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)

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, namely astaxanthin (AXN), canthaxanthin (CXN), adonirubin (ADR) and astaxanthin dimethyldisuccinate (AXN DMDS), and of six authorized carotenoids in poultry feed, 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 ca. 2 to ca. 4 500 mg/kg (depending on the carotenoid). Beta-carotene (BCAR), authorized in compound feed 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. The procedure applies to 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 method.

Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Carotinoiden in Mischfuttermitteln und Vormischungen für Tiere mittels Umkehrphasen-Hochleistungs-Flüssigchromatographie mit UV-Detektion (RP HPLC UV)

Dieses Analysenverfahren legt ein Verfahren der reversen Hochdruckphase-Flüssigchromatographie mit UV Detektion (RP HPLC UV) fest für die gleichzeitige Bestimmung von vier zugelassenen Carotinoiden in Fisch-Mischfuttermittel, und zwar Astaxanthin (AXN), Canthaxanthin (CXN), Adonirubin (ADR) und Astaxanthindimethyldisuccinat (AXN DMDS), und von sechs zugelassenen Carotinoiden in Geflügelfutter, und zwar Canthaxanthin (CXN), Capsanthin (CSN), Beta Apo 8' Carotinsäureethylester (BACARE), Citranaxanthin (CIXN), Lutein (LUT) und Zeaxanthin (ZEA) im Massenanteilbereich von etwa 2 mg/kg bis etwa 4 500 mg/kg (je nach Carotinoid). Beta Carotin (BCAR), in Mischfuttermittel für alle Tierspezies zugelassen, wurde ebenfalls in den Anwendungsbereich aufgenommen. Das Analysenverfahren ist für den Zweck der Quantifizierung der angegebenen Carotinoide und die Bestätigung der Kennzeichnung geeignet. Das Verfahren gilt für natürliche und synthetische Futtermittel-Zusatzstoffe.
Xanthophyllester, wie die von Lutein, Zeaxanthin und Capsanthin, die in Futtermitteln vorhanden sein könnten, sind keine zugelassenen Futtermittel-Zusatzstoffe und gehören daher nicht zum Anwendungs-bereich dieses Verfahrens.

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)

Le présent mode opératoire d’analyse spécifie une méthode par chromatographie liquide à haute performance en phase inverse couplée à une détection UV (CLHP-PI-UV) de quantification simultanée de quatre caroténoïdes autorisés dans les aliments composés pour poissons (à savoir, l’astaxanthine (AXN), la canthaxanthine (CXN), l’adonirubine (ADR) et le diméthyldisuccinate d’astaxanthine (DMDS AXN)), et de six caroténoïdes autorisés dans les aliments composés pour volaille (à savoir, la canthaxanthine (CXN) ; la capsanthine (CSN), l’ester éthylique de bêta-apo-8’-acide caroténoïque (BACARE), la citranaxanthine (CIXN), la lutéine (LUT) et la zéaxanthine (ZÉA)) présents à des teneurs allant d’environ 2 mg/kg à environ 4 500 mg/kg (selon le caroténoïde en question). Le bêta-carotène (BCAR), autorisé dans les aliments composés destinés à l’ensemble des espèces animales, est également couvert par le domaine d’application. Le mode opératoire d’analyse est applicable aux fins de la quantification des caroténoïdes déclarés et de la confirmation de l’étiquetage. Le mode opératoire s’applique aux additifs pour l’alimentation animale d’origine naturelle et de synthèse.
Les esters de xanthophylle tels que ceux de la lutéine, de la zéaxanthine et de la capsanthine qui pourraient être présents dans les matières premières pour aliments des animaux ne sont pas des additifs autorisés pour l’alimentation animale et ne sont donc pas couverts par le domaine d’application de la présente méthode.

Krma: metode vzorčenja in analize - Določevanje karotenoidov v krmnih mešanicah in premiksih s tekočinsko kromatografijo visoke ločljivosti z ultravijolično (UV) detekcijo (HPLC-UV)

Ta analitični postopek določa tekočinsko kromatografijo visoke ločljivosti z ultravijolično (UV) detekcijo (RP-HPLC-UV) z reverzno fazo za hkratno določevanje štirih dovoljenih karotenoidov v krmni mešanici za ribe, in sicer astaksantina (AXN), kantaksantina (CXN), adonirubina (ADR) in astaksantin dimetildisukcinata (AXN DMDS) ter šestih odobrenih karotenoidov v krmi za perutnino, in sicer kantaksantina (CXN); kapsantina (CSN), etil ester beta-apo-8'-karotenojske kisline (BACARE), citranaksantina (CIXN), luteina (LUT) in zeaksantina (ZEA), v ravneh od pribl. 2 do pribl. 4500 mg/kg (odvisno od karotenoida). V področje uporabe je dodan tudi beta-karoten (BCAR), dovoljen v krmnih mešanicah za vse živalske vrste. Analitski postopek je primeren za določanje količine navedenih karotenoidov in potrditev označevanja. Postopek se uporablja za naravne in sintetične krmne dodatke.
Ksantofilni estri, kot so lutein, zeaksantin in kapsantin, ki bi lahko bili prisotni v posamičnih krmilih, niso dovoljeni krmni dodatki in zato ne spadajo na področje uporabe te metode.

