FprEN 17547

Animal feeding stuffs: Methods of sampling and analysis - Determination of vitamin A, E and D content - Method using solid phase extraction clean-up and High Performance Liquid Chromatography

This European Standard specifies a method for the determination of the content of the total vitamin A (retinol), vitamin E (alpha-tocopherol) and vitamin D (D2 ergocalciferol or D3 cholecalciferol) content in animal feed using solid phase extraction (SPE) clean-up and high performance liquid chromatography (HPLC).
The limit of quantification is XXXX IU vitamin A/kg (using UV-detection), XX IU vitamin A/kg (using fluorescence detection), XX mg vitamin E/kg (using UV-detection), XX mg vitamin E/kg (using
fluorescence detection), XX IU vitamin D/kg (using UV-detection) and XX IU vitamin D/kg (using fluorescence detection).

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 1 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.
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.
ANMERKUNG 2 Die Bestimmungsgrenzen sind Mindestgrenzen, die nicht im Rahmen der Validierungsstudie bestimmt wurden. Niedrigere Bestimmungsgrenzen sind möglich, müssen jedoch 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 et la chromatographie liquide à haute performance

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 1 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.
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).
NOTE 2 Les limites de quantification sont les limites minimales qui n’ont pas été déterminées lors de l’étude de validation. Des limites de quantification inférieures peuvent être obtenues, 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 in tekočinsko kromatografijo visoke ločljivosti

General Information

• Status: Not Published
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 3 - Feed additives and drugs
• Current Stage: 5060 - Closure of Vote - Formal Approval
• Due Date: 26-Aug-2021
• Completion Date: 26-Aug-2021
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/523 - Mandate for standardisation addressed to CEN for methods of analysis in the field of animal nutrition - Part III
• Ref Project: kSIST FprEN 17547:2021 - Animal feeding stuffs: Methods of sampling and analysis - Determination of vitamin A, E and D content - Method using solid phase extraction clean-up and High Performance Liquid Chromatography

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN 17547:2020
01-oktober-2020
Krma: metode vzorčenja in analize - Določevanje vitaminov A, E in D - Metoda z
ekstrakcijo na trdno fazo in tekočinsko kromatografijo visoke ločljivosti

Animal feeding stuffs: Methods of sampling and analysis - Determination of vitamin A, E

and D content - Method using solid phase extraction clean-up and High Performance

Liquid Chromatography

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 et la chromatographie liquide à haute performance
Ta slovenski standard je istoveten z: prEN 17547
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 17547:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 17547:2020
DRAFT
EUROPEAN STANDARD
prEN 17547
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2020
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 clean-up and High
Performance Liquid Chromatography

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 et la chromatographie liquide à haute Festphasenextraktion und Hochleistungs-

performance Flüssigchromatographie

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 17547:2020 E

worldwide for CEN national Members.
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Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 Principle ............................................................................................................................................................. 7

5 Reagents and materials ................................................................................................................................. 9

6 Apparatus ........................................................................................................................................................ 10

7 Sampling .......................................................................................................................................................... 11

8 Sample preparation ..................................................................................................................................... 11

9 Procedure........................................................................................................................................................ 11

9.1 General ............................................................................................................................................................. 11

9.2 Preparation and standardization of standard solutions ............................................................... 11

9.2.1 Vitamin A (retinol) ....................................................................................................................................... 11

9.2.2 Vitamin E (α-tocopherol)........................................................................................................................... 13

9.2.3 Vitamin D ......................................................................................................................................................... 14

9.3 Calibration ...................................................................................................................................................... 15

9.3.1 General ............................................................................................................................................................. 15

9.3.2 Calibration solutions and plotting of calibration graph for vitamins A (retinol) and E

(α-tocopherol) ............................................................................................................................................... 15

9.3.3 Calibration solution and plotting of calibration graph for vitamins D2 and D3 ................... 16

9.4 Analysis of the sample ................................................................................................................................ 17

9.4.1 Preparation of test samples ..................................................................................................................... 17

9.4.2 Saponification ................................................................................................................................................ 18

9.5 Extraction of vitamin A (retinol), vitamin E (α-tocopherol) and vitamin D

(cholecalciferol) by SPE ............................................................................................................................. 18

9.6 Preparation of the test solution for HPLC separation ..................................................................... 18

9.6.1 Vitamin A (retinol) and vitamin E (α-tocopherol) ........................................................................... 18

