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

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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 23443:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 23443:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 23443:2020(E)
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

8.1.3 Chromatographic purity (P ) .............................................................................................................................10

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

© ISO 2020 – All rights reserved iii
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ISO 23443:2020(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.

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
---------------------- Page: 4 ----------------------
ISO 23443:2020(E)
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 .
© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
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

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]
© ISO 2020 – All rights reserved 1
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ISO 23443:2020(E)
4 Principle

Test samples (reconstituted powders, liquid ready-to-feed and liquid concentrates) are spiked with an

internal standard and treated with potassium hydroxide. Samples are then extracted with methyl tert-

butyl ether (MTBE) and tetrahydrofuran (THF), followed by hexane. The supernatants from the liquid-

liquid extraction are dried under nitrogen and reconstituted in 2-propanol. Separation is performed

by reversed-phase chromatography on a C30 column. All-trans β-carotene and lycopene are separated

from their major cis isomers, as well as from lutein, zeaxanthin and α-carotene. Although this method

does not involve the high system backpressure normally associated with UHPLC, the low system volume

is recommended for resolution with a 2,0 mm internal diameter (i.d.) column.

Throughout this method, estimated sample concentrations for standard and sample preparations are

stated per 100 g on a reconstituted basis (as is for ready-to-feed liquids, 25 g sample + 200 g water for

powder samples, or diluted 1:1 by weight for liquid concentrates) in accordance with References [8], [9]

and [10].
5 Reagents and materials

During the analysis, unless otherwise stated, only use reagents of recognized analytical grade and

distilled or demineralized water or water of equivalent purity. Reagent volumes may be scaled up or

down provided good laboratory practices are followed.
5.1 Reagents
5.1.1 Laboratory water, 18 megaohm-cm.
5.1.2 Methanol (MeOH), HPLC grade.
5.1.3 Methyl tert-butyl ether (MTBE), HPLC grade.
5.1.4 n-Hexane, HPLC grade.
5.1.5 Cyclohexane, HPLC grade.
5.1.6 Potassium hydroxide (KOH), pellets, ACS grade.
5.1.7 Reagent alcohol (ROH), denatured, 90 % ethanol, HPLC grade.
5.1.8 α-Tocopherol (Vitamin E), synthetic, 95 %.
5.1.9 Pyrogallic acid (Pyrogallol), ACS grade.
5.1.10 2-Propanol (IPA), HPLC grade.

5.1.11 Tetrahydrofuran (THF), 99,9 %, stabilized with butylated hydroxytoluene (BHT).

CAUTION — THF can form peroxides and only THF stabilized with BHT should be used. Refer

to safety data sheets when using any reagent. Use appropriate personal protective equipment

when performing analyses.
5.1.12 Ammonium acetate, HPLC grade, 98 %.
2 © ISO 2020 – All rights reserved
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ISO 23443:2020(E)
5.1.13 Potassium hydroxide solution, a mass fraction of 50 %.

Add 50 ml water to a 250 ml beaker. Weigh 50 g KOH and slowly transfer to the beaker under constant

stirring. When dissolved and cooled, transfer to a media bottle and store at room temperature for up to

six months.
5.1.14 Vitamin E solution in MTBE, substance concentration c = 10 mmol/l.

Dissolve 1,1 g α-tocopherol in 250 ml MTBE. Store in a refrigerator for up to one month.

5.1.15 Vitamin E solution in THF, c = 10 mmol/l.

Dissolve 1,1 g α-tocopherol in 250 ml THF. Store in a refrigerator for up to one month.

5.1.16 Pyrogallol solution, c = 0,2 mol/l pyrogallic acid in reagent alcohol.

Dissolve 6,3 g pyrogallic acid in 250 ml ROH. Store in a refrigerator for up to one month. Solution should

be clear at room temperature; discard if coloured.
5.1.17 Extraction solution, c = 1 mmol/l vitamin E in MTBE-THF (1 + 1).

