EN 17550:2021

SLOVENSKI STANDARD
01-februar-2022
Krma: metode vzorčenja in analize - Določevanje karotenoidov v krmnih
mešanicah in premiksih s tekočinsko kromatografijo visoke ločljivosti z
ultravijolično (UV) detekcijo (HPLC-UV)
Animal feeding stuffs: Methods of sampling and analysis - Determination of carotenoids
in animal compound feed and premixtures by high performance liquid chromatography -
UV detection (HPLC-UV)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von
Carotinoiden in Mischfuttermitteln und Vormischungen für Tiere mittels Umkehrphasen-
Hochleistungs-Flüssigchromatographie mit UV-Detektion (RP HPLC UV)
Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Détermination de la
teneur en caroténoïdes des aliments composés et des prémélanges pour animaux par
chromatographie liquide à haute performance couplée à une détection UV (CLHP-UV)
Ta slovenski standard je istoveten z: EN 17550:2021
ICS:
65.120 Krmila Animal feeding stuffs
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17550
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2021
EUROPÄISCHE NORM
ICS 65.120; 71.040.50
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of carotenoids in animal compound feed
and premixtures by high performance liquid
chromatography - UV detection (HPLC-UV)
Aliments des animaux - Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Détermination de la teneur en caroténoïdes Untersuchungsverfahren - Bestimmung von
des aliments composés et des prémélanges pour Carotinoiden in Mischfuttermitteln und
animaux par chromatographie liquide à haute Vormischungen für Tiere mittels Umkehrphasen-
performance couplée à une détection UV (CLHP-UV) Hochleistungs-Flüssigchromatographie mit UV-
Detektion (RP HPLC UV)
This European Standard was approved by CEN on 20 September 2021.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17550:2021 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Reagents and materials . 5
6 Apparatus . 9
7 Sampling . 11
8 Preparation of test sample . 11
9 Procedure . 11
10 Calculation . 16
11 Precision . 17
12 Test report . 17
Annex A (informative) Complementary instrumental and analytical information . 18
A.1 Absorption coefficient values . 18
A.2 Examples of chromatographic profile patterns of isomerized carotenoids . 19
A.3 Results of the collaborative study . 31
Bibliography . 48

European foreword
This document (EN 17550:2021) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by June 2022, and conflicting national standards shall be
withdrawn at the latest by June 2022.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
Introduction
The method described in this document aims at constituting a tool for the effective control of carotenoids
in feed by the competent authorities in the frame of Regulation (EC) No 1831/2003. Making use of the
properties of the isosbestic wavelength for quantification, the method allows, in laboratory routine
conditions, determining the sum of all isomers for each carotenoid authorized in poultry or fish feed. This
approach can result in higher analytical variation of quantitative results in some matrices, and analysis
should be repeated by an alternative method if the obtained variation significantly deviates from data
presented in A.3 of this document. These alternative methods may be those optimized for the
measurement of single carotenoids authorized under Regulation (EC) No 1831/2003 and available from
the European Union Reference Laboratory for Feed Additives.
1 Scope
This analytical procedure specifies a reverse phase high performance liquid chromatographic with UV
detection (RP-HPLC-UV) method for the simultaneous determination of four authorized carotenoids in
fish compound feed and fish premix, namely astaxanthin (AXN), canthaxanthin (CXN), adonirubin (ADR)
and astaxanthin dimethyldisuccinate (AXN DMDS), and of six authorized carotenoids in poultry feed and
poultry premix, namely canthaxanthin (CXN); capsanthin (CSN), ethyl ester of beta-apo-8'-carotenoic
acid (BACARE), citranaxanthin (CIXN), lutein (LUT) and zeaxanthin (ZEA) at levels ranging from
approximately 2 mg/kg to approximately 4 500 mg/kg (depending on the carotenoid). Beta-carotene
(BCAR), authorized in compound feed and premixes for all animal species, was also added to the scope.
The analytical procedure is fit for the purpose of quantitation of declared carotenoids and labelling
confirmation. This document is applicable to feed produced using natural and synthetic feed additives.
Xanthophyll esters like those of lutein, zeaxanthin and capsanthin that might be present in feed materials
are not authorized feed additives and therefore not part of the scope of this document.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle
The carotenoids are first disclosed through an enzymatic reaction at 50 °C. The samples are extracted
with acetone by means of a pressurized liquid extraction instrument or by liquid solid extraction. The
extracts are centrifuged and analysed by reverse phase HPLC with UV or Diode Array Detection. A
common isosbestic wavelength of 410 nm is selected for the determination of the target analytes, thus
ensuring that the various isomers of each of the carotenoids have similar absorbance coefficients. The
quantitation is performed through external calibration.