9.6.2 Vitamin D (cholecalciferol) ..................................................................................................................... 19

9.7 Semi-preparative HPLC clean-up for determination of vitamin D ........................................... 19

9.7.1 Determination of retention time window for vitamin D /D fraction ...................................... 19

2 3

9.7.2 Conditions for semi-preparative HPLC clean-up .............................................................................. 19

9.7.3 Collection of the vitamin D fraction ....................................................................................................... 20

9.8 High-performance liquid chromatography ........................................................................................ 20

9.8.1 Conditions for analytical HPLC ................................................................................................................ 20

9.8.2 Determination ............................................................................................................................................... 21

10 Expression of results ................................................................................................................................... 21

10.1 Result calculation for vitamin A (retinol) ........................................................................................... 21

10.2 Result calculation for vitamin E (α-tocopherol) ............................................................................... 22

10.3 Result calculation for vitamin D (cholecalciferol) .......................................................................... 22

10.4 Recovery of vitamin D................................................................................................................................. 23

11 Observations .................................................................................................................................................. 24

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12 Precision .......................................................................................................................................................... 24

12.1 Interlaboratory study ................................................................................................................................. 24

12.2 Repeatability .................................................................................................................................................. 25

12.3 Reproducibility .............................................................................................................................................. 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 ................................................................................................................................................................. 38

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European foreword

This document (prEN 17547:2020) 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 standardization request given to CEN by the European

Commission and the European Free Trade Association.
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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.
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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) content in animal feed using solid phase

extraction (SPE) clean-up and high-performance liquid chromatography (HPLC).

NOTE 1 The procedure enables also determination of vitamin D 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.

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.

NOTE 2 The limits of quantification are minimum limits which were not determined within the validation study.

Lower limits of quantification are possible but is 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)

EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/
3.1
vitamin A (retinol) content

content of all-trans-retinol and cis-isomers determined in accordance with this standard

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.

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3.2
vitamin E (α-tocopherol) content
content of α-tocopherol determined in accordance to this document

Note 1 to entry: The content of vitamin E (α-tocopherol) could 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 D3 (cholecalciferol) content
the content of cholecalciferol determined in accordance with this standard

Note 1 to entry: The content of vitamin D 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 separation between the vitamin D and vitamin D . Quantification of vitamin D is performed by

2 3 3

external standard calibration taking into account the recovery of the internal standard.

NOTE Figure 1 contains a flowchart for the determination of vitamins A, D and E.
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Figure 1 — Flowchart for the determination of vitamins A, D and E
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5 Reagents and materials
Use only reagents of recognized analytical grade.
5.1 Water, complying with at least grade 3 in accordance with EN ISO 3696:1995.
5.2 Potassium hydroxide (KOH), w ≅ 85 %.

5.3 Ethanol (C H OH), w = 95 % (by volume), or equivalent industrial methylated spirit (ethanol

2 5
denatured by methanol or hexane).
5.4 Ascorbic acid (C H O ).
6 8 6
5.5 Ascorbic acid, solution, ρ = 200 g/l.
5.6 Sodium sulphide (Na S × 9 H O).
2 2
5.7 Sodium sulphide, alkali solution (see 11.1 observations).

Dissolve 2 000 g of potassium hydroxide (5.2) in 1 200 ml of water (5.1). Dissolve 224 g of sodium

sulphide (5.6) in 800 ml of water (5.1) in ultrasonic bath. Mix both solutions together.

NOTE Dissolution of KOH is a slow procedure. It is necessary to mix the solution until as much as possible of

KOH is dissolved. After addition of sodium sulphide solution, the residuum of KOH is dissolved.

5.8 2,6-Di-tert-butyl-4-methylphenol (BHT), (see 11.2 observations).
5.9 Inert gas, e.g. nitrogen.
5.10 Methanol (CH OH), HPLC grade.
5.11 Ethanol (CH CH OH), HPLC grade.
3 2
5.12 Cyclohexane (C H ), HPLC grade.
6 12
5.13 2-Propanol (C H OH), HPLC grade.
3 7
5.14 n-Hexane (C H ), HPLC grade.
6 14
5.15 Mobile phase for semi-preparative HPLC-clean-up of vitamin D .

Mixture of n-hexane (5.14) and propanol (5.13) in the proportions e.g. 98 + 2 (by volume). The ratio of

the mixture shall be adapted to the HPLC-column employed. If necessary, filter through a membrane filter

(6.8).
5.16 Mobile phase for analytical HPLC.

