ISO 18329:2004

Milk and milk products — Determination of furosine content — Ion-pair reverse-phase high-performance liquid chromatography method

ISO 18329|IDF 193:2004 specifies a method for the quantitative determination of furosine in milk and milk products. The method is particularly applicable to raw or heat-treated milk and to cheese.

Lait et produits laitiers — Détermination de la teneur en furosine — Méthode par chromatographie liquide à haute performance en phase inverse par paire d'ions

General Information

• Status: Published
• Publication Date: 19-Oct-2004
• ICS: 67.100.01 - Milk and milk products in general
• Technical Committee: ISO/TC 34/SC 5 - Milk and milk products
• Drafting Committee: ISO/TC 34/SC 5 - Milk and milk products
• Current Stage: 9093 - International Standard confirmed
• Start Date: 22-Oct-2024
• Completion Date: 12-Feb-2026

Overview

ISO 18329:2004 (IDF 193:2004) defines a standardized analytical method for the quantitative determination of furosine (ε-furoylmethyl-lysine) in milk and milk products. The method uses ion-pair reverse-phase high-performance liquid chromatography (IP-RP HPLC) with UV detection at 280 nm to evaluate the early stage of the Maillard reaction - particularly useful for assessing effects of heat treatment on raw and processed milk and on cheese.

Key topics and technical requirements

• Analyte and units: Furosine content expressed as milligrams per 100 g of protein.
• Principle: Warm acid hydrolysis converts ε-lactulosyl-lysine into furosine; furosine is quantified by comparison with a certified furosine standard.
• Sample types: Raw or heat‑treated milk and cheese.
• Hydrolysis: Nitrogen-flushed vials; heat 23 h at 110 °C; filter hydrolysate; filtered hydrolysate may be stored at −20 °C.
• Protein determination: Nitrogen by Kjeldahl (ISO 8968-1) and conversion factor 6.38.
• Solid-phase extraction (SPE): C18 cartridge (≈500 mg) pre-wet with methanol then water; load 0.5 ml hydrolysate; elute with HCl solution III (3.0 mol/L). Purified hydrolysate stable ~1 week at −20 °C.
• HPLC conditions:
  • Column: Dedicated furosine RP column (4.6 × 250 mm, 5 µm) or equivalent.
  • Detector: UV at 280 nm; sensitivity such that 10 pmol injection S/N > 10; min AUFS 0.010.
  • Elution solvents: Dilute acetic acid (solvent A) and KCl in dilute acetic acid (solvent B).
  • Gradient (example): 0–12.5 min 100% A; 19.5 min to 50% A/50% B; hold to 22.0 min; return to 100% A by 24.0 min; total run 32 min.
  • Flow rate: 1.2 ml/min; column temp: 30–35 °C; injection volume: 20–50 µl.
• Quantification: Single-point comparison to furosine standard solution (approx. 1 nmol/ml); furosine peak should elute ~20–24 min and be baseline-resolved.
• Performance: Standard includes interlaboratory precision data (repeatability and reproducibility).

Applications and users

ISO 18329 is used by:

• Dairy quality-control and analytical laboratories monitoring heat treatment and Maillard reaction progression.
• Cheese and milk processors for process control, shelf‑life assessment, and product quality evaluation.
• Regulatory bodies and research labs studying thermal damage, protein modification, and food chemistry.

Practical uses include verifying pasteurization/sterilization impacts, comparing processing conditions, and supporting product specifications that reference furosine as a marker of protein glycation.

Related standards

• ISO 8968-1 / IDF 20-1 - Milk - Determination of nitrogen (Kjeldahl) (used to convert nitrogen to protein).
• ISO 707 - Recommended sampling for milk and milk products (sampling guidance).

Keywords: ISO 18329, furosine, milk analysis, HPLC, ion-pair reverse-phase, Maillard reaction, heat treatment, cheese testing, dairy quality control.