NOTE The maximum contents of the carotenoids, as established by the European regulations for the
authorization of feed additives ([3], [4]), are expressed in terms of the sum of the all-trans and cis isomers.
Therefore, from a legal point of view, it is important to sum up the areas of the corresponding isomers in the HPLC
chromatogram prior to quantification of the individual carotenoids.
5 Reagents and materials
WARNING 1 — Carotenoids are subject to light degradation. Protect analytical work adequately from
day light, and keep standard solutions protected from light by using amber glassware, amber vials or
aluminium foil.
WARNING 2 — Avoid inhalation of and exposure to the toxic standard materials and solutions thereof.
Work under fume hood when handling the solvents and solutions. Wear safety glasses and protective
clothing.
WARNING 3 — Always wear a safety mask when handling Hydromatrix™.
Unless otherwise specified, use only reagents of recognized analytical grade.
5.1 Protease with the ability to release carotenoids from their encapsulated form.
NOTE Suitable proteases are available .
5.2 Purified water, e.g. Milli-Q or equivalent .
5.3 Butylated hydroxytoluene BHT.
5.4 High purity diatomaceous earth suitable for Pressurized Liquid Extraction (PLE), e.g.
Hydromatrix™, bulk support material .
5.5 Acetone, HPLC grade.
5.6 Acetone, spectroscopic grade .
5.7 Acetonitrile, HPLC grade.
5.8 Methyl tert-butyl ether tBME, HPLC grade.
5.9 Tetrahydrofurane (THF) stabilized with 250 mg/l to 350 mg/l butylated hydroxytoluene
(BHT), HPLC grade.
5.10 n-Hexane, spectroscopic grade .
5.11 Ethanol, spectroscopic grade .
5.12 Cyclohexane, spectroscopic grade .
5.13 Mobile phase for HPLC
5.13.1 Phase A: acetonitrile + methyl tert-butyl ether + water mixture 70 + 20 + 10; V + V + V ,
1 2 3
stabilized with 1 000 mg/l BHT.
Using a graduated cylinder (6.17), transfer 700 ml of acetonitrile (5.7) into a 1 000 ml bottle. Measure
(6.17) and add 200 ml of methyl tert-butyl ether (5.8) and 100 ml water (5.2). Add 1,0 g of BHT (5.3).
Perform mixing and degassing for 10 min in an ultrasonic bath (6.11). This mobile phase is stable for 28
days.
NOTE The retention time of the carotenoids is strongly influenced by slight differences in the composition of
mobile phase A. The use of an HPLC quality control sample (9.1) is crucial for the correct signal allocation.

1 ®
Alcalase and Multifect PR 6L have been successfully used for the validation.
2 ®
Milli-Q, Hydromatrix™, Alcalase and Multifect PR 6L are examples of suitable products available commercially.
This information is given for the convenience of users of this document and does not constitute an endorsement by
CEN of these products. Equivalent products may be used if they can be shown to lead to the same results.
The exact spectroscopic grade depends on the carotenoid for which the UV standardisation of the standard
solution is performed (5.15.2).
Furthermore, the presence of non-targeted carotenoids might interfere with the signals of the targeted analytes
when the composition of mobile phase A deviates from 5.13.1.
5.13.2 Phase B: acetonitrile + methyl tert-butyl ether mixture (70 + 30; V + V ), stabilized with
1 2
1 000 mg/l BHT.
Using a graduated cylinder (6.17), transfer 700 ml of acetonitrile (5.7) into a 1 000 ml bottle. Measure
(6.17) and add 300 ml methyl tert-butyl ether (5.8). Add 1,0 g of BHT (5.3). Perform mixing and degassing
for 10 min in an ultrasonic bath (6.11). This mobile phase is stable for 28 days.