General Information

Status
Published
Public Enquiry End Date
19-Nov-2020
Publication Date
13-Jan-2022
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
29-Dec-2021
Due Date
05-Mar-2022
Completion Date
14-Jan-2022

Buy Standard

Standard
EN 17550:2022 - BARVE
English language
49 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day
Draft
prEN 17550:2020 - BARVE
English language
47 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN 17550:2022
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
SIST EN 17550:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN 17550:2022

---------------------- Page: 2 ----------------------
SIST EN 17550:2022


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.

---------------------- Page: 3 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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

2

---------------------- Page: 4 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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.
3

---------------------- Page: 5 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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.
4

---------------------- Page: 6 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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.
5

---------------------- Page: 7 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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.
1
NOTE Suitable proteases are available .
2
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.
2
Hydromatrix™, bulk support material .
5.5 Acetone, HPLC grade.
3
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.
3
5.10 n-Hexane, spectroscopic grade .
3
5.11 Ethanol, spectroscopic grade .
3
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.
3
The exact spectroscopic grade depends on the carotenoid for which the UV standardisation of the standard
solution is performed (5.15.2).
6

---------------------- Page: 8 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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.
7

---------------------- Page: 9 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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.
8

---------------------- Page: 10 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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
4
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.
9

---------------------- Page: 11 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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
4
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.
4
6.30 Vials block heater, Techne Dri-Block Heater or equivalent .
10

---------------------- Page: 12 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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.
11

---------------------- Page: 13 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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.
12

---------------------- Page: 14 ----------------------
SIST EN 17550:2022
EN 17550:2021 (E)
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.
...

SLOVENSKI STANDARD
oSIST prEN 17550:2020
01-november-2020
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 Reverse
Hochdruckphase-Flüssigchromatographie-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: prEN 17550
ICS:
65.120 Krmila Animal feeding stuffs
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
oSIST prEN 17550:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN 17550:2020

---------------------- Page: 2 ----------------------
oSIST prEN 17550:2020


DRAFT
EUROPEAN STANDARD
prEN 17550
NORME EUROPÉENNE

EUROPÄISCHE NORM

September 2020
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 Reverse
performance couplée à une détection UV (CLHP-UV) Hochdruckphase-Flüssigchromatographie-UV
Detektion (RP-HPLC-UV)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 327.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17550:2020 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Terms and definitions . 4
4 Principle . 4
5 Reagents and materials . 5
6 Apparatus . 8
7 Sampling . 10
8 Preparation of test sample . 10
8.1 General. 10
8.2 Laboratory sample . 10
8.3 Test sample . 10
8.4 Test portion . 10
9 Procedure . 11
9.1 General. 11
9.2 Enzymatic disclosure . 11
9.3 Extraction . 11
9.3.1 Extraction using PLE . 11
9.3.2 Conventional liquid solid extraction (SLE) . 12
9.4 Centrifugation . 12
9.5 HPLC analysis . 13
9.5.1 Analytical conditions . 13
9.5.2 HPLC determination . 13
9.5.3 Criteria for acceptance of the analytical result . 15
10 Calculation . 16
11 Test report . 16
11.1 Interlaboratory study . 16
11.2 Repeatability . 16
11.3 Reproducibility . 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 . 30
Bibliography . 46
2

---------------------- Page: 4 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
European foreword
This document (prEN 17550:2020) 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 document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
3

---------------------- Page: 5 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
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, namely astaxanthin (AXN), canthaxanthin (CXN), adonirubin (ADR) and astaxanthin
dimethyldisuccinate (AXN DMDS), and of six authorized carotenoids in poultry feed, 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 ca. 2 to ca. 4 500 mg/kg
(depending on the carotenoid). Beta-carotene (BCAR), authorized in compound feed 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. The procedure applies to 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 method.
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 http://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 ([1], [2]), are expressed in terms of the sum of the all-trans and cis isomers.
Therefore, the areas of the corresponding isomers in the HPLC chromatogram have to be summed up prior to
quantification of the individual carotenoids.
4

