Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance) (c. 152)

ANNEX IV U.K. METHODS OF ANALYSIS TO CONTROL THE LEVEL OF AUTHORISED ADDITIVES IN FEED

B. DETERMINATION OF VITAMIN E U.K.

5. Procedure U.K.

Note:

Vitamin E is sensitive to (UV-) light and to oxidation. All operations shall be carried out in the absence of light (using amber glassware, or glassware protected with aluminium foil) and oxygen (flush with nitrogen). During extraction air above the liquid shall be replaced by nitrogen (avoid excess pressure by loosening the stopper from time to time).

5.1. Preparation of the sample U.K.

Grind the sample so that it passes a 1 mm mesh sieve, taking care to avoid generation of heat. Grinding must be carried out immediately before weighing and saponification otherwise there may be losses of vitamin E.

5.2. Saponification U.K.

Depending on the vitamin E content weigh, to the nearest 0,01 g, 2 g to 25 g of the sample into a 500 ml flat bottom or conical flask (4.2.1). Add successively with swirling 130 ml ethanol (3.1), approximately 100 mg BHT (3.12), 2 ml sodium ascorbate solution (3.5) and 2 ml sodium sulphide solution (3.6). Fit the condenser (4.3) to the flask and immerse the flask in a water-bath with magnetic stirrer (4.7). Heat to boiling and allow to reflux for 5 minutes. Then add 25 ml potassium hydroxide solution (3.4) through the condenser (4.3) and allow to reflux for a further 25 min. with stirring under a slow stream of nitrogen. Then rinse the condenser with approximately 20 ml water and cool the content of the flask to room temperature.

5.3. Extraction U.K.

Transfer by decantation the saponification solution quantitatively by rinsing with a total volume of 250 ml water to a 1 000 ml separating funnel (4.2.3) or to the extraction apparatus (4.8). Rinse the saponification flask successively with 25 ml ethanol (3.1) and 100 ml light petroleum (3.2) and transfer the rinsings to the separating funnel or to the extraction apparatus. The proportion of water and ethanol in the combined solutions must be about 2:1. Shake vigorously for 2 min. and allow to settle for 2 minutes.

5.3.1. Extraction using a separating funnel (4.2.3) U.K.

When the layers have separated (see observation 7.3) transfer the light petroleum layer to another separating funnel (4.2.3). Repeat this extraction twice, with 100 ml light petroleum (3.2) and twice, with 50 ml light petroleum (3.2).

Wash the combined extracts in the separating funnel twice by gently swirling (to avoid formation of emulsions) with 100 ml portions of water and then by repeated shaking with further 100 ml portions of water until the water remains colourless on addition of phenolphthalein solution (3.7) (washing four times is usually sufficient). Filter the washed extract through a dry pleated filter for phase separation (4.4) to remove any suspended water into a 500 ml graduated flask (4.2.2). Rinse the separating funnel and the filter with 50 ml light petroleum (3.2), make up to the mark with light petroleum (3.2) and mix well.

5.3.2. Extraction using an extraction apparatus (4.8) U.K.

When the layers have separated (see observation 7.3) replace the stopper of the glass cylinder (4.8.1) by the ground glass insert (4.8.2) and position the U-shaped lower end of the adjustable tube so that it is just above the level of the interface. By application of pressure from a nitrogen line to the side-arm, transfer the upper light petroleum-layer to a 1 000 ml separating funnel (4.2.3). Add 100 ml light petroleum (3.2) to the glass cylinder, stopper and shake well. Allow the layers to separate and transfer the upper layer to the separating funnel as before. Repeat the extraction procedure with further 100 ml of light petroleum (3.2), then twice with 50 ml portions of light petroleum (3.2) and add the light petroleum layers to the separating funnel.

Wash the combined light petroleum extracts as described in 5.3.1 and proceed as described there.

5.4. Preparation of the sample solution for HPLC U.K.

Pipette an aliquot portion of the light petroleum solution (from 5.3.1 or 5.3.2) into a 250 ml pear shaped flask (4.2.4). Evaporate the solvent nearly to dryness on the rotary evaporator (4.1) with reduced pressure at a bath temperature not exceeding 40 ^oC. Restore atmospheric pressure by admitting nitrogen (3.9) and remove the flask from the rotary evaporator. Remove the remaining solvent with a stream of nitrogen (3.9) and dissolve the residue immediately in a known volume (10-100 ml) of methanol (3.3) (the concentration of DL-α-tocopherol must be in the range 5 μg/ml to 30 μg/ml).

5.5. Determination by HPLC U.K.

Vitamin E is separated on a C₁₈ reversed phase column (4.5.1) and the concentration is measured using a fluorescence detector (excitation: 295 nm, emission: 330 nm) or a UV detector (292 nm) (4.5.2).