5.14 Reference standards
Guaranteed purity is required for each lot of reference standard:
5.14.1 Astaxanthin (AXN), CAS N° 472-61-7, purity ≥ 970 g/kg.
5.14.2 Canthaxanthin (CXN), CAS N° 514-78-3, purity ≥ 970 g/kg.
5.14.3 Adonirubin (ADR), CAS N° 4418-72-8, purity ≥ 970 g/kg.
5.14.4 Astaxanthin dimethyldisuccinate (AXN DMDS), CAS N° 578006-46-9, purity ≥ 950 g/kg.
5.14.5 Capsanthin (CSN), CAS N° 465-42-9, purity ≥ 950 g/kg.
5.14.6 Ethyl ester of beta-apo-8‘-carotenoic acid (BACARE), CAS N° 1109-11-1, purity ≥ 950 g/kg.
5.14.7 Lutein (LUT), CAS N° 127-40-2, purity ≥ 950 g/kg.
5.14.8 Citranaxanthin (CIXN), CAS N° 3604-90-8, purity ≥ 950 g/kg.
5.14.9 Zeaxanthin (ZEA), CAS N° 144-68-3, purity ≥ 950 g/kg.
5.14.10 Beta-carotene (BCAR), CAS N° 7235-40-7, purity ≥ 950 g/kg.
5.15 Standard solutions
Protect all standard solutions from daily light.
5.15.1 Carotenoid stock standard solution, ca. 200 μg/ml.
Prepare fresh and measure immediately.
NOTE Possible carotenoids are astaxanthin (AXN), canthaxanthin (CXN), adonirubin (ADR), astaxanthin
dimethyldisuccinate (AXN DMDS), capsanthin (CSN), ethyl ester of beta-apo-8'-carotenoic acid (BACARE),
citranaxanthin (CIXN), lutein (LUT), zeaxanthin (ZEA) and beta-carotene (BCAR).
Accurately weigh 1,0 mg of the carotenoid standard (5.14) (note down the mass of standard) (or transfer
quantitatively the whole content of the container containing the standard substance (5.14)) in a 5,0 ml
volumetric flask.
Dissolve and make up to the mark with THF/BHT (5.9). Mix well using a vortex mixer (6.27) and an
ultrasonic bath (6.11). The accurate mass fraction needs to be standardized using the spectrophotometer
(6.2) as described in detail in 5.15.2. Reserve a 1 ml portion for the isomerisation procedure (5.15.4), if
needed. Reserve another aliquot for the identification of the analyte as described in 9.5.3.2 and store it in
the freezer.
5.15.2 UV standardization of the standard solutions
Pipette 100 μl of the selected carotenoid stock standard solution (5.15.1) into a 10 ml volumetric flask
(6.8) and make up to the mark with the appropriate solvent (Annex A). The nominal value of the obtained
solution is ca. 2 μg/ml. Scan the spectrum of this solution from 300 nm to 550 nm and measure the
absorption of this solution against the pure solvent, at the maximum, using the spectrophotometer (6.2).
The maximum is solvent specific and is given as an approximate value in Annex A.
The exact content of the selected carotenoid is given by Formula (1).
1 %
Carotenoid μg/ml = E × 10 000/E (1)
λmax 1 cm
EXAMPLE For all-E AXN, the solvent suggested in the table in Annex A is n-hexane. For this solvent, the
1 % 1 %
wavelength of measurement is approximately 470 nm and the E (or A ) is 2 100.
1 cm 1 cm
Pipette with a suited pipette 100 μl of the astaxanthin stock standard solution (5.15.1) into a 10 ml
volumetric flask and make up to the mark with n-hexane. The nominal value of the obtained standard
measuring solution is ca. 2 μg/ml. Measure the UV spectrum of this solution against pure n-hexane, using
the spectrophotometer (6.2) set at the wavelength of maximum absorption (approx. 470 nm).
NOTE The solvent of the measured solution is not pure as there is a small presence of THF/BHT (5.9). However,
the effect is considered as negligible and the same tabulated extinction coefficient is applied, given that the
contribution of THF/BHT (5.9) is very small (1 %).
The content of AXN is given by Formula (2).
AXN μg/ml = E × 10 000/2 100 (2)
max
A solution of the same concentration, 100 μl of the astaxanthin stock standard solution (5.15.1) pipetted
into a 10 ml volumetric flask and made up to the mark with acetone (5.5), shall be injected simultaneously
in the HPLC (see 5.15.3).
5.15.3 HPLC standard calibration curve
Pipette with a suited pipette 100 µl of carotenoid stock standard solution (5.15.1) into a 10 ml volumetric
flask and make up to the mark with acetone (5.5).
5.15.4 Isomerisation
5.15.4.1 General
It is recommended to perform this step when ambiguity occurs regarding the presence of possible
isomers.
5.15.4.2 Isomerisation of the standard solutions
The standards prepared according to 5.15.1 could be isomerised in order to have a profile of the all-trans
and cis isomers of each carotenoid, which are present in the equilibrium phase.
