SIST EN 17547:2022
(Main)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)
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
Standards Content (Sample)
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 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.
Figure 1 — Flowchart for the determination of vitamins A, D and E
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 ≈ 850 g/kg.
5.3 Ethanol (C H OH), w = 950 ml/l, or equivalent industrial methylated spirit (ethanol denatured by
2 5
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 sulfide (Na S ⋅ 9 H O).
2 2
5.7 Sodium sulfide, alkali solution (see 11.1 observations).
Dissolve 2 000 g of potassium hydroxide (5.2) in 1 200 ml of water (5.1) until as much as possible of KOH
is dissolved. In parallel dissolve 224 g of sodium sulfide (5.6) in 800 ml of water (5.1) in ultrasonic bath.
Mix both solutions together and stir the mixture until the potassium hydroxide (5.2) is dissolved
completely.
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. 980 + 20 (by volume). The ratio of
the mixture must 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 document 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.
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 28 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 Overhead rotating shaker (for cold saponification).
6.3 Amber glassware (see observation 11.3).
6.3.1 Flat bottom - or conical flasks, 250 ml 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
Chromabond XTR is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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 Syringe filter, with a Nylon or PVDF membrane, 0,2 μm (or 0,45 µm) pore size or equivalent, i.e.
fully chemical compatibility with methanol and adaptable on 2-5ml syringes .
6.10 HPLC system semi-preparative, for the clean-up of vitamin D, consisting of:
6.10.1 HPLC pump, set to deliver a constant eluent volume flow rate of e.g. 2,5 ml/min.
6.10.2 HPLC injection device, injection volume of 500 µl.
6.10.3 HPLC semi-preparative normal phase column with guard column (see 9.7.2).
6.10.4 Column oven, set to provide a constant column temperature.
6.10.5 UV-Detector
6.11 HPLC-system for analytical separation, consisting of the following:
6.11.1 HPLC-pump, set to deliver a constant eluent volume flow rate of e.g. 1 ml/min.
6.11.2 HPLC injection devices, injection volume of 20 µl and 100 µl.
6.11.3 HPLC reversed-phase column, with guard column (see 9.8.1).
6.11.4 Column oven, set to provide a constant column temperature.
6.11.5 Detectors for UV- and fluorescent detection.
6.11.6 Integrator / data handling system.
6.12 UV (or UV-Visible) spectrophotometer, capable of measuring absorbance at the wavelengths
defined in 9.2.1.4, 9.2.2.4 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
document. 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.
Chromafil PET is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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 representative portion of the dry laboratory sample so that it passes through a sieve with 1 mm
apertures. Prevent to heat up.
Grinding of sample(s) with adequate particle size distribution (e.g. premixtures and concentrates) may
not be necessary if homogeneity is ensured.
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 must 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).
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 has to be calculated from exact concentration of working standard solution of vitamin
A (retinol) (9.2.1.3) determined according to 9.2.1.4.
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 concentration of vitamin A (retinol) in working standard solution has to be determined
according to 9.2.1.4.
The working standard solution of vitamin A (retinol) is stable for at least 2 months in dark at 4°C but the
real concentration shall be checked before use.
9.2.1.4 Standardization of the vitamin A (retinol) working standard solution (9.2.1.3)
The exact concentration of vitamin A (retinol) in working standard solution (9.2.1.3) is determined by
spectrometric measurement. Measure the UV spectrum of this solution against cyclohexane (5.12) in the
spectrophotometer (6.12) at the absorption maximum between 325 nm and 327 nm.
The vitamin A (retinol) concentration can be calculated with Formula (1).
33 333××AA33 333
wSTD wSTD
CA19,212× (1)
A wSTD
A 1 735
where
C is the vitamin A (retinol) concentration, in IU/ml;
A
33 333 is the concentration of vitamin A (retinol) corresponding to 10000 µg/ml, in IU/ml;
A is the absorbance of working standard solution (9.2.1.3);
wSTD
A is the absorption coefficient of vitamin A (retinol) in cyclohexane at 325 nm.
1%
NOTE The absorption coefficient of vitamin A (retinol) in cyclohexane A at 325 nm is 1 735, i.e.
1cm
33 333 IU/ml provide absorbance 1 735, so the absorbance of 7,5 IU/ml is 0,3904.
9.2.2 Vitamin E (α-tocopherol)
9.2.2.1 General
For preparation of vitamin E (α-tocopherol) standard solutions use DL-α-tocopherol (5.18.1).
NOTE It is also possible to use as standard DL-α-tocopherol acetate, see Annex B. Preparation from DL-α-
tocopherol is preferable because in this case is not necessary to apply saponificantion.
