Animal feeding stuffs — Determination of vitamin E content — Method using high-performance liquid chromatography

This International Standard specifies a method for the determination of the vitamin E (DL-_-tocopherol) content of animal feeding stuffs and pet foods using high performance liquid chromatography.

Aliments des animaux — Détermination de la teneur en vitamine E — Méthode par chromatographie liquide à haute performance

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ISO 6867:2000 - Animal feeding stuffs -- Determination of vitamin E content -- Method using high-performance liquid chromatography
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First edition
Animal feeding stuffs — Determination of
vitamin E content — Method using high-
performance liquid chromatography
Aliments des animaux — Détermination de la teneur en vitamine E —
Méthode par chromatographie liquide à haute performance
Reference number
ISO 6867:2000(E)
ISO 2000

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ISO 6867:2000(E)
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ISO 6867:2000(E)
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO
member bodies). The work of preparing International Standards is normally carried out through ISO technical
committees. Each member body interested in a subject for which a technical committee has been established has
the right to be represented on that committee. International organizations, governmental and non-governmental, in
liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical
Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 6867 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee
SC 10, Animal feeding stuffs.
Annex A of this International Standard is for information only.
© ISO 2000 – All rights reserved iii

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Animal feeding stuffs — Determination of vitamin E content —
Method using high-performance liquid chromatography
1 Scope
This International Standard specifies a method for the determination of the vitamin E (DL-�-tocopherol) content of
animal feeding stuffs and pet foods using high performance liquid chromatography.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 3696:1987, Water for analytical laboratory use — Specifications and test methods.
ISO 6498, Animal feeding stuffs — Preparation of test samples.
A test portion of the sample is saponified with ethanolic potassium hydroxide solution and the vitamin E is extracted
into light petroleum. The light petroleum is removed by evaporation and the residue is dissolved in hexane. The
vitamin E concentration in the hexane extract is determined by normal-phase liquid chromatography using
conditions that separate DL-�-tocopherol from other tocopherols.
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise stated.
4.1 Water, complying with at least grade 3 in accordance with ISO 3696.
4.2 Potassium hydroxide solution.
Dissolve 500 g of potassium hydroxide in water (4.1) and dilute to 1 litre.
4.3 Ethanol, w(C H OH) = 95 % (by volume), or equivalent industrial methylated spirit.
2 5
4.4 Hexane, HPLC grade.
4.5 Light petroleum, boiling range 40 °Cto60 °C; the residue on evaporation shall be less than 20 mg/l.
4.6 Vitamin E standard substance: DL-�-tocopherol, minimum purity not less than 96,0 %.
The purity of the standard substance should be checked spectrophotometrically (see 8.5.2).
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ISO 6867:2000(E)
4.7 1,4-Dioxan, HPLC grade.
4.8 Sodium sulfate (Na SO ), anhydrous.
2 4
4.9 Sodium ascorbate solution, �=100g/l.
4.10 Inert gas,e.g.nitrogen.
4.11 Mobile phase for liquid chromatography.
Mix 30 ml 1,4-dioxan (4.7) with 970 ml hexane (4.4).
Filter through a membrane filter (5.5) before use.
4.12 Ethanol, w(C H OH) = 96 % (by volume).
2 5
4.13 Methanol (CH OH), HPLC grade.
5 Apparatus
Using laboratory apparatus and, in particular, the following.
5.1 High-performance liquid chromatograph, consisting of the following.
5.1.1 Pump, set to deliver a constant eluent volume flow rate of 1,5 ml/min.
5.1.2 HPLC injection device.
5.1.3 Column, length 250 mm, internal diameter 4,6 mm, packed with a stationary phase consisting of silica.
A column with at least 5 000 theoretical plates and a k� value of 0,8 m, both with respect to DL-�-tocopherol, has
been found to be satisfactory. The particle size should not be smaller than 5µm and not greater than 10µm. Other
systems may be used provided that a satisfactory separation of vitamin E from other co-extractives is achieved.
5.1.4 Detector, allowing the measurement of fluorescence emitted at a wavelength of 326 nm when the column
eluent is irradiated with ultraviolet light at a wavelength of 293 nm, with integrator/recorder.
5.2 Boiling water bath.
5.3 Rotary vacuum evaporator, with water bath at 40 °C.
5.4 Extraction apparatus (see Figure 1) consisting of the following:
� a cylinder of 1 litre capacity fitted with a ground glass neck and stopper;
� a ground glass joint, fitting the cylinder and equipped with an adjustable tube passing through the centre; and
� a side-arm.
The adjustable tube should have a U-shaped lower end and a jet at the opposite end so that the upper liquid layer
in the cylinder may be transferred to a separating funnel of 1 litre capacity.
Other extraction equipment such as conical flasks and separating funnels may be used in place of the apparatus
shown in Figure 1, provided that satisfactory recoveries of vitamin E are achieved.
5.5 Membrane filter, 0,45 m pore size, for filtration of mobile phase (4.11) and sample test solutions.
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ISO 6867:2000(E)
5.6 Grinding apparatus, capable of grinding the sample so that it passes through a sieve with 1 mm apertures.
5.7 UV (or UV/Visible) spectrometer, capable of measuring absorbance at the wavelengths defined in 8.5.2,
equipped with quartz cells of 10 mm path length.
1 Cylinder, of capacity 1 litre, with ground-glass neck 5 Bottle, of capacity 1 litre, with ground-glass joint
2 Light petroleum layer 6 Side-arm
3 Aqueous layer + saponified feed 7 Adjustable tube
Figure 1 — Example of extraction apparatus
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ISO 6867:2000(E)
6 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 International Standard. A recommended sampling method is
Store the sample in such a way that deterioration and change in its composition are prevented.
7 Preparation of test samples
Prepare the test sample in accordance with ISO 6498.
Just prior to starting the analysis, grind a portion of the well-mixed laboratory sample so that it passes through a
sieve with 1 mm apertures. Mix thoroughly.
Homogenize canned pet foods. Pass semi-moist pet foods through a mincer with 4-mm apertures.
8 Procedure
8.1 General
Because of the sensitivity of vitamin E 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 where possible.
Complete each assay within one working day.
8.2 Saponification
Weigh, to the nearest 0,1 g, approximately 50 g of the prepared sample (see clause 7) into a 1 litre conical flask.
Add to the test portion 200 ml of ethanol (4.3) whilst swirling the flask to disperse the sample. Add 2 ml of sodium
ascorbate solution (4.9), mix by swirling and then add 50 ml of potassium hydroxide solution (4.2) and swirl again.
Fit a reflux condenser to the flask and immerse the flask in the boiling water bath (5.2).
Allow the contents of the flask to reflux for 30 min, swirling occasionally.
NOTE In exceptional cases some products may require a longer saponification time.
Cool the flask to room temperature under a stream of cold water.
Transfer the contents of the flask into the extraction cylinder (see 5.4).
8.3 Extraction of vitamin E
Rinse the saponification flask with two 25 ml portions of ethanol (4.3) and transfer the rinsings to the cylinder.
Repeat the rinsing of the flask with two 125 ml portions of light petroleum (4.5) and one 250 ml portion of water
(4.1), each time transferring the rinsings to the cylinder.
Stopper the cylinder and shake well for 1 min, releasing the pressure from time to time.
Cool the cylinder under a stream of cold water while waiting for the two liquid phases to separate, before removing
the stopper.
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ISO 6867:2000(E)
When the layers have separated, remove the stopper, wash the sides of the stopper with a f

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