Animal feeding stuffs: Methods of sampling and analysis - Determination of vitamin A, E and D content - Method using solid phase extraction (SPE) clean-up and high-performance liquid chromatography (HPLC)

This document specifies a method for the determination of the content of the total vitamin A (retinol), vitamin E (α-tocopherol) and vitamin D3 (cholecalciferol) in animal feed using solid phase extraction (SPE) clean-up and high-performance liquid chromatography (HPLC).
NOTE   The procedure also enables determination of vitamin D2 but with the use of another internal standard. The method is fully validated only for vitamin D3.
The method has been successfully tested in collaborative trial for complete feed for broilers, pigs, and turkey, for premixture for broilers and piglets, for complementary feed for cows and mineral feed within the following ranges:
•   vitamin A: 4 365 IU/kg - 4 118 352 IU/kg;
•   vitamin E: 22 mg/kg - 13 800 mg/kg;
•   vitamin D3: 1 668 IU/kg - 1 638 150 IU/kg.
The limits of quantification were not determined within the validation study. Quantification limits of 1 100 IU for vitamin A/kg (using UV-detection), 4 mg for vitamin E/kg (using UV-detection), 2 mg for vitamin E/kg (using fluorescence detection) and 2 000 IU for vitamin D/kg (using UV-detection) should be normally achieved. Lower limits are possible provided they are validated by the user.

Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung des Gehalts an Vitamin A, E und D - Verfahren mittels Reinigung durch Festphasenextraktion und Hochleistungs-Flüssigchromatographie

Dieses Dokument legt ein Verfahren zur Bestimmung des Gesamtgehalts an Vitamin A (Retinol), Vitamin E (α Tocopherol) und Vitamin D3 (Cholecalciferol) in Futtermitteln mittels Reinigung durch Festphasenextraktion (SPE; en: solid phase extraction) und Hochleistungs-Flüssigchromatographie (HPLC; en: high performance liquid chromatography) fest.
ANMERKUNG Das Verfahren ermöglicht auch die Bestimmung von Vitamin D2, jedoch unter Verwendung eines anderen internen Standards. Das Verfahren ist nur für Vitamin D3 vollständig validiert.
Das Verfahren wurde in einem Ringversuch für Alleinfuttermittel für Hähnchen, Schweine und Puten, für Vormischungen für Hähnchen und für Ferkel, für Ergänzungsfuttermittel für Kühe sowie für mineralische Futtermittel innerhalb folgender Bereiche erfolgreich geprüft:
- Vitamin A: 4 365 IE/kg – 4 118 352 IE/kg;
- Vitamin E: 22 mg/kg – 13 800 mg/kg
- Vitamin D3: 1 668 IE/kg – 1 638 150 IE/kg.
Die Bestimmungsgrenzen wurden nicht im Rahmen der Validierungsstudie bestimmt. Üblicherweise sollten Bestimmungsgrenzen von 1 100 IE für Vitamin A/kg (mittels UV Detektion), 4 mg für Vitamin E/kg (mittels UV Detektion), 2 mg für Vitamin E/kg (mittels Fluoreszenzdetektion) und 2 000 IE für Vitamin D/kg (mittels UV Detektion) erreicht werden. Niedrigere Grenzen sind möglich, sofern sie vom Anwender validiert werden.

Aliments des animaux - Méthodes d'échantillonnage et d'analyse - Détermination de la teneur en vitamines A, E et D - Méthode utilisant la purification par extraction en phase solide (SPE) et la chromatographie liquide à haute performance (CLHP)

Le présent document spécifie une méthode de détermination de la teneur totale en vitamine A (rétinol), vitamine E (α-tocophérol) et vitamine D3 (cholécalciférol) dans les aliments pour animaux à l’aide d’une purification par extraction en phase solide (SPE) et d’une chromatographie liquide à haute performance (CLHP).
NOTE   Le mode opératoire permet également de déterminer la teneur en vitamine D2 mais en utilisant un autre étalon interne. La méthode est intégralement validée pour la vitamine D3 uniquement.
La méthode a été soumise à essai avec succès lors d’un essai interlaboratoires effectué sur un aliment complet pour poulets, porcs et dindes, sur un prémélange pour poulets et porcelets, sur un aliment complémentaire pour vaches ainsi que sur un aliment minéral dans les gammes suivantes :
•   vitamine A : 4 365 UI/kg - 4 118 352 UI/kg ;
•   vitamine E : 22 mg/kg - 13 800 mg/kg ;
•   vitamine D3 : 1 668 UI/kg - 1 638 150 UI/kg.
Les limites de quantification n’ont pas été déterminées lors de l’étude de validation. Il convient normalement d’atteindre des limites de quantification de 1 100 UI/kg pour la vitamine A (par détection UV), 4 mg/kg pour la vitamine E (par détection UV), 2 mg/kg pour la vitamine E (par détection fluorimétrique) et 2 000 UI/kg pour la vitamine D (par détection UV). Des limites inférieures sont envisageables, à condition qu’elles soient validées par l’utilisateur.