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 18329
IDF
First edition
2004-10-15
Milk and milk products — Determination
of furosine content — Ion-pair reverse-
phase high-performance liquid
chromatography method
Lait et produits laitiers — Détermination de la teneur en furosine —
Méthode par chromatographie liquide à haute performance en phase
inverse par paire d'ions
Reference numbers
IDF 193:2004(E)
©
ISO and IDF 2004
IDF 193:2004(E)
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ii © ISO and IDF 2004 – All rights reserved

IDF 193:2004(E)
Contents Page
Foreword. iv
Foreword. v
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle . 1
5 Reagents . 1
6 Apparatus. 2
7 Sampling . 3
8 Preparation of test portion. 3
8.1 Milk . 3
8.2 Cheese. 3
8.3 Hydrolysis. 4
8.4 Determination of protein content . 4
9 Procedure. 4
9.1 Solid-phase extraction (SPE) of the filtered hydrolysate. 4
9.2 HPLC determination. 4
10 Calculation and expression of results . 6
10.1 Calculation. 6
10.2 Expression of results. 6
11 Precision . 6
11.1 Interlaboratory test . 6
11.2 Repeatability. 6
11.3 Reproducibility . 6
12 Test report. 7
Annex A (informative) Examples of HPLC patterns . 8
Annex B (informative) Results of interlaboratory trials. 10
Bibliography . 11

IDF 193:2004(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 18329IDF 193 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International.
It is being published jointly by ISO and IDF and separately by AOAC International.
iv © ISO and IDF 2004 – All rights reserved

IDF 193:2004(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in
the development of standard methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50% of
IDF National Committees casting a vote.
International Standard ISO 18329|IDF 193 was prepared by Technical Committee ISO/TC 34, Food products,
Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with
AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International.
All work was carried out by the Joint ISO/IDF/AOAC Action Team, Characterization of heat treatment, of the
Standing Committee on Minor components and characterization of physical properties, under the aegis of its
project leader, Prof. P. Resmini (IT).

INTERNATIONAL STANDARD
IDF 193:2004(E)
Milk and milk products — Determination of furosine content —
Ion-pair reverse-phase high-performance liquid
chromatography method
1 Scope
This International Standard specifies a method for the quantitative determination of furosine (ε-furoylmethyl-
lysine) in milk and milk products. The method is particularly applicable to raw or heat-treated milk and to
cheese.
2 Normative references
The following referenced documents are indispensable for the application 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 8968-1IDF 20-1, Milk — Determination of nitrogen content — Part 1 — Kjeldahl method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
furosine content
mass fraction of substance determined by the procedure specified in this International Standard
NOTE Furosine content is expressed in milligrams per 100 g of protein.
4 Principle
The first stable “Maillard Reaction” (MR) product formed in milk and in cheese, ε-lactulosyl-lysine, is partially
converted by warm acid-hydrolysis into furosine, the determination of which allows the extent of the early
stage of MR to be evaluated. The MR extent is related to the type and intensity of heat treatments applied
both to raw material and in processing. The determination of furosine is performed by ion-pair reverse-phase
(IP-RP) HPLC with UV detection at 280 nm. Quantification of furosine is obtained by reference to a standard
sample of furosine.
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized
water or water of equivalent purity.
5.1 Hydrochloric acid (HCl), fuming, with a mass fraction 37 %.
IDF 193:2004(E)
5.2 Hydrochloric acid solution I, c(HCl) = 10,6 mol/l.
Mix 8 volumes of hydrochloric acid (5.1) with 1 volume of water to obtain hydrochloric acid solution I.
5.3 Hydrochloric acid solution II, c(HCl) = 8,0 mol/l.
Mix 2 volumes of hydrochloric acid (5.1) with 1 volume of water to obtain hydrochloric acid solution II.
5.4 Hydrochloric acid solution III, c(HCl) = 3,0 mol/l.
Mix 1 volume of hydrochloric acid (5.1) with 3 volumes of water to obtain hydrochloric acid solution III.
5.5 Methanol (CH OH).
5.6 HPLC elution solvents
Prepare HPLC elution solvents by using HPLC-grade reagents.
5.6.1 Water, of HPLC-grade.
5.6.2 Dilute acetic acid (CH CO H)
3 2
Dilute 4 ml of glacial acetic acid with water to 1 000 ml.
5.6.3 Potassium chloride solution, c(KCl) = 3 g/l.
Dissolve 3 g of potassium chloride in 1 000 ml of dilute acetic acid (5.6.2).
1)
5.7 Furosine, (e.g. Neosystem or equivalent).
5.8 Furosine standard solution, c[ε-N-(2-furoylmethyl)-L-lysine] = 1 nmol/ml (approx.).
Dissolve 15 mg of furosine (5.7) in 25 ml of hydrochloric acid solution III (5.4) and mix. Dilute 5 ml of this
solution with the hydrochloric acid solution III (5.4) to 100 ml. Mix again to obtain dilution 1. Dilute 1 ml of
dilution 1 with hydrochloric acid solution III (5.4) to 100 ml to obtain a f
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