Mix together methanol (5.10) and water (5.1) in the proportions 980 + 20 (by volume). The exact ratio

will be determined by the characteristics of the column employed. The use of other mobile phase

composition is allowed provided the separation of vitamins according the scope of this standard is

possible. If necessary, filter through a membrane filter (6.8).
5.17 Vitamin A standard substances.

5.17.1 All-trans-retinol acetate (C H O ), CAS = 127-47-9, MW = 328,49 g/mol, extra pure, of

22 32 2
certified activity, e.g. 2,80 × 10 IU/g.

5.17.2 All-trans-retinol palmitate (C H O ), CAS = 79-81-2, MW = 524,86 g/mol, extra pure, of

36 60 2
certified activity, e.g. 1,80 × 10 IU/g.
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5.18 Vitamin E standard substance.

5.18.1 DL-α-tocopherol (C H O ), CAS = 10191-41-0, MW = 430,72 g/mol, extra pure, of certified

29 50 2
purity.
5.19 Vitamin D standard substances.

5.19.1 Vitamin D (ergocalciferol; C H O), CAS = 50-14-6, MW = 384,62 g/mol, extra pure, of certified

2 27 44
activity, e.g. 40 × 10 IU/g.

5.19.2 Vitamin D (cholecalciferol; C H O), CAS = 67-97-0; MW = 384,62 g/mol, extra pure, of certified

3 27 44
activity, e.g. 40 × 10 IU/g.
5.20 Celite for SPE column

Base material coarse-grained kieselguhr (also known as diatomaceous earth, hydromatrix, celite);

particle size: max. 10 % < 100 μm, max. 90 % < 500 μm, max. 5 % > 800 μm; large pore size, high pore

volume, constantly high batch-to-batch quality.
6 Apparatus
Usual laboratory equipment and, in particular, the following:

6.1 Boiling water bath with magnetic stirrer or electrical heating device with stirring (for hot

saponification).
6.2 Overturning rotating stirrer (for cold saponification).
6.3 Amber glassware (see observation 11.3).
6.3.1 Flat bottom - or conical flasks, 250 and 500 ml, with ground-glass socket.

6.3.2 Allihn condenser, jacket length 300 mm, with ground-glass joint, with adapter for gas feed pipe.

6.3.3 Graduated flasks with ground-glass stoppers, narrow-necked, 20 ml, 25 ml, 50 ml and 100 ml.

6.3.4 Pear shaped flask with ground-glass stoppers, 100 ml.
6.4 Vials, suitable for sample concentrator.

6.5 Column for SPE, filled with celite (e.g. Chromabond XTR, 70 ml volume) which is able to adsorb

the water phase from the saponification solution (9.4.2) and release the vitamins A, E and D completely

by elution with organic solvents. The column shall have a capacity of not less than 20 ml aqueous solution

and possibly closed by a valve at the outlet.
6.6 Rotary vacuum evaporator, with water bath at 40 °C.
6.7 Sample nitrogen concentrator, heated to 50 °C.

6.8 Membrane filter, compatible with methanol, 0,45 μm pore size; e.g. Chromafil PET - 45/15 MS or

suitable filter with smaller pore size.
6.9 HPLC system semi-preparative, for the clean-up of vitamin D, consisting of:

6.9.1 HPLC pump, set to deliver a constant eluent volume flow rate of e.g. 2,5 ml/min.

6.9.2 HPLC injection device, injection volume of 500 µl.
6.9.3 HPLC semi-preparative normal phase column with guard column (see 9.7.2).
6.9.4 Column oven, set to provide a constant column temperature.
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6.9.5 UV-Detector
6.10 HPLC-system for analytical separation, consisting of the following:

6.10.1 HPLC-pump, set to deliver a constant eluent volume flow rate of e.g. 1 ml/min.

6.10.2 HPLC injection devices, injection volume of 20 µl and 100 µl.
6.10.3 HPLC reversed-phase column, with guard column (see 9.8.1).
6.10.4 Column oven, set to provide a constant column temperature.
6.10.5 Detectors for UV- and fluorescent detection.
6.10.6 Integrator / data handling system.

6.11 UV (or UV-Visible) spectrophotometer, capable of measuring absorbance at the wavelengths

defined in 9.2.1.3, 9.2.2.3 and 9.2.3.5, equipped with cells of 10 mm path length.