Dissolve 0,22 g α-tocopherol in 250 ml MTBE and 250 ml THF. Store in a refrigerator for up to one month.

5.1.18 Sample solution, c = 10 mmol/l vitamin E in IPA.

Dissolve 4,4 g α-tocopherol in 1 000 ml IPA. Store in a refrigerator for up to one month.

5.1.19 Mobile phase A for LC system, c = 20 mmol/l ammonium acetate in methanol–water (98 + 2).

Combine 980 ml MeOH, 20,0 ml water and 1,54 g ammonium acetate, and mix to dissolve.

5.1.20 Mobile phase B for LC system.
MTBE (5.1.3).
5.2 Standards
5.2.1 β-Carotene, USP (Rockville, MD) Part No. 1065480 or equivalent.
5.2.2 Apocarotenal (β-Apo-8′-carotenal), USP Part No. 1040854 or equivalent.

5.2.3 Lycopene, > 90 % by UV-Vis, Sigma-Aldrich (St. Louis, MO) Part No. L9879, Extrasynthese (Genay,

France) Part No. 0305 S , or equivalent.
5.2.4 β-Carotene system suitability reference standard, USP Part No. 1065491 .

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 and endorsement by ISO of the product named. Equivalent products

may be used if they can be shown to lead to the same results.
© ISO 2020 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 23443:2020(E)
5.3 Standards preparation

5.3.1 Standard solution preparation is summarized in Table 1 and detailed below. Use glass volumetric

pipettes and flasks for preparation of all standard solutions unless otherwise noted.

Table 1 — Composition and nominal concentrations of carotenoid standard solutions

Standard solution β-carotene Lycopene Apocarotenal
Stock solutions
Standard (mg) 5,0 2,5 5,0
Total volume (ml) 25 25 25
Concentration (mg/100 ml) 20 10 20
UV-Visible solutions (200 μg/100 ml)
Stock solution (ml) 1,0 2,0 —
Total volume (ml) 100 100 —
Working solutions
(200 μg/100 ml) in sample solvent
Stock solution (ml) 0,1 0,2 1,0
Total volume (ml) 10 10 100
Intermediate solutions in sample solvent
Stock solution (ml) 2,0 2,0 3,0
Total volume (ml) 100 — 50
Concentration (μg/100 ml) 400 200 1 200
5.3.2 Carotenoid stock solutions, ρ = 10 000 μg/100 ml to ρ = 20 000 μg/100 ml.

Weigh (to 0,01 mg) approximately 5 mg each of β-carotene (5.2.1) and apocarotenal (5.2.2) reference

standard into separate 25 ml volumetric flasks. Add approximately 20 ml vitamin E solution in MTBE

(5.1.14) to each, sonicate for 2 min to 3 min, and dilute to volume with vitamin E solution in MTBE.

Weigh (to 0,01 mg) approximately 2,5 mg lycopene (5.2.3) reference standard into a 25 ml volumetric

flask. Add approximately 20 ml vitamin E solution in THF (5.1.15), sonicate for 2 min to 3 min, and

dilute to volume with vitamin E solution in THF.

Store stock solutions at −20 °C for up to six months and check their purity each time new standard

solutions are made from them. When taken from the freezer, stock solutions should be sonicated for

2 min and vortexed to bring all carotenoids into solution.
5.3.3 UV-Visible solutions for spectroscopy potency check, ρ = 200 μg/100 ml.

Transfer 1,0 ml β-carotene standard stock solution (5.3.2) to a 100 ml volumetric flask and dilute to

volume with cyclohexane.

Transfer 2,0 ml lycopene standard stock solution (5.3.2) to a 100 ml volumetric flask and dilute to

volume with cyclohexane.

Immediately measure solutions by UV-visible spectroscopy and calculate purity according to 8.1.2.

5.3.4 Individual carotenoid working solutions for chromatographic purity check
(200 μg/100 ml).

5.3.4.1 Analyse working solutions by UHPLC on the same day they are prepared and calculate the

chromatographic purity of each according to 8.1.3.
4 © ISO 2020 – All rights reserved
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ISO 23443:2020(E)
5.3.4.2 β-carotene working solution.