---------------------- Page: 6 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
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
1
NOTE Suitable proteases are available.
2
5.2 Purified water, e.g. Milli-Q or equivalent
5.3 Butylated hydroxytoluene BHT
2

5.4 High purity diatomaceous earth suitable for PLE, e.g. Hydromatrix™, bulk support material
5.5 Acetone, HPLC grade
3
5.6 Acetone, spectroscopic grade
5.7 Acetonitrile, HPLC grade
5.8 Methyl tert-butyl ether tBME, HPLC grade
5.9 Tetrahydrofurane stabilized with 250-350 ppm BHT, HPLC grade
3
5.10 n-Hexane, spectroscopic grade
3
5.11 Ethanol, spectroscopic grade
3
5.12 Cyclohexane, spectroscopic grade

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.
3
The exact spectroscopic grade depends on the carotenoid for which the UV standardisation of the standard
solution is performed (5.15.2).
5

---------------------- Page: 7 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
5.13 Mobile phase for HPLC
5.13.1 Phase A: acetonitrile:methyl tert-butyl ether:water mixture 70:20:10; v:v:v, stabilized with
1 000 ppm 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.
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)
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), purity ≥ 97 %
5.14.2 Canthaxanthin (CXN), purity ≥ 97 %
5.14.3 Adonirubin (ADR), purity ≥ 97 %
5.14.4 Astaxanthin dimethyldisuccinate (AXN DMDS), purity ≥ 95 %
5.14.5 Capsanthin (CSN), purity ≥ 95 %
5.14.6 Ethyl ester of beta-apo-8‘-carotenoic acid (BACARE), purity ≥ 95 %
5.14.7 Lutein (LUT), purity ≥ 95 %
5.14.8 Citranaxanthin (CIXN), purity ≥ 95 %
5.14.9 Zeaxanthin (ZEA), purity ≥ 95 %
5.14.10 Beta-carotene (BCAR), purity ≥ 95 %
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).
6

---------------------- Page: 8 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
5.15.1.1 Storage container containing more than 1,0 mg
When the storage container contains more than 1,0 mg of the selected carotenoid (see 5.14.1 or 5.14.2 or
5.14.3 or 5.14.4 or 5.14.5 or 5.14.6 or 5.14.7 or 5.14.8 or 5.14.9 or 5.14.10, depending on the carotenoid
selected), make a 0,2 mg/ml standard solution by e.g. weighing 1,0 mg of this carotenoid (note down the
weight of standard) into a 5 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 a 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 paragraph 9.5.3.2 and
store it in the freezer.
5.15.1.2 Storage container containing less than 1,0 mg
When the storage container specifies a nominal content equal to or less than 1,0 mg of the selected
carotenoid (see 5.14.1 or 5.14.2 or 5.14.3 or 5.14.4 or 5.14.5 or 5.14.6 or 5.14.7 or 5.14.8 or 5.14.9 or
5.14.10, depending on the carotenoid selected), transfer quantitatively the whole content 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
paragraph 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. 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.
1cm 1cm
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,
we consider the effect negligible and we apply the same tabulated extinction coefficient 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 5.15.3.1).
7

---------------------- Page: 9 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
5.15.3 HPLC standard calibration curve
5.15.3.1 Carotenoid 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 5.15.1.1 or 5.15.1.2 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 v:v 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.
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)
8

---------------------- Page: 10 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
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
4
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
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
4
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 glass vials

4
Supelco® Suplex pKb-100, SUPELCOSIL™ Suplex™ pKb-100 Supelguard™ Cartridge, ASE 300 Dionex®, Büchi
SpeedExtractor E-914, Eppendorf® tubes and Techne Dri-Block Heater 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.
9

---------------------- Page: 11 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
6.21 HPLC glass vials crimper
6.22 Centrifuge
6.23 Centrifuge tubes, 50 ml or 100 ml
6.24 Ultraturrax 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
4
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 [3].
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).
10

---------------------- Page: 12 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
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.
NOTE Depending on the analytes to be determined a relevant carotenoid can be added to this mix.
The mixture is isomerised 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.
11

---------------------- Page: 13 ----------------------
oSIST prEN 17550:2020
prEN 17550:2020 (E)
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 ) of extract using a
ext
graduated cylinder (6.17).
9.3.2 Conventional liquid solid extraction (SLE)
Add 50 ml of acetone (5.5) and shake for 1 min. Centrifuge (6.22) at 1 800 g to 1 900 g 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 1 800 g to 1 900 g 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
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.