Fill a LC vial with 1 ml standard. Close tightly and check that the vial is well sealed and the cap does not
turn. Heat up the vial at about 80 °C for about 2 h to achieve the equilibrium between the isomers. A vials
block heater (6.30) can be used for this purpose.
Inject a dilution of the isomerised standard (e.g. 1 → 200).
The isomerised standard is stable for several months at room temperature.
5.16 HPLC positive quality control sample (QCS)
Weigh approximately 0,8 mg AXN (5.14.1), 0,4 mg CXN (5.14.2) and 0,5 g BHT (5.3) in a round bottom
flask (6.9). Add 200 ml of an acetone (5.5) + water (5.2) 75 + 25 V1 + V2 solution. Fit a condenser (6.10)
to the flask and immerse the flask in a heating mantle or heated bath (6.28) with a magnetic stirrer (6.29).
Heat to boiling and allow to refluxing for about 1,5 h to 2 h.
NOTE Alternatively, the mixture can be heated at 80 °C in a pressure-resistant tube.
This reaction will cause the formation of a stable isomers ratio. Transfer to a 500 ml volumetric flask (6.8)
and make up to volume with acetone (5.5). Mix well and transfer immediately in HPLC vials. The filling
should be fast and each vial should be immediately closed to avoid evaporation. Store the vials at room
temperature and away from light. This solution, when protected from oxygen, is stable for several
months.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 HPLC system, consisting of the following.
6.1.1 Pump, pulse free, capable of maintaining a volume flow rate from 0,1 ml/min to 2,0 ml/min.
6.1.2 Injection system, manual or autosampler.
If an autosampler is used, a cooled one is recommended although it was not used during the validation of
the method.
6.1.3 UV/VIS detector, variable wavelength, suitable for reliable measurements at 410 nm, or UV/VIS
photodiode array detector (DAD).
6.1.4 Computer data system.
® 4
6.1.5 Analytical column, Supelco Suplex pKb-100 5 µm, 250 mm × 4,6 mm or equivalent .
6.1.6 Guard column, 5 μm, 2 cm × 4 mm SUPELCOSIL™ Suplex™ pKb-100 Supelguard™ Cartridge or
equivalent .
6.2 Spectrophotometer, with 1 mm apertures.
6.3 Grinding instrument.
6.4 Sieve, with 1 mm apertures.
6.5 Balances, one analytical, of 10 g capacity or greater with 0,1 mg readability, and one, of 100 g
capacity or greater with 0,01 g readability.
6.6 Polypropylene containers, 100 ml with lids.

4 ® ®
Supelco Suplex pKb-100, SUPELCOSIL™ Suplex™ pKb-100 Supelguard™ Cartridge, ASE 300 Dionex , Büchi
® ®
SpeedExtractor E-914, Eppendorf tubes, Techne Dri-Block Heater and Ultra-Turrax mixer are examples of
suitable products available commercially. This information is given for the convenience of users of this document
and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be
shown to lead to the same results.
6.7 Variable-volume positive displacement piston pipettes, suitable for pipetting volumes ranging
from 50 μl to 100 μl.
6.8 Glass volumetric flasks of, 5 ml, 10 ml, 150 ml, 500 ml and 1 000 ml.
6.9 Round bottom flasks of 500 ml.
6.10 Allihn condenser.
6.11 Ultrasonic bath, temperature controlled.
6.12 Flat spatulas. ®
6.13 PLE, pressurized solvent extraction system, ASE 300 Dionex , Büchi SpeedExtractor E-914 or
equivalent .
6.14 PLE cells, suitable for the extraction unit used, 66 ml or larger.
6.15 Cellulose filters for PLE cells.
6.16 PLE vials, for the extraction unit used, at least 240 ml.
6.17 Graduated cylinder of 250 ml and 1 000 ml.
® 4
6.18 Microcentrifuge safe-lock tubes, 1,8 ml, Eppendorf tubes or equivalent .
6.19 Microcentrifuge.
6.20 1,5 ml HPLC amber glass vials.
6.21 HPLC glass vials crimper.
6.22 Centrifuge.
6.23 Centrifuge tubes, 50 ml or 100 ml.
® 4
6.24 Ultra-Turrax mixer .
6.25 PLE Funnels for PLE cells.
6.26 Powder funnels.
6.27 Vortex mixer.
6.28 Heating mantle or heated bath.
6.29 Magnetic stirrer.
6.30 Vials block heater, Techne Dri-Block Heater or equivalent .
7 Sampling
It is important that the laboratory receives a sample that is truly representative and has not been
damaged or changed during transport or storage.