With an adjustable pipet, transfer 100 μl of standard stock solution (5.3.2) to a 10 ml volumetric flask

and dilute to volume with sample solution.
5.3.4.3 Lycopene working solution.

With an adjustable pipet, transfer 200 μl standard stock solution (5.3.2) to a 10 ml volumetric flask and

dilute to volume with sample solution.
5.3.4.4 Apocarotenal working solution.

Transfer 1,0 ml standard stock solution (5.3.2) to a 100 ml volumetric flask and dilute to volume with

sample solution. Store at −20 °C for up to one month and use for internal standard (5.3.8).

5.3.5 Intermediate solutions, ρ = 200 μg/100 ml to 1 200 μg/100 ml.
5.3.5.1 Apocarotenal intermediate solution.

Transfer 3,0 ml apocarotenal stock solution (5.3.2) to a 50 ml volumetric flask and dilute to volume

with sample solution. Store at −20 °C for up to one month.
5.3.5.2 Mixed carotenoid intermediate solution.

Combine 2,0 ml each of β-carotene and lycopene standard stock solutions (5.3.2) in a 100 ml volumetric

flask and dilute to volume with sample solution. Store at −20 °C for up to one month.

5.3.6 Calibration solutions.

Transfer apocarotenal intermediate solution (5.3.5.1) and mixed carotenoid intermediate solution

(5.3.5.2) to volumetric flasks according to Table 2 and dilute to volume with sample solution. Store at

−20 °C for up to one month. Solutions may be aliquoted to HPLC vials prior to storing in the freezer.

Table 2 — Composition and nominal concentrations of carotenoid calibration solutions

Calibration solution C1 C2 C3 C4 C5
Apocarotenal intermediate (5.3.5.1) added (ml) 2,0 2,0 2,0 2,0 8,0
Mixed carotenoid intermediate (5.3.5.2) added (ml) 15,0 8,0 5,0 2,0 1,0
Total volume (ml) 25 25 25 25 100
Apocarotenal concentration (μg/100 ml) 96 96 96 96 96
β-carotene concentration (μg/100 ml) 240 128 80 32 4
Lycopene concentration (μg/100 ml) 120 64 40 16 2
5.3.7 β-Carotene system suitability solutions.

Preparation of β-carotene system suitability solutions is summarized in Table 3 and detailed below.

To make the stock solution, transfer approximately 20 mg β-carotene system suitability reference

standard (5.2.4) to a 50 ml volumetric flask. Add 1 ml water and 4 ml THF and sonicate for 5 min. Dilute

to volume with IPA and sonicate for 5 min. Cool to room temperature and filter the cloudy suspension

through a 0,2 μm polytetrafluoroethylene (PTFE) syringe filter.

To make the working solution, dilute 5 ml of the filtered stock solution to 25 ml with IPA. Store in a

refrigerator for up to three months or at −20 °C for up to six months.
© ISO 2020 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 23443:2020(E)
Table 3 — Composition of β-carotene system suitability solutions
Suitability solution β-carotene
Stock solution composition
Standard added (mg) 20
Total volume (ml) 50
Working solution composition
Stock solution added (ml) 5
Total volume (ml) 25
5.3.8 Internal standard solution (ISTD).

5.3.8.1 Prepare immediately before use. The apocarotenal solutions used to make the ISTD should be

made from the same apocarotenal stock solution (5.3.2) as that used to make the calibration solutions

(5.3.6).

5.3.8.2 Infant formula and samples with low carotenoid concentrations (up to 100 μg of an

individual carotenoid per 100 g).

Transfer 4,0 ml apocarotenal working solution (5.3.4.4) to a 50 ml volumetric flask and dilute to volume

with pyrogallol solution (5.1.16). This is enough solution for nine samples.
5.3.8.3 Samples with individual carotenoid concentrations > 100 μg/100 g.

