ISO 23443:2020

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
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
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

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
1)
5.2.1 β-Carotene, USP (Rockville, MD) Part No. 1065480 or equivalent.
1)
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,
1)
France) Part No. 0305 S , or equivalent.
1)
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.
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

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.
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;
2)
YMC, Kyoto, Japan) . Other columns may be used if the system suitability criteria (7.2.2) can be met.
3)
6.3 Guard column, C30 guard column, 10 mm × 2,1 mm × 3 μm (Part No. CT99S03-01Q1GC; YMC) .
3)
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

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.
7.1.3 Liquid ready-to-feed (RTF) with individual carotenoid concentrations ρ ≤ 200 μg/100 g
Shake bottle or can on a reciprocating shaker for 10 min before opening. Transfer approximately 5,25 g
sample into a 50 ml centrifuge tube.
7.1.4 RTF sample with individual carotenoid concentrations > 200 μg/100 g
Shake bottle or can on a reciprocating shaker for 10 min before opening. Transfer approximately 2 g
sample into a 50 ml centrifuge tube. Add 3 ml water, cap and vortex for 10 s. Let sit up to 15 min at room
temperature.
7.1.5 Infant formula concentrate
Shake bottle or can on a reciprocating shaker for 10 min before opening. Transfer approximately 2,5 g
sample into a 50 ml centrifuge tube. Add 2,5 ml water, cap and vortex for 10 s. Let sit up to 15 min at
room temperature.
7.1.6 Volumetrically pipet 5,0 ml of the appropriate ISTD solution from (5.3.8) to each tube.
7.1.7 Add 1,5 ml KOH solution (5.1.13) to each tube with a repeater pipet.
7.1.8 Shake on a reciprocating shaker for 5 min.
7.1.9 Add 8 ml extraction solution (5.1.17) to each tube with a repeater pipet.
7.1.10 Shake for 10 min.
7.1.11 With a repeater pipet or dispenser, add 10 ml water and 10 ml hexane to each tube.
7.1.12 Shake for 1 min.
7.1.13 Centrifuge at 1 000 rpm (or equivalent to 200g) for 5 min.
7.1.14 Transfer a portion of the supernatant to a 12 ml scintillation vial. An adjustable pipette may
be used.
For samples with individual carotenoid concentrations:
— ≤ 50 μg/100 g, use 10 ml supernatant;
— > 50 μg/100 g, use 3 ml supernatant.
7.1.15 Dry under nitrogen at ≤ 40 °C.
7.1.16 Reconstitute dried extract in sample solution (5.1.18) and vortex to mix, shaking if necessary to
include any residue on the sides of the vial. An adjustable pipette may be used for the sample solution.
Prepared sample extracts are stable for 24 h at room temperature.
7.1.17 For samples with individual carotenoid concentrations:
— ≤ 100 μg/100 g, add 0,5 ml;
— 100 μg/100 g to 500 μg/100 g, add 1 ml;
— 500 μg/100 g to 1 000 μg/100 g, add 2 ml;
8 © ISO 2020 – All rights reserved