9.2.2.2 Stock standard solution of vitamin E (α-tocopherol)
Weigh 50 mg of vitamin E (DL-α-tocopherol) (5.18.1) to the nearest 0,1 mg, into a 100 ml graduated flask
(6.3.3). Dissolve in ethanol (5.11) and make up to the mark with the same solvent.
The nominal concentration of this solution is 500 μg vitamin E (α-tocopherol) per ml.
The exact concentration of vitamin E (α-tocopherol) in stock standard solution (9.2.2.2) has to be
calculated from exact concentration of working standard solution of vitamin E (α-tocopherol) (9.2.2.3).
The stock standard solution of vitamin E (α-tocopherol) in ethanol is stable at least for six months in dark
at 4 °C and can be used for preparation of working standard solution according to 9.2.2.3 during this
period.
9.2.2.3 Working standard solution of vitamin E (α-tocopherol) in ethanol
Pipette 10,0 ml of the vitamin E (α-tocopherol) stock standard solution (9.2.2.2) into a 100 ml graduated
flask and fill up to the mark with ethanol (5.11).
The nominal concentration of the working standard solution is 50 μg vitamin E (α-tocopherol) per ml.
===
The exact concentration of vitamin E (α-tocopherol) in working standard solution has to be determined
according to 9.2.2.4.
The working standard solution of vitamin E (α-tocopherol) is stable for at least 2 months in dark at 4 °C
but the real concentration shall be checked before use.
9.2.2.4 Standardization of the vitamin E (α-tocopherol) working standard solution in ethanol
(9.2.2.3)
The exact concentration of vitamin E (α-tocopherol) in working standard solution (9.2.2.3) is determined
by spectrometric measurement absorbance of working standard solution (9.2.2.3) in ethanol (5.11).
Measure the UV spectrum of this solution against ethanol (5.11) in the spectrophotometer (6.12) at the
absorption maximum between 250 nm and 320 nm. The absorption maximum shall be at 292 nm for
ethanol (5.11).
The vitamin E (α-tocopherol) content in ethanol (5.11) can be calculated with Formula (2).
10 000× A
wSTD
C 131, 9× A (2)
E wSTD
A
where
C is the vitamin E concentration in ethanol (5.11), in μg/ml;
E
10 000 is the concentration of vitamin E (α-tocopherol) corresponding to 10000 µg/ml, in μg/ml;
A is the absorbance of working standard solution (9.2.2.3);
wSTD
A is the absorption coefficient of vitamin E (α-tocopherol) in ethanol at 292 nm.
1%
NOTE The absorption coefficient of vitamin E (α-tocopherol) in ethanol (5.11) at 292 nm A is 75,8, i.e.
1cm
1 000 mg/100 ml provide absorbance 75,8, so the absorbance of 50 µg/ml is 0,379.
9.2.3 Vitamin D
9.2.3.1 Stock internal standard solution of vitamin D (ergocalciferol)
Weigh 50 mg vitamin D (ergocalciferol) (5.19.1) to the nearest 0,1 mg into a 100 ml graduated flask
(6.3.3), dissolve it in ethanol (5.11) and fill up to 100 ml with ethanol (5.11).
Nominal concentration of this solution is 20 000 IU of vitamin D (ergocalciferol) per ml.
The exact content of vitamin D (ergocalciferol) in stock internal standard solution has to be determined
according to 9.2.3.5.
The stock internal solution of vitamin D (ergocalciferol) is stable for at least one month in dark at 4 °C
and can be used for preparation of internal working standard solution I according to 9.2.3.3 during this
period.
9.2.3.2 Stock standard solution of vitamin D (cholecalciferol)
Weigh 50 mg vitamin D (cholecalciferol) (5.19.2) to the nearest 0,1 mg into a 100 ml graduated flask
(6.3.3), dissolve it in ethanol (5.11) and fill up to 100 ml with ethanol (5.11).
Nominal concentration of this solution is 20 000 IU of vitamin D (cholecalciferol) per ml.
The exact concentration of vitamin D (cholecalciferol) in stock standard solution has to be determined
according to 9.2.3.5.
The stock solution of vitamin D (cholecalciferol) is stable for at least one month in dark at 4 °C and can
be used for preparation of working standard solution I according to 9.2.3.3 during this period.
= =
9.2.3.3 Working standard solution I of the vitamin D (cholecalciferol) and internal standard D
3 2
(ergocalciferol)
Transfer by pipette 5,0 ml of stock standard solutions of vitamin D (cholecalciferol) (9.2.3.2) and 5,0 ml
of stock internal standard solutions of vitamin D (ergocalciferol) (9.2.3.1) in the same 100 ml graduated
flask (6.3.3) and fill up to 100 ml with methanol (5.10).