Transfer 4,0 ml apocarotenal intermediate solution (5.3.5.1) to a 50 ml volumetric flask and dilute to

volume with pyrogallol solution.
6 Apparatus
Usual laboratory glassware and equipment and, in particular, the following.

6.1 UHPLC system, consisting of a binary or quaternary pump, autosampler, thermostatted column

compartment, UV-Vis detector and data acquisition software. A high sensitivity flow cell (e.g . 60 mm) in

the detector provides the best results, but a standard 10 mm flow cell may be used if system suitability

criteria can be met.

6.2 Analytical column, C30 carotenoid column, 250 mm × 2,0 mm × 3 μm (Part No. CT99S03-2502WT;

YMC, Kyoto, Japan) . Other columns may be used if the system suitability criteria (7.2.2) can be met.

6.3 Guard column, C30 guard column, 10 mm × 2,1 mm × 3 μm (Part No. CT99S03-01Q1GC; YMC) .

6.4 Guard cartridge holder, Part No. XPGCH-Q1 (YMC) .

6.5 Spectrophotometer, wavelength range of 200 nm to 700 nm, with 1 cm quartz cells.

6.6 Top loading balance, capable of weighing to 0,1 g.

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 of this product. Equivalent products may be

used if they can be shown to lead to the same results.

3) 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 of this product. Equivalent products may be

used if they can be shown to lead to the same results.
6 © ISO 2020 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 23443:2020(E)
6.7 Analytical balance, capable of weighing to 0,01 mg.
6.8 Ultrasonic water bath, 40 kHz.
6.9 Reciprocating shaker, capable of 200 rpm.
6.10 Evaporator, with pure nitrogen supply.
6.11 Laboratory centrifuge, with adapters for 50 ml centrifuge tubes.
6.12 Centrifuge tubes, 50 ml, polypropylene.
6.13 Syringes, 1 ml, disposable.
6.14 Syringe filters, 0,2 μm, PTFE.
6.15 Class A volumetric flasks, various sizes, clear and amber.
6.16 Scintillation vials, 12 ml, amber.
6.17 HPLC vials, amber, with 300 μl inserts.
6.18 Class A volumetric pipets, various sizes.
7 Procedure
7.1 Sample preparation
7.1.1 General

While this method can quantify carotenoids in the range of 1 μg/100 g to 1 300 μg/100 g, it is

recommended to only quantify a 100-fold difference with a single preparation. For example, the

range of 1 μg/100 g to 100 μg/100 g works well for infant formula, but the range of 15 μg/100 g to

1 500 μg/100 g would work best for samples with the highest carotenoid concentrations.

This method is not applicable to materials that contain measurable levels of β-apo-8′-carotenal

(apocarotenal). Because apocarotenal is used as an internal standard, its presence in the test material

would inflate the amount of internal standard measured in the samples, leading to artificially low

results for the analytes. Unknown samples should be prepared as blanks, using 5 ml pyrogallol solution

in place of ISTD solution in 7.1.6, to demonstrate that they do not contain apocarotenal.

Prepare up to 12 samples at a time. Weigh all samples (powders and liquids) to 0,1 mg. At several points

in the sample preparation, sample masses and dilutions may vary according to the concentration of an

individual carotenoid in the product. If carotenoids are present in different ranges in the same sample,

e.g. lycopene at ≤ 50 µg/100 g and β-carotene at 250 µg/100 g, then the sample should be prepared once

for each concentration.
7.1.2 Powders

Record masses of both powder sample and water to four significant figures. Reconstitute 25 g powder

sample with 200 ml 35 °C water in a reagent bottle and shake well. Mix on a spin plate for 1 min to 5 min

until completely dispersed and no solids are visible. To ensure homogeneity, shake again immediately

before transferring approximately 5,25 g reconstituted sample into a 50 ml centrifuge tube.

© ISO 2020 – All ri
...