— 1 000 μg/100 g to 1 500 μg/100 g, add 3 ml.
7.1.18 Filter through a 0,2 μm PTFE syringe filter prior to injection.
7.2 Chromatography
7.2.1 Chromatographic conditions
Set up the UHPLC system according to the specifications below and given in Table 4. Follow the
manufacturer’s instructions for column installation, cleaning and storage. 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 i.d. column. To minimize system dwell volume and extra-
column volume, 0,12 mm i.d. connecting tubing and a low volume flow cell designed for UHPLC systems
are recommended. On some LC systems, it is helpful to convert the pump to low delay volume mode.
Analytical column: YMC C30 3 μm, 250 mm × 2,0 mm
Guard column: YMC C30 3 μm, 10 mm × 2,0 mm
Column temperature: 30 °C
A: 20 mmol/l ammonium acetate in MeOH-Water (98+2)
Mobile phases:
B: MTBE
Flow rate: 0,25 ml/min
Backpressure: approximately 18 500 kPa (185 bar)
Injection volume: 5 μl
UV-Visible detection: 450 nm, ref = off
Table 4 — Gradient programme
Time (min) Mobile phase B %
0 3
1 8
8 15
25 100
25,5 3
32 3
7.2.2 System suitability checks
7.2.2.1 Resolution between β-carotene cis and trans isomers and α-carotene
Inject the β-carotene system suitability working solution (5.3.7), and determine the resolution between
the two major cis isomers of β-carotene, all-trans-β-carotene and α-carotene. Resolution should be ≥ 1,4
between 13-cis-β-carotene and cis/trans α-carotene, and ≥ 2,6 between all-trans- β-carotene and 9-cis-
β-carotene. See Figure A.1.
7.2.2.2 Inject the calibration solutions before and after each set of sample injections (up to 12 samples
in each set). Calculate the slope relative to the internal standard as shown in 8.3. The coefficient of
determination (R ) for each curve should be > 0,995. The slopes from the two curves should not differ
by more than 2 % for β-carotene and not by more than 10 % for lycopene. Use the average of the points
from the two curves bracketing the samples for calculations.
Representative sample chromatograms are shown in Figures A.2 to A.5.
8 Calculations
8.1 Determination of purity
8.1.1 General
Determine the purity of β-carotene and lycopene standards by first determining the spectrophotometric
purity and then the chromatographic purity of each. The overall purity is calculated as the product of
the two measured purities.
8.1.2 Spectrophotometric purity (P )
S
Measure each standard measuring solution (5.3.3) against the appropriate solvent blank at its
absorbance maximum (456 nm for β-carotene in cyclohexane and 476 for lycopene in cyclohexane).
Calculate the spectrophotometric purity of each reference standard as the observed absorbance over
the expected absorbance using Formula (1):
P = (A × V × 1 000)/(E × V × ρ ) (1)
S ms total stock m
where
A is the absorbance of the standard measuring solution;
ms
V is the total volume of standard measuring solution made;
total
1 000 is the factor for g to mg;
E is the extinction coefficient, E (β-carotene in cyclohexane: 2 505 at 456 nm, see
1 %,1cm
Reference [11], lycopene in cyclohexane: 3 310at 476 nm, see Reference [12]);
V is the volume of standard stock solution used, in ml;
stock
ρ is the stock concentration by mass, in mg/100 ml.
m
Spectrophotometric purity is typically greater than 0,90 (i.e. 90 %).
8.1.3 Chromatographic purity (P )
C
Inject standard working solutions (5.3.4) at least three times. Calculate the chromatographic purity
using Formula (2):
P = S /S (2)
C a s
where
S is the area of the all-trans-carotenoid peak;
a
S is the sum of areas of all relevant peaks.
s
Relevant peaks include all peaks in the HPLC chromatogram with the exception of solvent peaks.
Chromatographic purity is typically greater than 0,95 (i.e. 95 %).
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8.1.4 Reference standard purity (P)
Calculate the purity of each reference standard using Formula (3):
P = P × P × 100 (3)
S C
where 100 is the factor for converting decimal to per cent.
8.2 Concentration of each carotenoid in standard solutions
8.2.1 Stock solution concentrations
Calculate the mass concentration of each carotenoid (e.g. β-carotene, ρ ) in the all-trans form, in
bcs
μg/100 ml, in each standard stock solution (5.3.2) using Formula (4):
ρ = (m /V ) × (P /100) × 1 000 × 100 (4)
bcs bc bc bc
where
m is the mass of β-carotene used to make the stock solution, in mg;
bc