Sampling is not part of the method specified in this document. A recommended sampling method is given
in EN ISO 6497 [1].
8 Preparation of test sample
8.1 General
Prepare the test sample in accordance with EN ISO 6498.
8.2 Laboratory sample
Grind the laboratory sample (usually 50 g) so that it passes completely through a sieve with 1 mm
apertures (6.4) or until a fine paste is obtained. Mix thoroughly.
8.3 Test sample
The test sample consists of a representative and homogenized aliquot of the ground laboratory sample
(8.2) of at least 10 g.
8.4 Test portion
Accurately weigh 5,0 g to the nearest 0,1 g of the thoroughly mixed test sample (8.3) into a 100 ml
polypropylene container (6.6) for extraction procedure 9.3.1 or into a 50 ml or 100 ml centrifuge tube
(6.23) for extraction procedure 9.3.2. Note down the mass expressed in g. Submit it to the analysis
procedure (Clause 9).
9 Procedure
9.1 General
The complete procedure (9.2 to 9.4) should be applied to two test portions (8.4) of the same test sample
in order to perform two independent and parallel determinations.
The use of quality control samples is recommended.
The QCS (5.16) shall be injected before and after the complete sequence samples each day of analysis, in
order to assess the suitability of the HPLC/DAD system.
The QCS (5.16) contains two carotenoids:
1) CXN, a stable and more soluble carotenoid, and
2) AXN, a less stable and less soluble carotenoid.
Depending on the analytes to be determined, a relevant carotenoid may be added to this mix.
The mixture is isomerized until a constant ratio of the isomers is reached. This solution, when protected
from oxygen, is stable for several months.
9.2 Enzymatic disclosure
Add 0,2 g of BHT (5.3), 100 µl of enzyme (5.1) and 15 ml of purified water (5.2) to the test portion (8.4).
Close tightly and shake vigorously to ensure that all the feed is permeated with water. Place in an
ultrasonic bath (6.11) with the temperature set at 50 °C for 15 min to 20 min. Shake every 5 min.
9.3 Extraction
9.3.1 Extraction using PLE
Remove the lid of the test portion after step 9.2, taking care of not losing sample drops. Add
approximately 13 g of high purity diatomaceous earth suitable for PLE (5.4), close tightly and hand-shake
vigorously until the wet feed sample looks all adsorbed on the diatomaceous earth beadlets and detaches
well from the polypropylene container's wall.
NOTE Stomping the container on the table can help.
Open the containers carefully, remove the sample particles from the lid into the container using a flat
spatula (6.12). Mix well with the spatula (6.12), making sure that there are no sample clusters left. Place
two cellulose filters (6.15) on the bottom of the PLE cell (6.14). Transfer all the material in the PLE cell
(6.14) using a funnel (6.25). Top up with diatomaceous earth (5.4) if needed. Close the cell and extract
according to the parameters in Table 1.
Table 1 — Extraction parameters
Extraction solvent Acetone (5.5)
Pressure (bar) 103,4
Pressure (psi) 1 500
Temperature (°C) 58
Preheat time (min) 0
Heat time (min) 0
Static time (min) 7
Flush volume (%) 120
Purge time (s) 60
Static cycles 3
Each feed extraction lasts approximately 30 min. The instrument can run up to 12 samples in an
automated way, e.g. overnight.
Take the tube containing the raw extract. Mix well and record the total volume (V ) (10) of extract using
ext
a graduated cylinder (6.17).
9.3.2 Conventional liquid solid extraction (LSE)
Add 50 ml of acetone (5.5) to the test portion and shake for 1 min. Centrifuge (6.22) at approximately 1
800 g to 1 900 g (approximately 3 100 rpm) for 5 min. Collect the supernatant (first extract) and transfer
into a 150 ml volumetric flask (6.8). ®
Add 50 ml of acetone (5.5) to the solid residue of the first extraction. Use the Ultra-Turrax mixer (6.24)
if needed to achieve dissolution and shake for 1 min. Centrifuge (6.22) at approximately 1 800 g to 1 900 g
(approximately 3 100 rpm) for 5 min. Collect the supernatant (second extract) and pool with the first
extract in the 150-ml volumetric flask.
Add 40 ml of acetone (5.5) to the residue of the second extraction and shake for 1 min. Centrifuge (6.22)
at approximately 1 800 g to 1 900 g (approximately 3 100 rpm) for 5 min. Collect the supernatant (third
extract) and pool with the two first extracts in the 150 ml volumetric flask. Make up to volume (V ) (10)
ext
with acetone and shake vigorously.
