ISO 23443:2020
(Main)Infant formula and adult nutritionals — Determination of β-carotene, lycopene and lutein by reversed-phase ultra-high performance liquid chromatography (RP-UHPLC)
Infant formula and adult nutritionals — Determination of β-carotene, lycopene and lutein by reversed-phase ultra-high performance liquid chromatography (RP-UHPLC)
This document specifies a method for the quantitative determination of β-carotene and lycopene in infant formula and adult nutritionals in solid (i.e. powders) or liquid (i.e. ready-to-feed liquids and liquid concentrates) forms using reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) and UV-visible detection. The application range runs from 1 μg/100 g to 1 500 μg/100 g for lycopene and from 1 μg/100 g to 2 250 μg/100 g for β-carotene. Based on the single-laboratory validation, the limit of detection (LOD) was 0,1 μg/100 g and the limit of quantification (LOQ) was 0,3 μg/100 g for each carotenoid. The method does not apply to materials that contain measurable levels of β-apo-8′-carotenal. The reproducibility data meets the requirements given in References [8] and [10]. Annex C specifies the determination of lutein. The reproducibility data does not meet the requirements given in Reference [9].
Formules infantiles et produits nutritionnels pour adultes — Détermination du bêta-carotène, du lycopène et de la lutéine par chromatographie liquide ultra haute performance à phase inversée
General Information
Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 23443
First edition
2020-07
Infant formula and adult
nutritionals — Determination of
β-carotene, lycopene and lutein
by reversed-phase ultra-high
performance liquid chromatography
(RP-UHPLC)
Formules infantiles et produits nutritionnels pour adultes —
Détermination du bêta-carotène, du lycopène et de la lutéine par
chromatographie liquide ultra haute performance à phase inversée
Reference number
©
ISO 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and materials . 2
5.1 Reagents. 2
5.2 Standards . 3
5.3 Standards preparation . 4
6 Apparatus . 6
7 Procedure. 7
7.1 Sample preparation . 7
7.2 Chromatography . 9
7.2.1 Chromatographic conditions . 9
7.2.2 System suitability checks . 9
8 Calculations.10
8.1 Determination of purity .10
8.1.1 General.10
8.1.2 Spectrophotometric purity (P ) .10
S
8.1.3 Chromatographic purity (P ) .10
C
8.1.4 Reference standard purity (P) .11
8.2 Concentration of each carotenoid in standard solutions.11
8.2.1 Stock solution concentrations .11
8.2.2 Apocarotenal working solution concentration .11
8.2.3 Apocarotenal intermediate solution concentration .11
8.2.4 Carotenoid concentrations in mixed carotenoid intermediate solution.12
8.2.5 Concentrations of carotenoid analytes in calibrations solutions .12
8.2.6 Concentration of apocarotenal internal standard in calibrations solutions .12
8.3 Calculate calibration curve .12
8.4 Mass of apocarotenal .13
8.5 Contents of all-trans-β-carotene, cis isomers of β-carotene and total β-carotene . 13
8.6 Contents of total lycopene .14
9 Precision .15
9.1 General .15
9.2 Repeatability .15
9.3 Reproducibility .15
10 Test report .15
Annex A (informative) Example chromatograms .17
Annex B (informative) Precision data .22
Annex C (informative) Determination of lutein .25
Bibliography .32
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.
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 documents 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).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
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, 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 2016.13: Determination
of Lutein, β-Carotene, and Lycopene in Infant Formula and Adult Nutritionals by Ultra-High-Performance
Liquid Chromatography: Final Action (β-Carotene and Lycopene Only).
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 © ISO 2020 – All rights reserved
Introduction
Lutein, β-carotene and lycopene are among the carotenoids present in human milk and are added to
[1][2][3]
infant formula and adult nutritionals . Lutein may play a role in vision and cognitive function,
[4][5]
and β-carotene has provitamin A activity . Accurate and precise measurements of these added
ingredients are important for ensuring their presence in the allowable ranges.
This analytical method was originally presented to the Stakeholder Panel on Infant Formula and
Adult Nutritionals through AOAC International, and a single-laboratory validation was previously
[6]
published . It was recommended as an AOAC Final Action method for β-carotene and lycopene after
[7]
the collaborative study data was reviewed by the same panel .
INTERNATIONAL STANDARD ISO 23443:2020(E)
Infant formula and adult nutritionals — Determination of
β-carotene, lycopene and lutein by reversed-phase ultra-
high performance liquid chromatography (RP-UHPLC)
WARNING — The use of this method can involve hazardous materials, operations and equipment.
This method does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this method to establish appropriate safety and health practices.
1 Scope
This document specifies a method for the quantitative determination of β-carotene and lycopene in
infant formula and adult nutritionals in solid (i.e. powders) or liquid (i.e. ready-to-feed liquids and liquid
concentrates) forms using reversed-phase ultra-high performance liquid chromatography (RP-UHPLC)
and UV-visible detection. The application range runs from 1 μg/100 g to 1 500 μg/100 g for lycopene
and from 1 μg/100 g to 2 250 μg/100 g for β-carotene. Based on the single-laboratory validation, the
limit of detection (LOD) was 0,1 μg/100 g and the limit of quantification (LOQ) was 0,3 μg/100 g for
each carotenoid.
The method does not apply to materials that contain measurable levels of β-apo-8′-carotenal. The
reproducibility data meets the requirements given in References [8] and [10].
Annex C specifies the determination of lutein. The reproducibility data does not meet the requirements
given in Reference [9].
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 terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// 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
breas
...
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