Krma: metode vzorčenja in analize - Določevanje vitaminov A, E in D - Metoda z ekstrakcijo na trdno fazo (SPE) in tekočinsko kromatografijo visoke ločljivosti (HPLC)

Ta evropski standard določa metodo za določevanje vsebnosti vitamina A (retinol), vitamina E (alfa tokoferol) in vitamina D (D2 ergokalciferol ali D3 holekalciferol) v krmi z ekstrakcijo na trdno fazo (SPE) in tekočinsko kromatografijo visoke ločljivosti (HPLC).
Mejne vrednosti kvantifikacije so: XXXX IU vitamina A/kg (z detekcijo UV), XX IU vitamina A/kg (s fluorescenčno detekcijo), XX mg vitamina E/kg (z detekcijo UV), XX mg vitamina E/kg (s
fluorescenčno detekcijo), XX IU vitamina D/kg (z detekcijo UV) in XX IU vitamina D/kg (s fluorescenčno detekcijo).

General Information

Status
Published
Publication Date
09-Nov-2021
Withdrawal Date
30-May-2022
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
10-Nov-2021
Due Date
07-Jan-2021
Completion Date
10-Nov-2021

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SLOVENSKI STANDARD
01-januar-2022
Krma: metode vzorčenja in analize - Določevanje vitaminov A, E in D - Metoda z
ekstrakcijo na trdno fazo (SPE) in tekočinsko kromatografijo visoke ločljivosti
(HPLC)
Animal feeding stuffs: Methods of sampling and analysis - Determination of vitamin A, E
and D content - Method using solid phase extraction (SPE) clean-up and high-
performance liquid chromatography (HPLC)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung des Gehalts an
Vitamin A, E und D - Verfahren mittels Reinigung durch Festphasenextraktion und
Hochleistungs-Flüssigchromatographie
Aliments des animaux - Méthodes d'échantillonnage et d'analyse - Détermination de la
teneur en vitamines A, E et D - Méthode utilisant la purification par extraction en phase
solide (SPE) et la chromatographie liquide à haute performance (CLHP)
Ta slovenski standard je istoveten z: EN 17547:2021
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17547
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of vitamin A, E and D content - Method
using solid phase extraction (SPE) clean-up and high-
performance liquid chromatography (HPLC)
Aliments des animaux - Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Détermination de la teneur en vitamines A, Untersuchungsverfahren - Bestimmung des Gehalts an
E et D - Méthode utilisant la purification par extraction Vitamin A, E und D - Verfahren mittels Reinigung durch
en phase solide (SPE) et la chromatographie liquide à Festphasenextraktion und Hochleistungs-
haute performance (CLHP) Flüssigchromatographie
This European Standard was approved by CEN on 27 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 17547:2021 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 . 6
5 Reagents and materials . 8
6 Apparatus . 9
7 Sampling . 10
8 Sample preparation . 11
9 Procedure . 11
10 Expression of results . 21
11 Observations . 24
12 Precision . 25
13 Test report . 26
Annex A (informative) Examples of combinations of weighing, aliquot and dilution to reach
concentrations within the calibration curve . 27
Annex B (informative)  Preparation of stock standard solution of vitamin E (α-tocopherol)
from α-tocopherol acetate . 30
B.1 General. 30
B.2 Reagents . 30
B.3 Preparation of stock standard . 30
B.4 Standardization of the vitamin E (α-tocopherol) stock standard solution in
cyclohexane . 30
B.5 Calibration solutions and plotting of calibration graph for vitamins A (retinol) and E
(α-tocopherol) . 31
Annex C (informative) Results of the interlaboratory study . 32
Bibliography . 37