7 Sampling

It is important that the laboratory receive a sample which is truly representative and has not been

damaged or changed during transport or storage. Sampling is not part of the method specified in this

European Standard. A recommended sampling method is given in EN ISO 6497 [2].

Store the sample in such a way that deterioration and change in its composition are prevented.

8 Sample preparation

Samples are grinded at the day of analysis as recommended in the guidelines for sample preparation as

in EN ISO 6498.

Grind a portion of the well-mixed dry laboratory sample so that it passes through a sieve with 1 mm

apertures. Prevent to heat up.

Do not grind the sample(s) if the particle size distribution is adequate (e.g. premixtures and

concentrates).
Semi-moist pet foods (canned pet foods) can be homogenized by mincing.

Samples can be ground before the day of analysis. In this case the storage conditions shall prevent any

degradation, e.g. freeze the ground sample and defrost it in a fridge a night before analysis.

9 Procedure
9.1 General

Because of the sensitivity of vitamin A, E and D to UV radiation and air, perform all operations away from

natural and strong fluorescent light and as rapidly as is consistent with accurate working. Use amber

glassware (6.3) where possible (see observation 11.3).
9.2 Preparation and standardization of standard solutions
9.2.1 Vitamin A (retinol)
9.2.1.1 General

For preparation of vitamin A (retinol) standard solutions use all-trans-retinol acetate (5.17.1) or all-

trans-retinol palmitate (5.17.2).
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NOTE Standard substance of retinol itself is less stable then retinol palmitate or retinol acetate and therefor it

is usual to use these esters for preparation of standard solution of vitamin A. Nevertheless, use of standard

substance retinol is also possible.
9.2.1.2 Stock standard solution of vitamin A (retinol)

Weigh to the nearest 0,1 mg an amount of vitamin A (retinol acetate) (5.17.1) or vitamin A (retinol

palmitate) (5.17.2) containing approximately 100 000 IU of vitamin A (retinol) into a 250 ml flat bottom

or conical flask (6.3.1) and continue with saponification according to 9.4.2.1 or 9.4.2.2 and extraction

according to 9.5.

Collect the eluate from the SPE column (6.5) in a 100 ml graduated flask (6.3.3) and fill up to the mark

with cyclohexane (5.12).

The nominal concentration of stock standard solution of vitamin A (retinol) in cyclohexane is

approximately 75 IU per ml.

The exact content shall be calculated from exact concentration of working standard solution of vitamin A

(retinol) (9.2.1.2) determined according to 9.2.1.3.

The stock standard solution of vitamin A (retinol) is stable for 6 months in dark at 4°C and can be used

for preparation of working standard solution according to 9.2.1.2 during this period.

9.2.1.3 Working standard solution of retinol

Pipette 10,0 ml of the vitamin A (retinol) stock standard solution (9.2.1.2) into a 100 ml graduated flask

and fill up to the mark with cyclohexane (5.12).

The nominal concentration of the working standard solution is 7,5 IU vitamin A (retinol) per ml.

The exact content of vitamin A (retinol) in working standard solution shall be determined according to

9.2.1.3.

The working standard solution of vitamin A (retinol) is stable for at least 2 months in dark at 4°C but the

real shall be checked before use.

9.2.1.4 Standardization of the vitamin A (retinol) working standard solution (9.2.1.2)

The exact content of vitamin A (retinol) in working standard solution (9.2.1.2) is determined by

spectrometric measurement. Measure the UV spectrum of this solution against cyclohexane (5.12) in the

spectrophotometer (6.11) at the absorption maximum between 325 nm and 327 nm.
The vitamin A (retinol) content can be calculated with Formula (1).
33 333××AA33 333
wSTD wSTD
CA19,212× (1)
A wSTD
A 1 735
325
where
C is the vitamin A (retinol) content, in IU/ml;
33 333 is the concentration of retinol corresponding to 1 % solution, in IU/ml;
A is the absorption of working standard solution (9.2.1.3);
wSTD
A is the absorption coefficient of retinol in cyclohexane at 325 nm.
325

NOTE The absorption coefficient of vitamin A (retinol) in cyclohexane 𝐴𝐴 at 325 nm is 1 735, i.e.

1𝑐𝑐𝑐𝑐
33 333 IU/ml provide absorption 1 734, so the absorption of 7,5 IU/ml is 0,3904.
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