ISO 7102:2024

International
Standard
ISO 7102
IDF 257
First edition
Infant formula — Determination
2024-08
of β-galactooligosaccharides —
Ultra high performance liquid
chromatography (UHPLC) with
fluorescence detection after
pre-column derivatization
Préparation pour nourrissons — Détermination de la teneur
en β-galacto-oligosaccharides — Chromatographie liquide à
ultra-haute performance (CLUHP) couplée à une détection par
fluorescence après dérivation précolonne
Reference numbers
IDF 257:2024(en) © ISO and IDF 2024

IDF 257:2024(en)
© ISO and IDF 2024
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
ii
IDF 257:2024(en)
Contents Page
Forewords .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Chemicals and reagents . 2
5.1 List of chemicals and reagents .2
5.2 Preparation of reagents .3
5.3 Preparation of mobile phases .4
5.4 Preparation of standard solutions .4
6 Apparatus . 4
7 Procedure . 5
7.1 Sample preparation .5
7.1.1 Powdered or concentrated products on a ready-to feed (RTF) basis .5
7.1.2 Reconstituted products as prepared in 7.1.1 or products sold as RTF .6
7.1.3 Homogeneous powdered products without prior reconstitution .6
7.1.4 Calibration curve.6
7.1.5 Dextran ladder .6
7.1.6 Hydrolysis of maltodextrins and GOS .6
7.1.7 Reagent blank .6
7.1.8 Internal standard addition .6
7.1.9 Derivatization .6
7.1.10 Dilution .7
7.2 Chromatographic conditions .7
7.3 System suitability test .7
7.4 Calibration .7
8 Calculation . 8
9 Precision data . 8
9.1 General .8
9.2 Repeatability .9
9.3 Reproducibility .9
10 Test report . 9
Annex A (normative) Assessment of maltotriose concentration .10
Annex B (informative) Determination of molecular weight using dextran ladder .12
Annex C (informative) Determination of molecular weight using LC-MS .13
Annex D (informative) Example chromatograms .15
Annex E (informative) Precision data . 17
Bibliography . 19

iii
IDF 257:2024(en)
Forewords
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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), in collaboration with AOAC INTERNATIONAL. It
is being published jointly by ISO and IDF and separately by AOAC INTERNATIONAL. The method described
in this document is equivalent to the AOAC Official Method 2021.01.
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
IDF 257:2024(en)
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.
IDF draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). IDF takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, IDF had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. IDF shall not be held responsible for identifying any or all such patent rights.
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 Composition
and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, in
collaboration with AOAC INTERNATIONAL. It is being published jointly by ISO and IDF and separately by
AOAC INTERNATIONAL. The method described in this document is equivalent to the AOAC Official Method
2021.01.
The work was carried out by the IDF/ISO Action Team on C42 of the Standing Committee on Analytical
Methods for Composition under the aegis of its project leader Mr S. Austin (CH).

v
International Standard
IDF 257:2024(en)
Infant formula — Determination of β-galactooligosaccharides
— Ultra high performance liquid chromatography (UHPLC)
with fluorescence detection after pre-column derivatization
1 Scope
This document specifies a method for the determination of β-galactooligosaccharides (GOS) in infant
formula (both powder and liquid) containing 0,2 g/100 g to 3,0 g/100 g of GOS in the product as prepared
ready for consumption.
The method has been validated in a multi-laboratory study with reconstituted infant formula at levels
of 0,236 g/100 g, 0,594 g/100 g, 0,616 g/100 g and 0,688 g/100 g and infant formula RTF at levels of
[1]
0,316 g/100 g and 0,858 g/100 g. During the single laboratory validation study spike-recovery experiments
were performed up to 3 g/100 g in reconstituted infant formula powders (milk-based, partially hydrolysed
milk-based and soy-based), and reconstituted adult nutritional powders.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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/
3.1
infant formula
breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants
during the first months of life up to the introduction of appropriate complementary feeding
[SOURCE: Codex Standard 72-1981]
4 Principle
Powdered or concentrated samples are reconstituted in water and oligosaccharides present in samples
are extracted at 70 °C. The ready-to-feed products (including reconstituted concentrates or powders) are
diluted and two aliquots of the diluted sample are taken and both are treated with amyloglucosidase to
hydrolyse any maltooligosaccharides present (Assay 1), one of the two aliquots is additionally treated
with β-galactosidase (Assay 2) to hydrolyse all the GOS present. An internal standard (laminaritriose) is
added to both aliquots and the oligosaccharides are fluorescently labelled with 2-aminobenzamide (2AB).
Labelled extracts are diluted with acetonitrile prior to injection on an UHPLC-FLD system equipped with a
hydrophilic interaction liquid chromatography (HILIC) analytical column. The analytes are separated using
a gradient of aqueous ammonium formate in acetonitrile and detected with a fluorescence detector.
An external maltotriose calibration curve is prepared in the same way as the samples but without
enzymatic treatment. Since it is the 2AB label that is detected, each oligosaccharide has an equivalent molar
response. The maltotriose calibration curve can thus be used to determine the molar concentrations of the