INTERNATIONAL ISO
STANDARD 23443
First edition
2020-05
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
PROOF/ÉPREUVE
Reference number
ISO 23443:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 23443:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 23443:2020(E)
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 .......................................................................................................................................10

8 Calculations.............................................................................................................................................................................................................10

8.1 Determination of purity ...............................................................................................................................................................10

8.1.1 General...................................................................................................................................................................................10

8.1.2 Spectrophotometric purity (P ) .......................................................................................................................10

8.1.3 Chromatographic purity (P ) .............................................................................................................................11

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 .......................................................................12

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 ........................................................................................................................................................13

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 ................................................................................................................................................................................................................16

Annex A (informative) Example chromatograms .................................................................................................................................17

Annex B (informative) Precision data ..............................................................................................................................................................22

Annex C (informative) Determination of lutein .....................................................................................................................................25

Bibliography .............................................................................................................................................................................................................................32

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ISO 23443:2020(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.

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.
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ISO 23443:2020(E)
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 .
© ISO 2020 – All rights reserved PROOF/ÉPREUVE v
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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

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]
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ISO 23443:2020(E)
4 Principle

Test samples (reconstituted powders, liquid ready-to-feed and liquid concentrates) are spiked with an

internal standard and treated with potassium hydroxide. Samples are then extracted with methyl tert-

butyl ether (MTBE) and tetrahydrofuran (THF), followed by hexane. The supernatants from the liquid-

liquid extraction are dried under nitrogen and reconstituted in 2-propanol. Separation is performed

by reversed-phase chromatography on a C30 column. All-trans β-carotene and lycopene are separated

from their major cis isomers, as well as from lutein, zeaxanthin and α-carotene. Although this method

does not involve the high system backpressure normally associated with UHPLC, the low system volume

is recommended for resolution with a 2,0 mm internal diameter (i.d.) column.

Throughout this method, estimated sample concentrations for standard and sample preparations are

stated per 100 g on a reconstituted basis (as is for ready-to-feed liquids, 25 g sample + 200 g water for

powder samples, or diluted 1:1 by weight for liquid concentrates) in accordance with References [8], [9]

and [10].
5 Reagents and materials

During the analysis, unless otherwise stated, only use reagents of recognized analytical grade and

distilled or demineralized water or water of equivalent purity. Reagent volumes may be scaled up or

down provided good laboratory practices are followed.
5.1 Reagents
5.1.1 Laboratory water, 18 megaohm-cm.
5.1.2 Methanol (MeOH), HPLC grade.
5.1.3 Methyl tert-butyl ether (MTBE), HPLC grade.
5.1.4 n-Hexane, HPLC grade.
5.1.5 Cyclohexane, HPLC grade.
5.1.6 Potassium hydroxide (KOH), pellets, ACS grade.
5.1.7 Reagent alcohol (ROH), denatured, 90 % ethanol, HPLC grade.
5.1.8 α-Tocopherol (Vitamin E), synthetic, 95 %.
5.1.9 Pyrogallic acid (Pyrogallol), ACS grade.
5.1.10 2-Propanol (IPA), HPLC grade.

5.1.11 Tetrahydrofuran (THF), 99,9 %, stabilized with butylated hydroxytoluene (BHT).

CAUTION — THF can form peroxides and only THF stabilized with BHT should be used. Refer

to safety data sheets when using any reagent. Use appropriate personal protective equipment

when performing analyses.
5.1.12 Ammonium acetate, HPLC grade, 98 %.
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ISO 23443:2020(E)
5.1.13 Potassium hydroxide solution, a mass fraction of 50 %.

Add 50 ml water to a 250 ml beaker. Weigh 50 g KOH and slowly transfer to the beaker under constant

stirring. When dissolved and cooled, transfer to a media bottle and store at room temperature for up to

six months.
5.1.14 Vitamin E solution in MTBE, substance concentration c = 10 mmol/l.

Dissolve 1,1 g α-tocopherol in 250 ml MTBE. Store in a refrigerator for up to one month.

5.1.15 Vitamin E solution in THF, c = 10 mmol/l.

Dissolve 1,1 g α-tocopherol in 250 ml THF. Store in a refrigerator for up to one month.