Inject an aliquot portion (e.g. 20 μl) of the methanolic solution obtained in 5.4 and elute with the mobile phase (3.8). Calculate the mean peak heights (areas) of several injections of the same sample solution and the mean peak heights (areas) of several injections of the calibration solutions (5.6.2).

HPLC conditions U.K.

The following conditions are offered for guidance; other conditions may be used provided that they give equivalent results.

Liquid chromatographic column (4.5.1):	250 mm × 4 mm, C18, 5 or 10 μm packing, or equivalent
Mobile phase (3.8):	Mixture of methanol (3.3) and water e.g. 980 + 20 (v + v).
Flow rate:	1-2 ml/min.
Detector (4.5.2)	Fluorescence detector (excitation: 295 nm/emission: 330 nm) or UV detector (292 nm)

5.6. Calibration (DL-α-tocopherol acetate or DL-α-tocopherol) U.K.

5.6.1. DL-α-tocopherol acetate standard U.K.

5.6.1.1. Preparation of the working standard solution U.K.

Transfer by pipette 25 ml of the DL-α-tocopherol acetate stock solution (3.10.1) into a 500 ml flat bottom or conical flask (4.2.1) and hydrolyse as described under 5.2. Subsequently extract with light petroleum (3.2) according to 5.3 and make up to 500 ml with light petroleum. Evaporate 25 ml of this extract on the rotary evaporator (see 5.4) nearly to dryness, remove the remaining solvent with a stream of nitrogen (3.9) and redissolve the residue in 25,0 ml of methanol (3.3). The nominal concentration of this solution is 45,5 μg DL-α-tocopherol per ml, equivalent to 50 μg DL-α-tocopherol acetate per ml. The working standard solution has to be freshly prepared before use.

5.6.1.2. Preparation of the calibration solutions and calibration graph U.K.

Transfer 1,0, 2,0, 4,0 and 10,0 ml of the working standard solution into a series of 20 ml graduated flasks, make up to the mark with methanol (3.3) and mix. The nominal concentrations of these solutions are 2,5, 5,0, 10,0 and 25,0 μg/ml DL-α-tocopherol acetate, i.e. 2,28, 4,55, 9,1 μg/ml and 22,8 μg/ml DL-α-tocopherol.

Inject 20 μl of each calibration solution several times and determine the mean peak heights (areas). Using the mean peak heights (areas) plot a calibration graph.

5.6.1.3. UV standardisation of the DL-α-tocopherol acetate stock solution (3.10.1) U.K.

Dilute 5,0 ml of the DL-α-tocopherol acetate stock solution (3.10.1) to 25,0 ml with ethanol and measure the UV spectrum of this solution against ethanol (3.1) in the spectrophotometer (4.6) between 250 nm and 320 nm.

The absorption maximum shall be at 284 nm:

= 43,6 at 284 nm in ethanol

At this dilution an extinction value of 0,84 to 0,88 must be obtained.

5.6.2. DL-α-tocopherol standard U.K.

5.6.2.1. Preparation of the working standard solution U.K.

Transfer by pipette 2 ml of the DL-α-tocopherol stock solution (3.11.1) into a 50 ml graduated flask, dissolve in methanol (3.3) and make up to the mark with methanol. The nominal concentration of this solution is 40 μg DL-α-tocopherol per ml, equivalent to 44,0 μg DL-α-tocopherol acetate per ml. The working standard solution has to be freshly prepared before use.

5.6.2.2. Preparation of the calibration solutions and calibration graph U.K.

Transfer 1,0, 2,0, 4,0 and 10,0 ml of the working standard solution into a series of 20 ml graduated flasks, make up to the mark with methanol (3.3) and mix. The nominal concentrations of these solutions are 2,0, 4,0, 8,0 and 20,0 μg/ml DL-α-tocopherol, i.e. 2,2, 4,4, 8,79 μg/ml and 22,0 μg/ml DL-α-tocopherol acetate.

Inject 20 μl of each calibration solution several times and determine the mean peak heights (areas). Using the mean peak heights (areas) plot a calibration graph.

5.6.2.3. UV standardisation of the DL-α-tocopherol stock solution (3.11.1) U.K.

Dilute 2,0 ml of the DL-α-tocopherol stock solution (3.11.1) to 25,0 ml with ethanol and measure the UV spectrum of this solution against ethanol (3.1) in the spectrophotometer (4.6) between 250 nm and 320 nm. The absorption maximum shall be at 292 nm:

= 75,8 at 292 nm in ethanol

At this dilution an extinction value of 0,6 must be obtained.