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: 19-Apr-2025

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 furosine standard solution of about
1 nmol/ml furosine.
Calculate the exact concentration of furosine in the final furosine standard solution on the basis of the net
content declared for the commercial product.
The furosine standard solution remains stable for 24 months, if stored at −20 °C.
5.9 Nitrogen, gas chromatography purity.
6 Apparatus
Usual laboratory equipment and, in particular, the following.
6.1 C 18 cartridge, or minicolumn (500 mg), used in the solid-phase extraction.

1) Furosine is supplied by Neosystem, Rue de Bologne 7, 67100 Strasbourg, France. This is an example of a suitable
product available commercially.
This information is given for the convenience of users of this International Standard and does not constitute an
endorsement by ISO or IDF of this product.
2 © ISO and IDF 2004 – All rights reserved

IDF 193:2004(E)
6.2 HPLC equipment, provided with gradient pumping system, metal-free injector with injection loop
between 20 µl and 50 µl, and a thermostatic column oven.
6.3 UV-detector, capable of operating at 280 nm wavelength with minimum AUFS of 0,010 or lower.
Under these chromatographic conditions and with an injection of 10 pmol of furosine, the signal-to-noise ratio
shall be higher than 10.
6.4 “Furosine dedicated” column, of diameter 4,6 mm and of length 250 mm, 5 µm particle size (e.g.
2)
Alltech ), or an equivalent column capable of separating the furosine peak on the baseline without interfering
with other peaks.
6.5 Integrator, or data-reprocessing software, capable of measuring the peak areas.
6.6 Analytical balance, capable of weighing to the nearest 1 mg.
...