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International
Standard
ISO 20631
First edition
Infant formula and adult
2024-07
nutritionals — Determination
of total folate content by
trienzyme extraction and
ultra high performance liquid
chromatography tandem mass
spectrometry (UHPLC-MS/MS)
Préparations pour nourrissons et produits nutritionnels pour
adultes — Détermination de la teneur en folates totaux par
extraction trienzymatique et chromatographie liquide à ultra
performance (CLUHP) couplée à une spectrométrie de masse en
tandem (SM/SM)
Reference number
© ISO 2024
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ii
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents and materials . 2
5.1 List of reagents .2
5.2 Preparation of solvent for standard solutions .3
5.3 Preparation of folate standard solutions .4
5.4 Preparation of folate internal standard stock solutions .5
5.5 Preparation of calibration standard solutions .6
5.6 Preparation of substrate solution to test plasma conjugase activity .7
5.7 Reagent for folate analysis .8
6 Apparatus . 9
7 Procedure .10
7.1 Sample preparation .10
7.1.1 Sample processing to make them homogenous .10
7.1.2 Reconstitution of powder sample into liquid .11
7.2 Extraction .11
7.3 Extract purification .11
7.4 Instrumental analysis . 12
7.4.1 Analysis of instrument blank, calibration standards, method blank and samples . 12
7.4.2 UHPLC conditions and MS parameters for different systems . 12
8 Calculations .13
8.1 Calculation of ratios of peak areas of the folate compounds to the respective internal
standard peak areas . 13
8.2 Calibration curve . 13
8.3 Calculation of concentration of folate compounds in the method blank . 13
8.4 Calculation of concentration of folate compounds in the sample extracts . 13
8.5 Calculation of folate compounds and total folate in reconstituted or RTF samples
(μg/100 g) .14
8.6 Calculation of total folate in powder samples (μg/100 g) as-is basis . 15
8.7 Calculation of folic acid nanograms released in the conjugase test . 15
8.8 Calculation of per cent conversion of Pte-Glu3 to folic acid in the conjugase assay . 15
9 Precision .15
9.1 General . 15
9.2 Repeatability . 15
9.3 Reproducibility .16
Annex A (informative) Precision data . 17
Bibliography .18
iii
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
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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
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Any trade name used in this document is information given for the convenience of users and does not
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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, in collaboration with
AOAC INTERNATIONAL. It is being published by ISO and separately by AOAC INTERNATIONAL. The method
described in this document is equivalent to the AOAC Official Method 2011.06 Total Folate in Infant Formula
and Adult Nutritionals by Trienzyme Extraction and LC-MS/MS Quantitation.
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
International Standard ISO 20631:2024(en)
Infant formula and adult nutritionals — Determination
of total folate content by trienzyme extraction and ultra
high performance liquid chromatography tandem mass
spectrometry (UHPLC-MS/MS)
1 Scope
This document specifies a method for the analysis of total folate in infant formula and adult nutritionals
using trienzyme extraction and ultra high performance liquid chromatography tandem mass spectrometry
(UHPLC-MS/MS).
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
adult nutritional
nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole
source of nourishment, made from any combination of milk, soy, rice, whey, hydrolysed protein, starch and
amino acids, with and without intact protein
3.2
infant formula
breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants
during the first month of life up to the introduction of appropriate complementary feeding
[SOURCE: Codex Standard 72-1981]
4 Principle
Folates in a sample are extracted in a buffer (pH = 6,0) containing internal standards by treatments with
protease, amylase and rat plasma conjugase (trienzyme digestion). The extract is purified and concentrated
using a weak anion exchange (WAX) solid phase extraction (SPE). Polyglutamate forms of folates in the
sample are deconjugated to monoglutamates during the extraction and are analysed by UHPLC-MS/MS. Folic
acid, 5-methyl-tetrahydrofolate (5-CH -THF) and, 5-formyl-tetrahydrofolate (5-CHO-THF) are quantified,
and total folate is estimated and expressed as folic acid. Isotopically labelled folic acid ( C-folic acid), 5-CH -
13 13
THF ( C-5-CH -THF) and 5-CHO-THF ( C-5-CHO-THF) are used as the internal standards (IS).
5 Reagents and materials
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled
or demineralized water or water of equivalent purity.
Most of the chemicals used are LC-MS grade unless specified. Product numbers and suppliers, when listed,
reflect those used in validation. Equivalent chemicals can be used.
All chemicals used in preparation of standards were stored at a minimum at −20 °C or as directed by the
manufacturer. Because folate compounds are light sensitive, all samples and standards shall be prepared,
handled and stored in the dark or under yellow-shielded lighting or UV-filtered lighting. If standards and
samples shall be transported through or into an area without UV-filtered lighting, they shall be tightly
wrapped in foil.
Store chemicals per manufacturer guidelines or s
...