The nominal concentration of working solution I of each vitamin D form (vitamin D (cholecalciferol) and
vitamin D (ergocalciferol)) is 1 000 IU per ml.
This working standard solution has to be freshly prepared before use.
9.2.3.4 Working standard solution II of the vitamin D (cholecalciferol) and internal standard D
3 2
(ergocalciferol)
Pipette 1,0 ml of the working standard solution I (9.2.3.3) into 100 ml graduated flask (6.3.3), make up to
the mark with methanol (5.10) and mix.
The nominal concentration of working solution II of each vitamin D form (vitamin D (cholecalciferol)
and vitamin D (ergocalciferol)) is 10 IU per ml.
The exact concentration has to be calculated from exact concentration of stock internal standard solution
of vitamin D (ergocalciferol) (9.2.3.1) or stock standard solution D (cholecalciferol) (9.2.3.2).
2 3
This working standard solution II has to be freshly prepared before use.
9.2.3.5 Standardization of vitamins D (ergocalciferol) internal stock standard solution and D
2 3
(cholecalciferol) stock standard solution
Dilute 2 ml of the vitamin D (ergocalciferol) and D (cholecalciferol) stock standard solutions (9.2.3.1
2 3
and 9.2.3.2) in separate graduated flasks (6.3.3) with ethanol (5.11) to 100 ml and measure the UV-
spectrum from 220 nm to 320 nm against ethanol (5.11) on the spectrophotometer (6.12). The
absorption maximum should be at 263 nm – 265 nm.
The vitamin D (ergocalciferol) content in ethanol (5.11) can be calculated with Formula (3).
400 000× AA400 000×
STD STD
CA869,6× (3)
D STD
A 460
where
C is the vitamin D (ergocalciferol) concentration in ethanol (5.11), in IU /ml;
D2 2
400 000 is the concentration of vitamin D (ergocalciferol) corresponding to 10000 µg/ml, in
IU/ml;
A is the absorbance of stock standard solution (9.2.3.1) diluted according to 9.2.3.5;
STD
A is the absorption coefficient of vitamin D (ergocalciferol) in ethanol at 264 nm.
264 2
1%
NOTE 1 The absorption coefficient of vitamin D (ergocalciferol) in ethanol (5.11) at 264 nm A is 460, i.e.
1cm
1 000 mg/100 ml (= 400 000 IU/ml) provide absorbance 460, so the absorbance of 400 IU/ml is 0,460.
The vitamin D (cholecalciferol) concentration in ethanol (5.11) can be calculated with Formula (4).
===
400 000× AA400 000×
STD STD
CA824,7× (4)
D STD
A 485
where
C is the vitamin D (cholecalciferol) concentration in ethanol (5.11), in IU /ml;
D3 3
400 000 is the concentration of vitamin D (cholecalciferol) corresponding to 10000 µg/ml, in
IU/ml;
A is the absorbance of stock standard solution (9.2.3.1) diluted according to 9.2.3.5;
STD
A is the absorption coefficient of vitamin D (cholecalciferol) in ethanol at 264 nm.
264 3
1%
NOTE 2 The absorption coefficient of vitamin D (cholecalciferol) in ethanol (5.11) at 264 nm A is 485, i.e.
1cm
1 000 mg/100 ml (= 400 000 IU/ml) provide absorbance 485, so the absorbance of 400 IU/ml is 0,485.
9.3 Calibration
9.3.1 General
Preferably carry out the preparation of the calibration solutions and the feedstuff samples at the same
time. Nevertheless, the calibrations for vitamin A (retinol), vitamin E (α-tocopherol) and vitamin D
(cholecalciferol) can be used over a long time provided that the intermediate precision conditions in the
laboratory are stable. This shall be checked regularly by appropriate quality assurance procedures.
The preparation of calibration solutions containing all vitamins (retinol, ergocalciferol, cholecalciferol
and tocopherol) together is possible but the use of such calibration solutions is to be validated by the
user.
9.3.2 Calibration solutions and plotting of calibration graph for vitamins A (retinol) and E (α-
tocopherol)
9.3.2.1 Evaporation
Evaporate a defined volume stock standard solution of vitamin A (retinol) (9.2.1.2) and working standard
solution of vitamin A (retinol) (9.2.1.3) near to dryness under vacuum using a rotary vacuum evaporator
(6.6) at a temperature not exceeding 40 °C and 50 °C respectively. Restore atmospheric pressure by
admitting inert gas (5.9) finally. Remove the remaining solvent with a stream of nitrogen (5.9). Dissolve
the residue immediately in the same volume of methanol (5.10) taking for evaporation.
