 ml glass flask

(6.2) and add by weighing an amount of methanol (5.1.4) 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.

1) CAS Registry Number® (CAS RN®) is the trademark of a product supplied by CAS corporation. This information

is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of the

product named. Equivalent products may be used if they can be shown to lead to the same results.

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ISO 23970:2021(E)
IDF 252:2021(E)
5.2.4 C melamine stock solution, ρ = 20 µg/ml.

Pipette 1,0 ml of C melamine (5.1.8) into a 50 ml volumetric flask (6.3). 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 (6.2).

The solution is stable under these conditions during at least 1 year.
5.2.5 C cyanuric acid stock solution, ρ = 20 µg/ml.

Pipette 1,0 ml of C cyanuric acid (5.1.9) into a 50 ml volumetric flask (6.3). 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 (6.2).

The solution is stable under these conditions during at least 1 year.
5.3 Preparation of standard solutions
5.3.1 Melamine standard solution I, ρ = 20 µg/ml.

Pipette 1,0 ml of the melamine stock solution (5.2.1) into a 50 ml volumetric flask (6.3). Make up to the

mark with the dilution solution (5.4.6) and mix. The concentration is 20 µg/ml. Transfer this solution

into a 100 ml glass flask (6.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable under

these conditions during at least 1 month.
5.3.2 Cyanuric acid standard solution I, ρ = 20 µg/ml.

Pipette 1,0 ml of the cyanuric acid stock solution (5.2.2) into a 50 ml volumetric flask (6.3). Make up

to the mark with the dilution solution (5.4.6) and mix. The concentration is 20 µg/ml. Transfer this

solution into a 100 ml glass flask (6.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable

under these conditions during at least 1 month.
5.3.3 Melamine standard solution II, ρ = 0,2 µg/ml.

Pipette 1,0 ml of the melamine standard solution I (5.3.1) into a 100 ml volumetric flask (6.3). Make

up to the mark with the dilution solution (5.4.6) and mix. The concentration is 0,2 µg/ml. Transfer this

solution into a 100 ml glass flask (6.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable

under these conditions during at least 1 month.
5.3.4 Cyanuric acid standard solution II, ρ = 0,2 µg/ml.

Pipette 1,0 ml of the cyanuric acid standard solution I (5.3.2) into a 100 ml volumetric flask (6.3). Make

up to the mark with the dilution solution (5.4.6) and mix. The concentration is 0,2 µg/ml. Transfer this

solution into a 100 ml glass flask (6.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable

under these conditions during at least 1 month.
5.3.5 Benzoguanamine standard solution I, ρ = 20 µg/ml.

Pipette 1,0 ml of the benzoguanamine stock solution (5.2.3) into a 50 ml volumetric flask (6.3). Make

up to the mark with the dilution solution (5.4.6) and mix. The concentration is 20 µg/ml. Transfer this

solution into a 100 ml glass flask (6.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is stable

under these conditions during at least 1 month.
5.3.6 Benzoguanamine standard solution II, ρ = 2 µg/ml.

Pipette 1,0 ml of the benzoguanamine standard solution I (5.3.5) into a 10 ml volumetric flask (6.3).

Make up to the mark with the dilution solution (5.4.6) and mix. The concentration is 2 µg/ml. Transfer

this solution into a 20 ml glass flask (6.2) and store it in a refrigerator at 4 °C (±3 °C). The solution is

stable under these conditions during at least 1 month.
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ISO 23970:2021(E)
IDF 252:2021(E)
13 13
5.3.7 C melamine and C cyanuric acid standard solution, ρ = 2 µg/ml.
13 13

Pipette 0,5 ml of the C melamine stock solution (5.2.4) and 0,5 ml of the C cyanuric acid stock solution

(5.2.5) into a 5 ml volumetric flask (6.3). Make up to the mark with water and mix. The concentration

for both compounds is 2 µg/ml. Transfer this solution into a 20 ml glass flask (6.2) 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.
5.4 Preparation of calibration solutions

5.4.1 Pipette the volumes given in Table 1 into a 10 ml volumetric flask (6.3) and make up to the mark

with the dilution solution (5.4.6).
Table 1 — Preparation of the calibration solutions
Calibration solution number
Calibration solution
1 2 3 4 5 6 7
Melamine standard solution I (5.3.1), in ml 0 0 0 0 0 0,05 0,25
Cyanuric acid standard solution I (5.3.2), in ml 0 0 0 0 0 0,05 0,25
Melamine standard solution II (5.3.3), in ml 0 0,05 0,25 1,25 2,5 0 0
Cyanuric acid standard solution II (5.3.4), in ml 0 0,05 0,25 1,25 2,5 0 0
Benzoguanamine standard solution II (5.3.6), in ml 0,5 0,5 0,5 0,5 0,5 0,5 0,5
13 13
C melamine and C cyanuric acid standard solu-
0,5 0,5 0,5 0,5 0,5 0,5 0,5
tion (5.3.7), in ml
13 13
Concentration C melamine – C cyanuric acid, in
0,1 0,1 0,1 0,1 0,1 0,1 0,1
µg/ml
Concentration native melamine – cyanuric acid, in
0 0,001 0,005 0,025 0,05 0,1 0,5
µg/ml

5.4.2 Mobile phase A for HPLC, substance concentration c(ammonium acetate) = 10 mmol/l.

Dissolve 0,77 g of ammonium acetate (5.1.3) in 1 000 ml of water.
5.4.3 Mobile phase B for HPLC.
Acetonitrile (5.1.2).
5.4.4 Mobile phase A for UHPLC.
Mix 3 parts per volume of formic acid (5.1.1) with 97 parts per volume of water.