5.1.16 Pyrogallol solution, c = 0,2 mol/l pyrogallic acid in reagent alcohol.

Dissolve 6,3 g pyrogallic acid in 250 ml ROH. Store in a refrigerator for up to one month. Solution should

be clear at room temperature; discard if coloured.
5.1.17 Extraction solution, c = 1 mmol/l vitamin E in MTBE-THF (1 + 1).

Dissolve 0,22 g α-tocopherol in 250 ml MTBE and 250 ml THF. Store in a refrigerator for up to one month.

5.1.18 Sample solution, c = 10 mmol/l vitamin E in IPA.

Dissolve 4,4 g α-tocopherol in 1 000 ml IPA. Store in a refrigerator for up to one month.

5.1.19 Mobile phase A for LC system, c = 20 mmol/l ammonium acetate in methanol–water (98 + 2).

Combine 980 ml MeOH, 20,0 ml water and 1,54 g ammonium acetate, and mix to dissolve.

5.1.20 Mobile phase B for LC system.
MTBE (5.1.3).
5.2 Standards
5.2.1 β-Carotene, USP (Rockville, MD) Part No. 1065480 or equivalent.
5.2.2 Apocarotenal (β-Apo-8′-carotenal), USP Part No. 1040854 or equivalent.

5.2.3 Lycopene, > 90 % by UV-Vis, Sigma-Aldrich (St. Louis, MO) Part No. L9879, Extrasynthese (Genay,

France) Part No. 0305 S , or equivalent.
5.2.4 β-Carotene system suitability reference standard, USP Part No. 1065491 .

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 and endorsement by ISO of the product named. Equivalent products

may be used if they can be shown to lead to the same results.
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ISO 23443:2020(E)
5.3 Standards preparation

5.3.1 Standard solution preparation is summarized in Table 1 and detailed below. Use glass volumetric

pipettes and flasks for preparation of all standard solutions unless otherwise noted.

Table 1 — Composition and nominal concentrations of carotenoid standard solutions

β-carotene Lycopene Apocarotenal
Stock solutions
Standard (mg) 5,0 2,5 5,0
Total volume (ml) 25 25 25
Concentration (mg/100 ml) 20 10 20
UV-Visible solutions (200 μg/100 ml)
Stock solution (ml) 1,0 2,0 —
Total volume (ml) 100 100 —
Working solutions
(200 μg/100 ml) in sample solvent
Stock solution (ml) 0,1 0,2 1,0
Total volume (ml) 10 10 100
Intermediate solutions in sample solvent
Stock solution (ml) 2,0 2,0 3,0
Total volume (ml) 100 — 50
Concentration (μg/100 ml) 400 200 1 200
5.3.2 Carotenoid stock solutions, ρ = 10 000 μg/100 ml to ρ = 20 000 μg/100 ml.

Weigh (to 0,01 mg) approximately 5 mg each of β-carotene (5.2.1) and apocarotenal (5.2.2) reference

standard into separate 25 ml volumetric flasks. Add approximately 20 ml vitamin E solution in MTBE

(5.1.14) to each, sonicate for 2 min to 3 min, and dilute to volume with vitamin E solution in MTBE.

Weigh (to 0,01 mg) approximately 2,5 mg lycopene (5.2.3) reference standard into a 25 ml volumetric

flask. Add approximately 20 ml vitamin E solution in THF (5.1.15), sonicate for 2 min to 3 min, and

dilute to volume with vitamin E solution in THF.

Store stock solutions at −20 °C for up to six months and check their purity each time new standard

solutions are made from them. When taken from the freezer, stock solutions should be sonicated for

2 min and vortexed to bring all carotenoids into solution.
5.3.3 UV-Visible solutions for spectroscopy potency check, ρ = 200 μg/100 ml.

Transfer 1,0 ml β-carotene standard stock solution (5.3.2) to a 100 ml volumetric flask and dilute to

volume with cyclohexane.