P is the reference standard purity of all-trans-β-carotene calculated in (8.1.4);
bc
100 is the conversion from per cent to decimal;
1 000 is the conversion of mg to μg;
100 is the conversion from µg/ml to µg/100 ml;
V is the dilution volume of the stock solution.
bc
8.2.2 Apocarotenal working solution concentration
Calculate the mass concentration of apocarotenal, ρ , in the all-trans form, in μg/100 ml, in the
aws
apocarotenal working solution (5.3.4.4) using Formula (5):
ρ = ρ × (1/100) (5)
aws as
where
ρ is the concentration of apocarotenal stock solution calculated in 8.2.1;
as
1/100 is the dilution of stock solution to working solution.
8.2.3 Apocarotenal intermediate solution concentration
Calculate the mass concentration of apocarotenal, ρ , in the all-trans form, in μg/100 ml, in the
ai
apocarotenal intermediate solution (5.3.5.1) using Formula (6):
ρ = ρ × (3/50) (6)
ai as
where
ρ is the concentration of apocarotenal stock solution calculated in 8.2.1;
as
3/50 is the dilution of stock solution to intermediate solution.
8.2.4 Carotenoid concentrations in mixed carotenoid intermediate solution
Calculate the mass concentration of each carotenoid analyte (e.g. β-carotene, ρ ) in the all-trans form,
bci
in μg/100 ml, in the mixed carotenoid intermediate solution (5.3.5.2) using Formula (7):
ρ = ρ × (V /V) (7)
bci bcs bcs mc
where
ρ is the concentration of β-carotene stock solution calculated in 8.2.1;
bcs
V is the volume of β-carotene stock solution used;
bcs
V is the total volume of mixed carotenoid intermediate solution.
mc
8.2.5 Concentrations of carotenoid analytes in calibrations solutions
Calculate the mass concentration of each carotenoid analyte (e.g. β-carotene, ρ ) in the all-trans form,
bc
in μg/100 ml, in each calibration solution (5.3.6) using Formula (8):
ρ = ρ × (V /V ) (8)
bc bci mci t
where
ρ is the concentration of β-carotene in the mixed carotenoid solution calculated in 8.2.4;
bci
V is the volume of mixed carotenoid intermediate solution used;
mci
V is the total volume of the calibration solution.
t
8.2.6 Concentration of apocarotenal internal standard in calibrations solutions
Calculate the mass concentration of apocarotenal, ρ , in μg/100 ml, in each calibration solution (5.3.6)
a
using Formula (9):
ρ = ρ × (V / V ) (9)
a ai ai t
where
ρ is the concentration of the apocarotenal intermediate solution calculated in 8.2.3;
ai
V is the volume of apocarotenal intermediate solution used;
ai
V is the total volume of the calibration solution.
t
8.3 Calculate calibration curve
For each calibration solution calculate:
a) the peak area ratio for each analyte (peak area [all-trans β-carotene or lycopene])/(peak area
[internal standard]);
b) the concentration ratio (concentration [all-trans β-carotene or lycopene])/(concentration [internal
standard]).
Build a 5-point calibration curve with internal standard by plotting peak area ratios against
concentration ratios, with relative concentration on the x-axis.
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The accuracy on calibration points should be 100 % ± 10 % for calibration solutions C1 to C4, and the
coefficient of determination (R ) should be greater than 0,995.
The calibration and calculation may be achieved through data processing within the instrument
software or off-line.
8.4 Mass of apocarotenal
Calculate the mass of apocarotenal, m , in μg, added to the test samples using Formula (10):
a
m = (ρ /100) × V × (4/50) (10)
a a a
where
ρ is the mass concentration of apocarotenal in the intermediate (8.2.3) or working solution
a
(8.2.2) used in the ISTD solution, in μg/100 ml;
100 is the conversion from µg/100 ml to µg/ml
V the volume of ISTD solution added to each sample, in ml;
a
4 the volume of apocarotenal intermediate or WS used in the ISTD solution, in ml;
50 is the volume of ISTD solution made, in ml.
8.5 Contents of all-trans-β-carotene, cis isomers of β-carotene and total β-carotene
Test sample concentrations calculated in this clause are expressed per 100 g on an as is basis for all
samples, allowing each laboratory to apply appropriate reconstitution factors.
Calculate the contents of all-trans-β-carotene, cis isomers of β-carotene and total β-carotene in the test
samples. For peak identification, refer to relative retention times of peaks in Figures A.1 to A.4.
Calculate the mass fraction of all-trans-β-carotene w , in μg/100 g, using Formula (11):
atbc
m A
 
