EN ISO 6867:2000 pdf download

EN ISO 6867:2000 pdf download.Animal feeding stuffs – Determination ofvitamin E content- Method using high-performance liquid chromatography.
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 given in ISO 6497 [1].
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 mm, 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 mm, 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.
When the layers have separated, remove the stopper, wash the sides of the stopper with a few millilitres of light petroleum (4.5) and insert the adjustable tube (see 5.4), positioning the lower open end so that it is just above the level of the interface.
By application of a slight pressure of inert gas (4.10) to the side arm tube, transfer the upper, light petroleum layer to a 1 litre separating funnel (see 5.4).
Add 125 ml of light petroleum (4.5) to the cylinder, stopper and shake well for 1 mm.
Allow the layers to separate and transfer the upper layer to the separating funnel using the adjustable tube (see 5.4) as before.
Again, add 125 ml of light petroleum (4.5) to the cylinder, stopper and shake well for 1 mm.
Again, allow the layers to separate and transfer the upper layer to the separating funnel using the adjustable tube as before.
Wash the combined light petroleum extracts with four 100 ml portions of water using at first only gentle inversion then only gentle shaking in order to keep emulsion formation to a minimum.
Transfer the washed extract through a medium/fast filter paper containing 30 g of anhydrous sodium sulfate (4.8) into a 1 litre flask suitable for vacuum evaporation (5.3).
Rinse the separating funnel with two 20 ml portions of light petroleum (4.5) and add the rinsings through the filter to the evaporation flask.
Wash the filter further with two 25 ml portions of light petroleum (4.5) and collect the washings in the evaporation flask.
Evaporate the light petroleum extract to dryness under vacuum at a temperature not exceeding 40 °C.
Care should be taken to ensure that the flask is removed from the rotary evaporator immediately after reaching the point of dryness; prolonged drying may lead to loss of vitamin E from the extract residue.
If the vitamin E concentration of the light petroleum extract is sufficiently high, the extract may be made up to a fixed volume with light petroleum and an aliquot part taken for the rotary evaporation stage.
Restore atmospheric pressure by admitting inert gas (4.10).
8.4 Determination
8.4.1 Dissolve the residue from 8.3 in a minimum volume of hexane (4.4) and transfer quantitatively to a 25 ml volumetric flask.
Rinse the evaporation flask with three small portions of hexane (4.4), transferring the rinsings to the volumetric flask. Dilute to volume with hexane and mix.
If necessary, filter the sample extract through a membrane filter (5.5) or centrifuge.
8.4.2 Inject 20 p1 of the sample extract onto the column of the liquid chromatograph (5.1) and measure the area of the DL-cx-tocopherol peak. The following HPLC conditions are offered for guidance; other conditions may be used
provided that they give equivalent results:
liquid chromatographic column (5.1.3): 250 mm x 4,6 mm, silica 5 pm or 10 pm packing, or equivalent;
— mobile phase (4.11): mixture of hexane (4.4) and 1 ,4-dioxan (4.7), 970:30 (by volume);
— flow rate: 1,5 ml/mm;
— detector (5.1.4): fluorescence detector (excitation 295 nm, emission 330 nm).