Transfer 2,0 ml lycopene standard stock solution (5.3.2) to a 100 ml volumetric flask and dilute to

volume with cyclohexane.

Immediately measure solutions by UV-visible spectroscopy and calculate purity according to 8.1.2.

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ISO 23443:2020(E)
5.3.4 Individual carotenoid working solutions for chromatographic purity check
(200 μg/100 ml).

5.3.4.1 Analyse working solutions by UHPLC on the same day they are prepared and calculate the

chromatographic purity of each according to 8.1.3.
5.3.4.2 β-carotene working solution.

With an adjustable pipet, transfer 100 μl of standard stock solution (5.3.2) to a 10 ml volumetric flask

and dilute to volume with sample solution.
5.3.4.3 Lycopene working solution.

With an adjustable pipet, transfer 200 μl standard stock solution (5.3.2) to a 10 ml volumetric flask and

dilute to volume with sample solution.
5.3.4.4 Apocarotenal working solution.

Transfer 1,0 ml standard stock solution (5.3.2) to a 100 ml volumetric flask and dilute to volume with

sample solution. Store at −20 °C for up to one month and use for internal standard (5.3.8).

5.3.5 Intermediate solutions, ρ = 200 μg/100 ml to 1 200 μg/100 ml.
5.3.5.1 Apocarotenal intermediate solution.

Transfer 3,0 ml apocarotenal stock solution (5.3.2) to a 50 ml volumetric flask and dilute to volume

with sample solution. Store at −20 °C for up to one month.
5.3.5.2 Mixed carotenoid intermediate solution.

Combine 2,0 ml each of β-carotene and lycopene standard stock solutions (5.3.2) in a 100 ml volumetric

flask and dilute to volume with sample solution. Store at −20 °C for up to one month.

5.3.6 Calibration solutions.

Transfer apocarotenal intermediate solution (5.3.5.1) and mixed carotenoid intermediate solution

(5.3.5.2) to volumetric flasks according to Table 2 and dilute to volume with sample solution. Store at

−20 °C for up to one month. Solutions may be aliquoted to HPLC vials prior to storing in the freezer.

Table 2 — Composition and nominal concentrations of carotenoid calibration solutions

Calibration solution C1 C2 C3 C4 C5
Apocarotenal intermediate (5.3.5.1) added (ml) 2,0 2,0 2,0 2,0 8,0
Mixed carotenoid intermediate (5.3.5.2) added (ml) 15,0 8,0 5,0 2,0 1,0
Total volume (ml) 25 25 25 25 100
Apocarotenal concentration (μg/100 ml) 96 96 96 96 96
β-carotene concentration (μg/100 ml) 240 128 80 32 4
Lycopene concentration (μg/100 ml) 120 64 40 16 2
5.3.7 β-Carotene system suitability solutions.

Preparation of β-carotene system suitability solutions is summarized in Table 3 and detailed below.

To make the stock solution, transfer approximately 20 mg β-carotene system suitability reference

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ISO 23443:2020(E)

standard (5.2.4) to a 50 ml volumetric flask. Add 1 ml water and 4 ml THF and sonicate for 5 min. Dilute

to volume with IPA and sonicate for 5 min. Cool to room temperature and filter the cloudy suspension

through a 0,2 μm polytetrafluoroethylene (PTFE) syringe filter.

To make the working solution, dilute 5 ml of the filtered stock solution to 25 ml with IPA. Store in a

refrigerator for up to three months or at −20 °C for up to six months.
Table 3 — Composition of β-carotene system suitability solutions
β-carotene
Stock solution composition
Standard added (mg) 20
Total volume (ml) 50
Working solution composition
Stock solution added (ml) 5
Total volume (ml) 25
5.3.8 Internal standard solution (ISTD).

5.3.8.1 Prepare immediately before use. The apocarotenal solutions used to make the ISTD should be

made from the same apocarotenal stock solution (5.3.2) as that used to make the calibration solutions

(5.3.6).