If it is not possible to find or use 100 ml centrifuge tubes, the more common 50 ml centrifuge tubes may
be used. In this case however, the three extraction steps with 50 ml acetone shall be replaced by four
extraction steps with 35 ml to 37 ml acetone (5.5) each.
Similarly, if a 150 ml volumetric flask (6.8) is not available, record the final volume V (10) using a
ext
graduated cylinder (6.17).
Transfer a 1,8 ml aliquot of the raw extract into a microcentrifuge safe-lock tube (6.18).
9.4 Centrifugation
Centrifuge (6.19) for 1 min at high relative centrifugal acceleration (e.g. 13 000 g, approximately 11 800
rpm).
NOTE Alternatively, filter the extract through a 0,45 µm membrane filter (suitable for acetone).
Transfer the supernatant to a HPLC amber vial (6.20) and proceed to the HPLC analysis (9.5).
9.5 HPLC analysis
9.5.1 Analytical conditions
The following conditions are provided for guidance. Other conditions may be used provided they yield to
equivalent results.
9.5.1.1 HPLC column as in 6.1.5.
9.5.1.2 Guard column as in 6.1.6.
9.5.1.3 Mobile phase as in 5.13, flow rate: 0,5 ml/min.
9.5.1.4 Injection volume, 5 µl.
9.5.1.5 Column temperature, 20 °C.
9.5.1.6 Detection wavelength, isosbestic wavelength 410 nm [5].
9.5.2 HPLC determination
9.5.2.1 Carotenoids elution
Perform the elution of the carotenoids in gradient mode. The gradient to be applied is displayed in
Table 2.
9.5.2.2 External calibration curves
Inject each calibration solution described in 5.15.3 immediately after preparation in the HPLC system
(6.1) and in parallel with the spectrophotometric standardization (5.15.2). Perform, at least, two parallel
determinations (on separate 1 → 100 dilutions) or inject at least twice the external calibrant (5.15.3).
Note the mean area of the peak, A. The concentration of this solution, c, correlated to the area of the peak
is the exact content determined using the spectrophotometer (in µg/ml).
The calibration regression line is plot with the following two points:
• Calibration point 1: concentration 0 µg/ml; area 0 (Arbitrary Unit, AU);
• Calibration point 2: concentration c = exact content determined using the spectrophotometer in
µg/ml; determined area A, in AU.
Determine the equation of the calibration regression line according to Formula (3).
A
a= (3)
c
where
is the slope coefficient of the calibration curve obtained from the standard solutions;
a
A is the area of the peak;
c is the concentration of the solution, in µg/ml.
Table 2 — Elution gradient
Time % of mobile phase A % of mobile phase B
min
0 100 0
10 100 0
14 40 60
30 40 60
30,1 100 0
45 100 0
Using these conditions the retention times for various carotenoids should approximately be as in Table 3.

Table 3 — Approximate retention times with the conditions as in 9.5.1
Carotenoid retention time
min
AXN DMDS, astaxanthin dimethyldisuccinate 8,0
AXN, astaxanthin 13,5
ADR, adonirubin 14,5
CXN, canthaxanthin 15,8
CSN, capsanthin 18,6
CIXN, citranaxanthin 20,9
BACARE, ethyl ester of beta-apo-8'-carotenoic acid 21,4
LUT, lutein 24,5
ZEA, zeaxanthin 25,8
BCAR, beta-carotene 32,0
9.5.2.3 Quality control sample
Just after preparation, inject one vial containing the quality control sample QCS (5.16) and verify the
suitability of the HPLC system as a whole. Record the areas of the peaks of the two carotenoids in the
sample, and their respective retention time. These values are the REFERENCE (areareference i and
Rtreference i with i being 1 or 2 (2 being the total number of carotenoids in the QCS solution)).
Before analysis of an unknown sample, inject a QCS vial, record and plot the areas and retention times
obtained for the peaks of the two analytes in the QCS and compare to the reference values (area and
retention times). Inject a new QCS vial at the end of the sample sequence to verify that the suitability of
the HPLC is still acceptable.
9.5.2.4 Sample extracts
Inject each sample extract obtained in (9.3.1) or (9.3.2). Record the peak area for the carotenoids signals.
9.5.3 Criteria for acceptance of the analytical result
9.5.3.1 Run acceptance
A QCS shall always be injected before and after an analysis is performed. To accept the experiments the
following criteria shall be met:
a) The areas of the QCS AXN and CXN peaks shall be equal (±8 %) to the respective areas of the reference
sample (9.5.2.3).
b) The retention time of the QCS AXN and CXN peaks shall be equal (±8 %) to the respective retention
times of the reference sample (9.5.2.3).