5.4.5 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 (5.1.3) in 30 ml of water. Add 970 ml of acetonitrile (5.1.2) to the

mixture and shake firmly. The turbid mixture will clear overnight.

The optimal choice for the mobile phases A and B can depend on the instrument configuration (see 6.10

to 6.12), particularly the type of column used. Equivalent products may be used if they can be shown to

lead to the same results.
5.4.6 Dilution solution.

Transfer, using measuring cylinders, 70 ml of acetonitrile (5.1.2) and 30 ml of water into a 100 ml

volumetric flask (6.3) and mix. Store at room temperature for no longer than 1 month.

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ISO 23970:2021(E)
IDF 252:2021(E)
6 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 shall be used.

6.1 Disposable polypropylene carbonate tube, of approximately 50 ml.
6.2 Glass flask, with volume of 20 ml and 100 ml.
6.3 One-mark volumetric flask, with volumes of 5 ml, 10 ml, 50 ml and 100 ml.
6.4 Shaking machine, adjustable from 0 strokes/min to 300 strokes/min.
6.5 Ultra sonication bath.

6.6 Centrifuge, with the capability to centrifuge 50 ml tubes (6.1) and a maximum g force of at least

4 000g.

6.7 Centrifuge, with the capability to centrifuge standard micro test tubes (6.8), chilled and a

maximum g force of at least 6 000g.
6.8 Standard micro test tubes, 1,5 ml.
6.9 Vials, for LC.
6.10 Liquid chromatograph triple quadrupole mass spectrometer consisting of:
a) a pump system, capable of delivering a gradient at the required flow;
b) an injector, capable of injecting 5 µl.

6.11 TSKgel® Amide-80 HILIC column, length of 100 mm, internal diameter of 3,0 mm and particle

size of 3 µm.
TM 3)

6.12 UHPLC BEH (bridged ethyl hybrid) HILIC column, length of 150 mm, internal diameter of

2,1 mm and particle size of 1,7 µm.
6.13 Liquid nitrogen.
6.14 Milling equipment, capable of milling to a final particle size of <300 µm.

2) TSKgel is the trademark of a product supplied by Tosoh Corporation. This information is given for the

convenience of users of this document and does not constitute an endorsement by ISO or IDF of the product named.

Equivalent products may be used if they can be shown to lead to the same results.

3) BEH is the trademark of a product supplied by Waters Corporation. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO or IDF of the product named. Equivalent

products may be used if they can be shown to lead to the same results.
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ISO 23970:2021(E)
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7 Procedure
7.1 Sample preparation

Warm all milk-based liquid products, e.g. full fat milk, skimmed milk, raw milk and chocolate milk, to

40 °C and shake gently.

If necessary, mill all dry products based on milk, soy or wheat, e.g. milk powder, infant formula and

biscuits, to a particle size of <1 mm.

Mill all milk- or cocoa-based products, e.g. chocolate, candy and sweets (toffee), cryogenically using, for

example, liquid nitrogen after cutting in small cubes of ≤1 cm.
7.2 Extraction
7.2.1 General
The following samples shall be included in each series:
— calibration solutions;
— procedure blank (n = 1);

— (certified) reference material at an appropriate level or a home-made reference sample;

— all samples;
— calibration solution number 3, 4 or 5 (see Table 1).

The procedure blank shall be free of contaminants at or above the limits of quantification. A sample

matrix can be used where previous analysis showed that no cyanuric acid (CYA) and melamine (MEL)

were present in the sample above the LOQ. The sample amount used for extraction is 1 g of blank

sample. Deviations in sample mass shall be taken into account in Formula (5) (m = sample mass in g).

The calibration curve is used for identification and quantification. Calibration solution 3, 4 or 5 after

the series is used for control of the stability. The areas of melamine and cyanuric acid obtained with

this standard shall be equal to the areas obtained with the same standard in the calibration curve with

a maximum deviation of 10 %.
7.2.2 Extraction procedure

Weigh, to the nearest 0,01 g, 1 g of sample into a disposable tube (6.1). Add 250 µl of C melamine

stock solution (5.2.4) and 250 µl of C cyanuric acid stock solution (5.2.5). Incubate for at least 1 h at

ambient temperature. Add 5 ml of hot water (96 °C to 100 °C) and shake manually for 30 s to obtain a

slurry. Add 5 ml of acetonitrile (5.1.2) and shake again. Mix the slurry with 30 ml of acetonitrile (5.1.2)

and 10 ml of water. Shake well for 5 min a
...