IDF 257:2024(en)
oligosaccharides in the two assays. It is then necessary to know the molecular weight of each signal in the
chromatogram to convert the molar concentrations to mass concentrations. This can be done by coupling a
mass spectrometer. The molecular weight of each oligosaccharide may also be estimated by comparing the
relative retention time of the oligosaccharide against that of a dextran ladder. The GOS content is obtained
by subtracting the OS content obtained in Assay 2 from the OS content obtained in Assay 1.
5 Chemicals and reagents
5.1 List of chemicals and reagents
Use only reagents of recognized analytical/HPLC grade, unless otherwise specified.
5.1.1 Deionized water, purified with resistivity ≥ 18 MΩ.
1)
5.1.2 Maltotriose, with accurately known purity ≥ 99,0 %, e.g. ultrapure, Carbosynth, Newbury, UK . In
case of issues, check the moisture content and purity following the procedure described in Annex A.
5.1.3 Laminaritriose (50 mg), purity > 90 % (e.g. Megazyme, Bray, Ireland) .
5.1.4 Acetic acid, glacial 100 %.
5.1.5 Sodium hydroxide pellets.
5.1.6 Acetonitrile, HPLC grade.
5.1.7 Dimethylsulfoxide.
5.1.8 2-aminobenzamide (2AB, anthranilic acid amide)
The 2AB should be a white to off-white crystalline powder. If the 2 AB is not white it is recommended to
recrystallize twice from ethanol (95 %) to obtain a white crystalline powder before use.
5.1.9 2-methylpyridine borane complex (2-picoline borane), purity 95 %.
5.1.10 Amyloglucosidase (Aspergillus niger), 9 U/mg (e.g. Roche Diagnostics: 11 202 367 001 ).
5.1.11 β-galactosidase (Aspergillus niger), 4 000 U/ml (e.g. Megazyme, Bray, Ireland E-BGLAN ).
5.1.12 Formic acid, 100 %.
5.1.13 Ammonium hydroxide solution, 25 % to 30 %.
5.1.14 Dextran, with average molecular weight of 1 000 Da.
5.1.15 Isomaltose.
1) This is an example of a suitable product available commercially. 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.

IDF 257:2024(en)
5.2 Preparation of reagents
5.2.1 Maltotriose (malto-3) stock solution, substance concentration c = 10 µmol/ml.
Weigh 50 mg of maltotriose (5.1.2) into a weighing boat and record the mass to 0,1 mg. Transfer quantitatively
into a 10 ml volumetric flask with water (5.1.1) and dilute to the volume with the same solvent.
5.2.2 Laminaritriose internal standard working solution, c = 2 µmol/ml.
Weigh the whole content of a 50 mg laminaritriose (5.1.3) vial into a weighing boat and record the mass to
0,1 mg. Transfer quantitatively into a 50 ml volumetric flask and complete to the mark with water (5.1.1).
5.2.3 Sodium hydroxide solution, c = 1 mol/l.
Dissolve 10 g ± 0,2 g of sodium hydroxide pellets (5.1.5) in 200 ml of water (5.1.1) in a 250 ml volumetric
flask. After cooling down to room temperature, make up to the mark with demineralised water and mix well.
5.2.4 Sodium acetate buffer solution, c = 0,2 mol/l, pH = 4,5.
Into a large beaker (>500 ml) containing 400 ml of demineralised water (5.1.1), pipette 5,8 ml of glacial
acetic acid (5.1.4). Adjust to a pH of 4,5 with sodium hydroxide solution (5.2.3). Transfer the solution to a
500 ml volumetric flask and make up to the mark with water (5.1.1).
5.2.5 Mixture of 25 parts per volume of water and 75 parts per volume of acetonitrile.
Add 50 ml ± 1 ml of water (5.1.1) to 150 ml ± 1 ml of acetonitrile (5.1.6) in a glass bottle and mix.
5.2.6 2AB labelling reagent, c = 2AB (0,35 mol/l) + 2-picoline borane (1 mol/l) in a mixture of 70 parts
per volume of dimethylsulfoxide (DMSO) and 30 parts per volume of acetic acid.
Pipette the volume of DMSO (5.1.7) and glacial acetic acid (5.1.4) in a 20 ml glass tube according to the
number of tests to perform (see Table 1 for quantities). Mix the solution using a vortex mixer. Weigh the
amount of 2AB (5.1.8) and 2-picoline borane (5.1.9) in another 20 ml glass tube (see Table 1), then add the
corresponding volume of a mixture of 30 parts per volume of acetic acid and 70 parts per volume of DMSO.
Mix (vortex) and use an ultrasonic bath for complete dissolution if necessary.
Table 1 — Example of quantities for 2AB reagent preparation
DMSO - Acetic acid 2AB (0,35 mol/l) + 2-picoline borane (1 mol/l)
(70 + 30) in DMSO - Acetic acid (70 + 30)
Max.
DMSO Acetic acid DMSO - 2AB 2-picoline borane
number
ml ml Acetic acid mg mg
of tests
ml
50 4,7 2,0 6,00 286 ± 5 642 ± 5
100 9,3 4,0 12,0 572 ± 10 1 284 ± 10
250 23,3 10,0 30,0 1 430 ± 15 3 209 ± 15
5.2.7 Amyloglucosidase solution, (60 U/ml in 0,2 mol/l sodium acetate buffer pH = 4,5).
Weigh an amount of amyloglucosidase (5.1.10) corresponding to 600 U ± 20 U and dissolve with 10,0 ml of
sodium acetate buffer (5.2.4). This solution is prepared on the day of use and kept at 4 °C until use.
IMPORTANT — For the development and validation of this method, the amyloglucosidase (Product
N° 11202367001) available from Roche Diagnostics , was used. Enzyme activities may vary
slightly from one batch to the other (units per mg are mentioned on the label). Adapt the mass of
enzyme in order to reach a concentration of 60 U/ml ± 2 U/ml. Another amyloglucosidase (Product