5.3.8.2 Infant formula and samples with low carotenoid concentrations (up to 100 μg of an

individual carotenoid per 100 g).

Transfer 4,0 ml apocarotenal working solution (5.3.4.4) to a 50 ml volumetric flask and dilute to volume

with pyrogallol solution (5.1.16). This is enough solution for nine samples.
5.3.8.3 Samples with individual carotenoid concentrations > 100 μg/100 g.

Transfer 4,0 ml apocarotenal intermediate solution (5.3.5.1) to a 50 ml volumetric flask and dilute to

volume with pyrogallol solution.
6 Apparatus
Usual laboratory glassware and equipment and, in particular, the following.

6.1 UHPLC system, consisting of a binary or quaternary pump, autosampler, thermostatted column

compartment, UV-Vis detector and data acquisition software. A high sensitivity flow cell (e.g . 60 mm) in

the detector provides the best results, but a standard 10 mm flow cell may be used if system suitability

criteria can be met.

6.2 Analytical column, C30 carotenoid column, 250 mm × 2,0 mm × 3 μm (Part No. CT99S03-2502WT;

YMC, Kyoto, Japan) . Other columns may be used if the system suitability criteria (7.2.2) can be met.

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 of this product. Equivalent products may be

used if they can be shown to lead to the same results.
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ISO 23443:2020(E)

6.3 Guard column, C30 guard column, 10 mm × 2,1 mm × 3 μm (Part No. CT99S03-01Q1GC; YMC) .

6.4 Guard cartridge holder, Part No. XPGCH-Q1 (YMC) .

6.5 Spectrophotometer, wavelength range of 200 nm to 700 nm, with 1 cm quartz cells.

6.6 Top loading balance, capable of weighing to 0,1 g.
6.7 Analytical balance, capable of weighing to 0,01 mg.
6.8 Ultrasonic water bath, 40 kHz.
6.9 Reciprocating shaker, capable of 200 rpm.
6.10 Evaporator, with pure nitrogen supply.
6.11 Laboratory centrifuge, with adapters for 50 ml centrifuge tubes.
6.12 Centrifuge tubes, 50 ml, polypropylene.
6.13 Syringes, 1 ml, disposable.
6.14 Syringe filters, 0,2 μm, PTFE.
6.15 Class A volumetric flasks, various sizes, clear and amber.
6.16 Scintillation vials, 12 ml, amber.
6.17 HPLC vials, amber, with 300 μl inserts.
6.18 Class A volumetric pipets, various sizes.
7 Procedure
7.1 Sample preparation
7.1.1 General

While this method can quantify carotenoids in the range of 1 μg/100 g to 1 300 μg/100 g, it is

recommended to only quantify a 100-fold difference with a single preparation. For example, the

range of 1 μg/100 g to 100 μg/100 g works well for infant formula, but the range of 15 μg/100 g to

1 500 μg/100 g would work best for samples with the highest carotenoid concentrations.

This method is not applicable to materials that contain measurable levels of β-apo-8′-carotenal

(apocarotenal). Because apocarotenal is used as an internal standard, its presence in the test material

would inflate the amount of internal standard measured in the samples, leading to artificially low

results for the analytes. Unknown samples should be prepared as blanks, using 5 ml pyrogallol solution

in place of ISTD solution in 7.1.6, to demonstrate that they do not contain apocarotenal.

3) 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 of this product. Equivalent products may be

used if they can be shown to lead to the same results.
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ISO 23443:2020(E)

Prepare up to 12 samples at a time. Weigh all samples (powders and liquids) to 0,1 mg. At several points

in the sample preparation, sample masses and dilutions may vary according to the concentration of an

individual carotenoid in the product. If carotenoids are present in different ranges in the same sample,

e.g. lycopene at ≤ 50 µg/100 g and β-carotene at 250 µg/100 g, then the sample should be prepared once

for each concentration.
7.1.2 Powders

Record masses of both powder sample and water to four significant figures. Reconstitute 25 g powder

sample with 200 ml 35 °C
...

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