If the criteria are not met, a new calibration is needed.
NOTE When the system is stable, it can be run smoothly without performing a new calibration for at least 3
months.
c) The retention time of the AXN and CXN peaks in the sample extract (9.5.2.4) shall be equal (±5 %) to
the respective retention times in the QCS (9.5.2.3).
d) The retention time of the AXN and CXN peaks in the first sample extract (9.5.2.4) in the sequence
shall be equal (±2 %) to the respective retention times in the last sample extract (9.5.2.4).
For very long sequences, a QCS should be inserted as a new reference for retention times' comparison.
The next sample extract injected would therefore also become the new reference for retention times'
comparison. The criterion is valid when no change is made, e.g. the same mobile phase batch is used along
the whole sequence.
The samples should pass the identification criteria (9.5.3.2).
9.5.3.2 Identification criteria
Various isomers may exist for a given analyte (see Annex A).
Inject an appropriate dilution of the standard stored in the freezer as described in 5.15.1. The analytes
present in the sample can be injected one at a time or as a mixture.
The identity of the analytes (including all isomers; see Annex A) is confirmed if the following two criteria
are satisfied:
1) The retention time of the sample peaks shall be equal (±8 %) to the retention time of the standard
peaks.
2) The difference spectra of the sample and standard peaks recorded at the peak apex shall not be
visually different for those parts of the spectra with a relative absorption of at least 10 % (between
10 % and 100 % in a normalized spectrum). This criterion is met when:
a) the same maxima are present within a margin determined by the resolution of the detection
system (4 nm), and
b) at those particular maxima wavelengths, the relative deviation, in comparison with the
absorbance of the standard analyte, between the two spectra (sample – standard) does not
exceed 25 %.
Additionally and if the necessary software and expertise are available, the following criterion can also be
applied:
3) Assess the purity of the sample peak on the basis of the conformity of the difference spectra, recorded
at the apex and at up-slope and down-slope inflection points. For those parts of the spectra with a
relative absorption of at least 10 % (between 10 % and 100 % in a normalized spectrum), at each
wavelength the relative absorption shall be equal (within 25 %) for all spectra.
10 Calculation
Calculate the target carotenoid content in the unknown samples by comparing the peak area of the
injected sample extract with the peak area of the standards used for the external calibration curve
(9.5.2.2).
Use Formula (4) for the calculation of target carotenoid content (CTC) of the sample in milligrams per
kilogram (mg/kg).
AV
TC ext
(4)
C = *
TC
am
where
C is the initial mass fraction of the target carotenoid in the feed sample, in mg/kg;
TC
A is the sum of the areas of the HPLC peaks for the various isomers of the target carotenoid (AU);
TC
a is the slope coefficient of the calibration curve obtained from the standard solutions (see
Formula (3));
V is the final volume of extract, in ml;
ext
EXAMPLE This is 150 ml if the 150 ml-volumetric flask is used.
m is the test portion mass, in g.
11 Precision
11.1 Interlaboratory study
Details of the interlaboratory study on the precision of the method, organized by the European
Commission, Joint Research Centre (EC, JRC, Geel, Belgium) are given in A.3.
11.2 Repeatability
The absolute difference between two parallel independent single test results, expressed as a percentage
of the mean determined value, obtained using the same method on identical test material in the same
laboratory by the same operator using the same equipment within a short interval of time, will in no more
than 5 % of cases exceed the repeatability limit (r) derived from Formula (5).
r=2 ××2 RSD=2,8× RSD
(5)
rr
where
r is the repeatability limit;
RSD is the repeatability standard deviation.
r
11.3 Reproducibility
The absolute difference between two parallel independent single test results, expressed as a percentage
of the mean determined value, obtained using the same method on identical test material on different
days by different operators using different equipment, will in no more than 5 % of cases exceed the
reproducibility limit (R) derived from Formula (6).
R=2 ××2 RSD=2,8× RSD
(6)
RR
where
R is the reproducibility limit;
RSD is the reproducibility standard deviation.
R
12 Test report
The test report shall specify the following information:
a) all information necessary for the complete identification of the sample;
b) the test method used, with reference to this document EN 17550:2021;
c) the test result(s) obtained and the units in which the test result(s) have been expressed according to
Clause 10 of this document EN 17550:2021;
d) all operating details not specified in this document, or regarded as optional, together with details of
any incidents which may have influenced the test result(s);
e) the date of the test;
f) the sampling method used, if known.