IDF 257:2024(en)
2)
N° 10102857001) also available from Roche Diagnostics has also been tested and found to be
suitable. This enzyme is already in suspension (140 U/ml) and can be diluted with 0,2 mol/l sodium
acetate buffer pH = 4,5 in order to be in working concentration (60 U/ml). When enzymes from
another source are used it is imperative to ensure the enzyme employed will completely hydrolyse
any maltodextrins in the product without hydrolysing any analytes, as well as not showing any
interference in the chromatogram. This can be checked by performing an analysis with maltodextrin
as a sample, a GOS ingredient as a sample, and running a blank with the amyloglucosidase only.
5.2.8 β-Galactosidase solution (4 000 U/ml).
Use the solution as is.
IMPORTANT — For the development and validation of this method, the β-galactosidase E-BGLAN
available from Megazyme International , was used. When enzyme from another source is used it is
imperative to ensure the enzyme employed will completely hydrolyse the galacto-oligosaccharides
without hydrolysing any other oligosaccharides that may be present in the sample.
5.2.9 Dextran solution.
Weigh about 20 mg of isomaltose (5.1.15) and about 50 mg of dextran 1 000 (5.1.14) into a weighing boat.
Transfer into a 50 ml volumetric flask with water (5.1.1) and dilute up to the mark.
5.3 Preparation of mobile phases
5.3.1 Eluent A, acetonitrile.
5.3.2 Eluent B, ammonium formate solution, (c = 0,1 mol/l, pH = 4,4).
Add 4,6 g ± 0,1 g (3,78 ml) of formic acid (5.1.12) in a beaker containing 800 ml of water (5.1.1). Adjust the pH
to 4,40 ± 0,05 with ammonium hydroxide solution (5.1.13). Transfer quantitatively to a 1 000 ml volumetric
flask and dilute to the volume with water (5.1.1). This solution is stable for seven days at room temperature.
5.4 Preparation of standard solutions
Prepare a six-level calibration curve by diluting the maltotriose stock solution (5.2.1) as described in Table 2
using volumetric flasks made up to the final volume with water (5.1.1).
Table 2 — Dilution scheme for the preparation of the standard curve
Standard solution Volume of maltotriose Final volume Maltotriose
stock solution (5.2.1) ml concentration
µl nmol/ml
Level 1 80 20 40
Level 2 200 10 200
Level 3 400 10 400
Level 4 800 10 800
Level 5 1 200 10 1 200
Level 6 1 600 10 1 600
6 Apparatus
The usual laboratory apparatus and, in particular, the following shall be used.
2) This is an example of a suitable product available commercially. 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.

IDF 257:2024(en)
6.1 Analytical balance, capable of weighing to the nearest 0,1 mg.
6.2 Weighing boats.
6.3 Volumetric flasks, 10 ml to 1 000 ml.
6.4 Glass tubes, 10 ml or 20 ml.
6.5 pH-meter, readability of pH = 0,1.
6.6 Microcentrifuge tubes, volume of 1,5 ml and 2 ml, with locking cap or screw cap.
6.7 Floating rack for microtubes.
6.8 Water bath, at 70 °C ± 1 °C, with magnetic stirring if available.
6.9 Water bath, at 65 °C ± 1 °C.
6.10 Microcentrifuge, 10 000g.
6.11 Micropipettes with tips, volume of 0,02 ml to 10 ml.
6.12 Vortex mixer.
6.13 Ultrasonic bath.
3)
6.14 Amide LC column, 1,7 µm, 2,1 mm × 150 mm (e.g. Waters BEH Amide Glycan , or equivalent)
6.15 Liquid chromatography instrument, consisting of:
— pump able to deliver a binary gradient at a flow of up to 0,6 ml/min and a backpressure of 103 420 kPa
(15 000 psi);
— autosampler, able to maintain a temperature around 10 °C ± 2 °C, and deliver an injection volume of 2 µl;
— column compartment able to maintain a temper
...