a atbc
w =× −I × (11)
 
atbc atbc
m A S
 
s a atbc
where
m is the mass of apocarotenal added to the test sample, in μg;
a
m is the mass of the test sample, in g;
s
A is the peak area of all-trans-β-carotene in the sample chromatogram, in arbitrary units;
atbc
A is the peak area of apocarotenal in the sample chromatogram, in arbitrary units;
a
I is the y-intercept of the calibration curve for all-trans-β-carotene;
atbc
S is the slope of the calibration curve for all-trans-β-carotene.
atbc
Calculate the mass fraction of cis isomers of β-carotene, w , in μg/100 g, using Formula (12):
cisbc
m  AA×14,,+×12 +A 
()() () 100
a 15cisbc 13cisbcc9 isbc
w =× −I × (12)
 
cisbc atbcc
m A S
 
s a atbc
where
m is the mass of apocarotenal added to the test sample, in μg;
a
m is the mass of the test sample, in g;
s
A is the peak area of 15-cis-β-carotene in the sample chromatogram, in arbitrary units;
15cisbc
1,4 is the response factor for 15-cis-β-carotene relative to all-trans-β-carotene;
A is the peak area of 13-cis-β-carotene in the sample chromatogram, in arbitrary units;
13cisbc
1,2 is the response factor for 13-cis-β-carotene relative to all-trans-β-carotene;
A is the peak area of 9-cis-β-carotene in the sample chromatogram, in arbitrary units;
9cisbc
A is the peak area of apocarotenal in the sample chromatogram, in arbitrary units;
a
I is the y-intercept of the calibration curve for all-trans-β-carotene;
atbc
S is the slope of the calibration curve for all-trans-β-carotene.
atbc
Calculate the mass fraction of total β-carotene w , in μg/100 g, using Formula (13):
tbc
w =+w w (13)
tbcatbccisbc
8.6 Contents of total lycopene
Test sample concentrations calculated in this clause are expressed per 100 g on an as is basis for all
samples, allowing each laboratory to apply appropriate reconstitution factors.
Calculate the content of total lycopene in the test samples. For peak identification, refer to relative
retention times of peaks in Figure A.5. The first cis lycopene peak has a relative retention time of 0,87
compared to all-trans-lycopene and any peaks between the first cis peak and all-trans-lycopene are also
included as cis lycopene.
Calculate the mass fraction of total trans and cis isomers of lycopene, w , in μg/100 g, using
tlyc
Formula (14):
 
AA+
m ()
atlcyc islyc
a
 
w =× −I × (14)
tlyc atlyc
m  A  S
s a atlyc
 
where
m is the mass of apocarotenal added to the test sample, in μg;
a
m is the mass of the test sample, in g;
s
A is the peak area of all-trans-lycopene in the sample chromatogram, in arbitrary units;
atlyc
A is the peak area of cis isomers of lycopene in the sample chromatogram, in arbitrary units;
cislyc
A is the peak area of apocarotenal in the sample chromatogram, in arbitrary units;
a
I is the y-intercept of the calibration curve for all-trans-lycopene;
atlyc
S is the slope of the calibration curve for all-trans-lycopene.
atlyc
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9 Precision
9.1 General
Details of the interlaboratory test of the precision of the method are summarized in Annex B. The values
derived from the interlaboratory test may not be applicable to analyte concentration ranges and/or
matrices other than
...