When using a nitrogen sample concentrator (6.7) for evaporation the solvent can be fully removed in one
step till the sample is dried. As described before dissolve the residue immediately in the same volume of
methanol (5.10) that was volume taking for evaporation.
9.3.2.2 Transfer different volumes of the stock standard solution and working standard solution of
vitamin A (retinol) (prepared according 9.2.1.2 or 9.2.1.3) and vitamin E (α-tocopherol) (9.2.2.2 or
9.2.2.3) into a series of 20 ml graduated flasks (6.3.3). Make up to the mark with methanol (5.10) and mix.
Table 1 provides examples of calibration solutions for determination of vitamins A and E.
The exact concentration of the calibration solutions should be calculated from concentration of working
standard solutions of vitamin A (retinol) (9.2.1.3) and vitamin E (α-tocopherol) (9.2.2.3).
===
Table 1 — Calibration solutions for vitamins A and E
Calibration Volume of vitamin A Nominal Volume of vitamin E Nominal
solution (retinol) working concentration of (α-tocopherol) concentration of
standard solution vitamin A (retinol) working standard vitamin E (α-
(prepared according solution (9.2.2.3) tocopherol)
[IU/ml]
to 9.2.1.3)
[ml] [μg/ml]
[ml]
0 0,0 0,000 0,0 0,0
1 0,3 0,113 0,2 0,5
3 0,8 0,300 0,8 2,0
4 1,6 0,600 1,6 4,0
5 4,0 1,500 3,0 7,5
Calibration Volume of vitamin A Nominal Volume of vitamin E Nominal
solution (retinol) stock concentration of (α-tocopherol) stock concentration of
standard solution in vitamin A (retinol) standard solution vitamin E (α-
methanol (9.2.1.2) (9.2.2.2) tocopherol)
[IU/ml]
[ml] [ml] [μg/ml]
6 0,8 3,0 0,6 15,0
7 1,6 6,00 1,2 30,0
8 4,8 18,00 2,4 60,0
NOTE The calibration curve is linear for vitamin A (retinol) in the range 0,1 IU/ml – 30 IU/ml and for vitamin E
(α-tocopherol) 0,5 μg/ml – 60 μg/ml but it is not necessary to prepare all concentrations in one calibration curve.
It depends on the expected concentration of the test solution (9.6).
Inject 20 μl of each calibration solution several times and determine the mean peak areas or the mean
peak heights.
Use a linear regression as mathematic model (y = ax + b), where x = exact content of vitamin in the
calibration solutions and y = the corresponding mean peak height or area.
9.3.3 Calibration solution and plotting of calibration graph for vitamins D and D
2 3
Transfer different volumes of working standard solutions I (9.2.3.3) and working standard solutions II
(9.2.3.4) into a series of 20 ml graduated flasks (6.3.3). Make up to the mark with methanol (5.10) and
mix. The exact concentration of the calibration solutions should be calculated from concentration of stock
standard solutions of vitamin D and stock internal standard solution of vitamin D (9.2.3.1 and 9.2.3.2).
3 2
Table 2 — Calibration solutions for vitamin D
Calibration Volume of working standard solution II Nominal concentration of vitamin D2 or D3
solution (9.2.3.4) (IU/ml) in the calibration solution
[ml]
0 0 0
1 2,00 1,00
2 6,00 3,00
3 12,0 6,00
Calibration Volume of working standard solution I Nominal concentration of vitamin D2 or D3
solution (9.2.3.3) (IU/ml) in the calibration solution
[ml]
4 0,2 10,0
5 0,4 20,0
6 1,0 50,0
7 1,5 75,0
8 2,0 100
Inject 100 μl of each calibration solution several times and determine the mean peak areas or the mean
peak heights.
Use a linear regression as mathematic model (y = ax + b), where x = exact content of vitamin in the
calibration solutions and y = the corresponding mean peak height or area.
9.4 Analysis of the sample
9.4.1 Preparation of test samples
Mix the grinded sample (8) thoroughly and depending on the vitamin A (retinol) and/or vitamin E (α-
tocopherol) and/or vitamin D content weigh a test portion of the sample 2 g to 30 g to the nearest 1 mg
of the sample (see observation 11.4) into a 500 ml flat bottom or conical flask (6.3.1).
Use vitamin D as internal standard (ISTD).