Annex A
(informative)
Complementary instrumental and analytical information
A.1 Absorption coefficient values
Carotenoids in solution obey the Beer-Lambert law, namely their absorbance is proportional to the
concentration.
1 % 1 %
Values of absorption coefficients E , (or A ) specified for a particular solvent, are published
1 cm 1 cm
in literature (e.g. [6], USP Monographs and JECFA monographs ([7], [8], [9])).
For convenience, the absorption coefficient values used during the in-house validation, the respective
solvent, the λ and the typical coefficient of the calibration curve obtained are reported in Table A.1.
max
Other values, extracted from published articles or review or experimentally determined, may be equally
suitable to determine the exact concentration.
Table A.1 — Absorption coefficient values
1 % a
Analyte Solvent used
A or λ nm
1 cm max
1 %
E
1 cm
b
AXN 2 100 470
n-Hexane
c
AXN DMDS 1 519 470
n-Hexane
d
CXN 2 200 466
Cyclohexane
e
ADR 2 150 476
Cyclohexane
f
CIXN 2 680 473
Cyclohexane
g
CSN 2 157 470
Acetone
h
LUT 2 550 445
Ethanol
i
ZEA 2 480 450
Ethanol
j
BACARE 2 550 449
Cyclohexane
f
BCAR 2 500 455
Cyclohexane
a
These are approximate values.
b
From Britton et al. [6], no 404 - 406.
c 1 %
Derived from the A of astaxanthin considering the different molecular
1 cm
masses.
d
From JECFA Monograph, 51st JECFA [7].
e 1 %
Derived from the A of astaxanthin and canthaxanthin.
1 cm
f
From JECFA Monograph, 31st JECFA [8].
g 1 %
Alternative to the use of benzene (E = 2 072, approx. λ = 483 nm.
1 cm max
h
US Pharmacopeia, 2009 USPC Official 8/1/09 – 11/30/09 Dietary Supplements:
Lutein.
i
From Britton et al. [6], no 119.
j
From JECFA Monograph, 74th JECFA [9].
A.2 Examples of chromatographic profile patterns of isomerized carotenoids
Figure A.1 to Figure A.10 show examples of profiles obtained if isomerization occurs. In Figure A.11 and
Figure A.12, the profiles of isomerized carotenoids are combined.
Key
x retention time, in min
y signal intensity, in AU
1 before isomerization
2 after isomerization of the same solution of the given carotenoid
NOTE The line starting under the other line is number 1; the line starting on top is number 2. In the peak around
15 min, line 1 exceeds line 2.
Figure A.1 — Chromatographic profile of pure standard solutions of ADR, including peak
identification
Key
x retention time, in min
y signal intensity, in AU
1 before isomerization
2 after isomerization of the same solution of the given carotenoid
NOTE The line starting under the other line is number 1; the line starting on top is number 2. In the peak around
15 min, line 1 exceeds line 2.
Figure A.2 — Chromatographic profile of pure standard solutions of AXN, including peak
identification
Key
x retention time, in min
y signal intensity, in AU
1 before isomerization
2 after isomerization of the same solution of the given carotenoid
NOTE The line starting under the other line is number 1; the line starting on top is number 2. In the peak around
8 min, line 1 exceeds line 2.
Figure A.3 — Chromatographic profile of pure standard solutions of AXN DMDS, including peak
identification
Key
x retention time, in min
y signal intensity, in AU
1 before isomerization
2 after isomerization of the same solution of the given carotenoid
NOTE The line starting under the other line is number 1; the line starting on top is number 2. In the peak around
22 min, line 1 exceeds line 2.
Figure A.4 — Chromatographic profile of pure standard solutions of BACARE, including peak
identification
Key
x retention time, in min
y signal intensity, in AU
1 before isomerization
2 after isomerization of the same solution of the given carotenoid
NOTE The line starting under the other line is number 1; the line starting on top is number 2. In the peak around
33 min, line 1 exceeds line 2.
Figure A.5 — Chromatographic profile of pure standard solutions of BCAR, including peak
identification
Key
x retention time, in min
y signal intensity, in AU
1 before isomerization
2 after isomerization of the same solution of the given carotenoid
NOTE The line starting under the other line is number 1; the line starting on top is number 2. In the peak around
22 min, line 1 exceeds line 2.
Figure A.6 — Chromatographic profile of pure standard solutions of CIXN, including peak
identification
Key
x retention time, in min
y signal intensity, in AU
1 before isom
...