ISO/FDIS 25712

IUC 443:2025(en)
ISO/IULTCS
Secretariat: ISO
Date: 20252026-01-12-18
Chemicals for the leather tanning industry — Determination of the
total content of melamine
Produits chimiques pour l'industrie du tannage du cuir — Détermination de la teneur totale en
melaminemélamine
FDIS stage
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Contents
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Apparatus . 1
6 Reagents . 2
7 Sampling and sample preparation . 4
8 Procedure . 4
8.1 Extraction . 4
8.2 Instrumental analysis . 5
9 Expression of results . 5
9.1 Calculation without internal standard . 5
9.2 Calculation with internal standard . 5
10 Precision . 6
11 Test report . 6
Annex A (informative) Chromatographic analysis operating parameters for LC-MS/MS . 8
Annex B (informative) Chromatographic analysis operating parameters for LC-UV or LC-DAD . 10
Annex C (informative) Precision . 11
Bibliography . 12

iii
Foreword
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IULTCS, originally formed in 1897, is a world-wide organization of professional leather societies to further the
advancement of leather science and technology. IULTCS has three Commissions, which are responsible for
establishing international methods for the sampling and testing of leather. ISO recognizes IULTCS as an
international standardizing body for the preparation of test methods for leather.
This document was prepared by the Chemical Test Commission of the International Union of Leather
Technologists and Chemists Societies (IUC Commission, IULTCS), in collaboration with the European
Committee for Standardization (CEN) Technical Committee CEN/TC 289, Leather, in accordance with the
Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
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© ISO #### 2026 – All rights reserved
iv
Introduction
This document includes a procedure for analysing melamine in leather tanning chemicals using liquid
chromatography (LC) equipment.
In the leather industry, melamine can be present as a residual monomer in resins based on the polymerisation
of melamine, where melamine-formaldehyde resin is the more typical. This type of resin is present in chemical
products such as melamine-formaldehyde resin or compound products and is used in the retanning stage of
the tanning process.
At present, the official European Chemicals Agency (ECHA) classification recognized in the European Union
(EU) for Melaminemelamine is the following:
— — Substancesubstance of very high concern (SVHC) and included in the candidate list for
[1[1] ]
authorisation ; ;
[2[2] ]
— — Suspectedsuspected to be Carcinogenic ; ;
[2[2] ]
— — Underunder assessment as Persistent, Bioaccumulative and Toxic ; ;
[2[2] ]
— — Underunder assessment as Endocrine Disrupting . .
v
IUC 443:20252026(en)
Chemicals for the leather tanning industry — Determination of the
total content of melamine
1 Scope
This document specifies a method for determining the total content (solvent extractable) of melamine in
chemicals for the leather tanning industry.
This method requires the use of liquid chromatography (LC) with a triple quadrupole mass spectrometer
(MS/MS), an ultraviolet (UV) detector, or diode array detector (DAD) to identify and quantify the melamine.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— — ISO Online browsing platform: available at https://www.iso.org/obp
— — IEC Electropedia: available at https://www.electropedia.org/
4 Principle
The chemical sample is extracted in a 50:50 (volume fraction) mixture of acetonitrile and water. Subsequently,
an aliquot of the extract is diluted in the appropriate mobile phase and analysed using LC-MS, LC-MS/MS, or
LC with a UV (LC-UV) or diode array detector (DAD; LC-DAD).
Melamine is a highly polar substance. From a chromatographic perspective, hydrophilic interaction liquid
chromatography (HILIC) provides good separation and quantification of melamine using LC-MS, LC-MS/MS,
LC-UV, and LC-DAD. However, because HILIC-based columns are not widely available in laboratories,
reversed-phase (RP) chromatography using C8 or C18 columns can be a suitable alternative, offering good
separation with LC-MS and LC-MS/MS detection. Therefore, this document describes two alternative
chromatographic approaches: one using a HILIC column and the other using an RP (C18 or C8) column.
NOTE Melamine is water-soluble but has low solubility in organic solvents. Therefore, care is taken to ensure
complete solubilization of melamine during all analytical steps.
5 Apparatus
The usual laboratory apparatus and, in particular, the following shall be used:
5.1 5.1 Ultrasonic bath, with controllable heating capable of maintaining a temperature of
(30 ± 2) °C.
5.2 5.2 Glass container with a screw cap, for example volume of 20 ml.
5.3 5.3 Suitable syringe membrane filters, for example PTFE with pore size 0,2 µm.
5.4 5.4 Volumetric flasks, for example volume of 10 ml and 100 ml.
5.5 5.5 LC vials, with cap, for example volume of 2 ml.
5.6 5.6 Analytical balance, with a resolution of 0,1 mg.
5.7 5.7 Pipettes, various sizes, for example volume of 0,1 ml to 20 ml.
5.8 5.8 Instrumental equipment, LC-MS/MS.
5.9 5.9 Alternative instrumental equipment, LC-MS, LC-UV, LC-DAD.
NOTE If two detectors are used, they are arranged in series on the same LC system.
6 Reagents
If not otherwise specified, analytical reagent grade chemicals shall be used.
® 11)
6.1 6.1 Acetonitrile, CAS Registry Number (CAS RN) 75-05-8. For LC-MS/MS, LC/MS quality shall
be used HPLC quality methanol is suitable for LC-UV or LC-DAD.
6.2 6.2 Water, LC-MS quality, HPLC grade for LC-DAD.
6.3 6.3 Extraction mix.
Mix 500 ml of acetonitrile (6.1(6.1)) with 500 ml of water (6.2(6.2),), to obtain an acetonitrile:water (50:50,
volume fraction) mixture. ®
6.4 6.4 Glacial acetic acid, CAS RN 64-19-7, minimum 99,0 % (mass fraction). ®
6.5 6.5 Ammonium acetate, CAS RN 631-61-8, quality for LC-MS.
6.6 6.6 10 mmol/l ammonium acetate buffer pH 5,9.
A 10 mmol/l ammonium acetate buffer (pH 5,9) is prepared fresh daily by dissolving approximately 0,771 g
of ammonium acetate (6.5(6.5)) in 1 l of water (6.2(6.2).). After dissolution, add 40 ml of glacial acetic acid
(6.4(6.4)) and adjust the pH to 5,9 if needed.
6.7 6.7 Solution A, mobile phase for HILIC column.
Mix 920 ml of acetonitrile (6.1(6.1)) with 80 ml of 10 mmol/l ammonium acetate buffer (6.6(6.6)) to obtain
an acetonitrile:10 mmol/l ammonium acetate buffer (92:8, volume fraction) mixture.
6.8 6.8 Solution B, mobile phase for HILIC column.
Mix 600 ml of acetonitrile (6.1(6.1)) with 400 ml of 10 mmol/l ammonium acetate buffer (6.6(6.6)) to obtain
an acetonitrile:10 mmol/l ammonium acetate buffer (60:40, volume fraction) mixture.
6.9 6.9 Solution C, mobile
...