European foreword
This document (EN 17547:2021) 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 May 2022, and conflicting national standards shall be
withdrawn at the latest by May 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
WARNING — The method described in this document implies the use of reagents that pose a hazard to
health. The standard does not claim to address all associated safety problems. It is the responsibility of
the user of this document to take appropriate measures for the health and safety protection of the
personnel prior to use of the standard and to ensure that regulatory and legal requirements are complied
with.
1 Scope
This document specifies a method for the determination of the content of the total vitamin A (retinol),
vitamin E (α-tocopherol) and vitamin D (cholecalciferol) in animal feed using solid phase extraction
(SPE) clean-up and high-performance liquid chromatography (HPLC).
NOTE The procedure also enables determination of vitamin D2 but with the use of another internal standard.
The method is fully validated only for vitamin D .
The method has been successfully tested in collaborative trial for complete feed for broilers, pigs, and
turkey, for premixture for broilers and piglets, for complementary feed for cows and mineral feed within
the following ranges:
• vitamin A: 4 365 IU/kg – 4 118 352 IU/kg;
• vitamin E: 22 mg/kg – 13 800 mg/kg;
• vitamin D : 1 668 IU/kg – 1 638 150 IU/kg.
The limits of quantification were not determined within the validation study. Quantification limits of
1 100 IU for vitamin A/kg (using UV-detection), 4 mg for vitamin E/kg (using UV-detection), 2 mg for
vitamin E/kg (using fluorescence detection) and 2 000 IU for vitamin D/kg (using UV-detection) should
be normally achieved. Lower limits are possible provided they are validated by the user.
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 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
3.1
vitamin A content
retinol
content of all-trans- and cis-isomers of retinol determined in accordance with this document
Note 1 to entry: The vitamin A (retinol) content is expressed in International Units per kilogram (IU/kg).
Note 2 to entry: 1 IU of vitamin A (retinol) is equal to 0,300 µg of all-trans-retinol or 0,344 µg all-trans-retinol
acetate or 0,546 µg all-trans-retinol palmitate or 0,359 µg all-trans-retinol propionate.
3.2
vitamin E content
α-tocopherol
content of α-tocopherol determined in accordance with this document
Note 1 to entry: The content of vitamin E (α-tocopherol) can be also expressed as mg α-tocopherol acetate per kg.
Note 2 to entry: 1 mg vitamin E (α-tocopherol acetate) corresponds to 0,91 mg vitamin E (α-tocopherol).
Note 3 to entry: In samples can also be present β-, γ-, δ-tocopherol and α-, β-, γ-, δ-tocotrienol. This method uses
reverse phase separation which does not separate individual forms of tocopherol. Therefore, the content of
vitamin E expressed as α-tocopherol or α-tocopherol acetate includes all forms without taking into account
differences in vitamin activities and the respective proportions of each form. Using a normal phase-column the
separation of α-, β-, γ- and δ-tocopherol is possible (see observation 11.6).
3.3
vitamin D content
cholecalciferol
the content of cholecalciferol determined in accordance with this document
Note 1 to entry: The content of vitamin D3 is expressed in International Units per kg (IU/kg). 1 IU corresponds to
an activity of 0,025 µg vitamin D (cholecalciferol).
Note 2 to entry: For feeding stuffs, only vitamin D3 is authorized as feed additive pursuant to Regulation
(EC) No 1831/2003 [1]. Addition of vitamin D2 is not allowed. Therefore, the vitamin D2 can be used as internal
standard.
Note 3 to entry: For accurate calculation of the results it is important that the sample does not contain any other
vitamin D2 than that added as internal standard.
4 Principle
The sample is saponified with ethanolic potassium hydroxide solution. In case that vitamin D
(cholecalciferol) is to be determined the internal standard is added before saponification. The vitamins
are extracted and purified by SPE column eluting with cyclohexane. The cyclohexane is removed by
evaporation and the residue is dissolved in methanol (for determination of vitamin A (retinol) and
vitamin E (α-tocopherol)) or in n-hexane (for determination of vitamin D (cholecalciferol)).
The vitamin A (retinol) and vitamin E (α-tocopherol) concentrations in the methanolic extract are
determined by reversed-phase liquid chromatography using external calibration and HPLC conditions
that give a single peak for all retinol isomers as well as for all tocopherols.
The n-hexane extract for vitamin D determination is purified by semi-preparative normal-phase HPLC
on silica gel. The purified extract is separated by reversed phase HPLC using conditions that give a
baseline separatio
...

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