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 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.

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 Umkehrphasen-Hochleistungs-Flüssigchromatographie mit UV-Detektion (RP HPLC UV) fest für die gleichzeitige Bestimmung von vier zugelassenen Carotinoiden in Fisch-Mischfuttermittel und Fischfutter-Vormischung, und zwar Astaxanthin (AXN), Canthaxanthin (CXN), Adonirubin (ADR) und Astaxanthindimethyldisuccinat (AXN DMDS), und von sechs zugelassenen Caroti¬noiden in Geflügelfutter und Geflügelfutter-Vormischung, 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 und Vormischungen für alle Tierspezies zugelassen, wurde ebenfalls in den Anwendungs¬bereich aufgenommen. Das Analysenverfahren ist für den Zweck der Quantifizierung der angegebenen Carotinoide und die Bestätigung der Kennzeichnung geeignet. Das Verfahren ist auf Futtermittel anzuwenden, die unter Verwendung von natürlichen und synthetischen Futtermittel-Zusatzstoffen hergestellt werden.
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 Dokuments.

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 et les prémélanges 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 et les prémélanges 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 et les prémélanges 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 présent document s’applique aux aliments pour animaux produits à partir d’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 du présent document.

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
Publication Date
21-Dec-2021
Withdrawal Date
29-Jun-2022
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
22-Dec-2021
Due Date
21-Jul-2020
Completion Date
22-Dec-2021

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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).
...

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