Dilute the vitamin D internal stock standard solution (9.2.3.1) so that 1 ml of this diluted solution
contains the same amount of internal standard (IU) as the expected vitamin D amount (IU) in the test
portion. Pipette 1,0 ml of this diluted solution of vitamin D into the flask with test portion.
Use the added units of ISTD as ISTD for recovery calculation according Formula (7) (10.3). Close the
spiked
saponification flask and wait for 10 min.
The addition of internal standard is unnecessary if only the vitamins A (retinol) and/or E (α-tocopherol)
are to be determined. In this case add 1 ml of ethanol (5.11) instead of diluted internal standard solution.
Nevertheless, the addition of internal standard does not affect the analysis of vitamin A (retinol) and/or
vitamin E (α-tocopherol).
Proceed with 9.4.2.1 or 9.4.2.2.
NOTE Annex A suggests examples for possible amounts of weighing combined with further aliquots and
dilutions depending on the declared content in order to reach a concentration in the test solution which is within
the calibration curve.
9.4.2 Saponification
9.4.2.1 Cold saponification
Add to the sample in conical or flat bottom flask approximately 1 g of ascorbic acid (5.4) and 100 mg BHT
(5.8). Then add successively with swirling accurate volume of 130,0 ml ethanol (5.3) and finally exactly
30,0 ml alkali sodium sulfide solution (5.7) and mix well. Remove the air in the flask with a stream of
nitrogen, close the flask and put it into overturning rotating stirrer (6.2) for at least 12 h (preferably
overnight) at the laboratory temperature.
9.4.2.2 Hot saponification
Add to the sample in conical or flat bottom flask approximately 1 g of ascorbic acid (5.4) and 100 mg BHT
(5.8). Then add successively with swirling accurate volumes of 130,0 ml ethanol (5.3) and finally exactly
30,0 ml alkali sodium sulfide solution (5.7) and mix well. Fit a condenser (6.3.2) to the flask and immerse
the flask in a water-bath with magnetic stirrer (6.1). Heat to boiling and allow refluxing for 30 min with
stirring under a slow stream of inert gas (5.9). After that time separate the flask from the condenser and
let the saponification solution cool to room temperature, e.g. in ice bath.
NOTE In exceptional cases some products can require divergent saponification conditions (see observation
11.4) or a longer saponification time (see observation 11.5).
9.5 Extraction of vitamin A (retinol), vitamin E (α-tocopherol) and vitamin D3
(cholecalciferol) by SPE
Be sure that the sample particles in the saponification solution (prepared according 9.4.2.1 or 9.4.2.2)
were settling down and the supernatant is clear as far as possible. Transfer 40,0 ml of the supernatant
into a 50 ml graduated flask (6.3.3). Fill up to the volume with ascorbic acid solution (5.5).
NOTE 1 The ascorbic acid solution (5.5) is to be prepared freshly just before use
If the solid part does not settle well centrifugation of a sufficiently large volume of the saponification
solution seems possible but this step is to be validated by the user.
Shake very thoroughly to form a visually homogeneous emulsion and transfer 15,0 ml of this solution
immediately to each of two SPE columns (6.5). One column is used for the determination of vitamin A
(retinol) and/or vitamin E (α-tocopherol), the other one for vitamin D (cholecalciferol).
NOTE 2 The emulsion tends to rapidly disintegrate. Therefore, it is very important to transfer the aliquot of the
well-shaken emulsion to the column immediately after the mixing.
Allow the solution to penetrate slowly into the column by gravity. After complete penetration of the
extract into the column wait for 15 min. After this time add cyclohexane (5.12) on the column till the
cyclohexane-front reach almost the outlet. Stop the elution flow with the valve and wait for 5 min. Open
the valve and apply further volumes of cyclohexane (5.12) onto the column and collect the eluate that
pass through the column by gravity into a 100 ml graduated flask (6.3.3). The flow rate should not be
faster than 1 drop/s. Stop eluting just before the defined volume (100 ml) is reached. Fill up to the volume
with cyclohexane (5.12).
NOTE 3 For samples with low content of vitamin D3 can be that the concentration in the eluate is too low for
further determination. The situation can be improved by using two 70 ml CHROMABOND columns and pooling of
the eluates of the two columns.
9.6 Preparation of the test solution for HPLC separation
9.6.1 Vitamin A (retinol) and vitamin E (α-tocopherol)
Evaporate a defined volume of the cyclohexane extract (9.5) near to dryness under vacuum using a rotary
vacuum evaporator (6.6) at a temperature not exceeding 40 °C and 50 °C respectively. Restore
atmospheric pressure by admitting inert gas (5.9) finally. Remove the remaining solvent with a stream of
nitrogen (5.9). Dissolve the residue immediatel
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