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)

Assemble the heating unit (point 4.1) and the filter crucible (point 4.2), then attach the cylinder (point 4.3) to the crucible. Pour 150 ml of boiling sulphuric acid (point 3.1) into the assembled cylinder and crucible and if necessary add a few drops of antifoaming agent (point 3.2).

Bring the liquid to the boil within 5 ± 2 minutes and boil vigorously for exactly 30 minutes.

Open the tap to the discharge pipe (point 4.1) and, under vacuum, filter the sulphuric acid through the filter crucible and wash the residue with three consecutive 30 ml portions of boiling water, ensuring that the residue is filtered dry after each washing.

Close the outlet tap and pour 150 ml boiling potassium hydroxide solution (point 3.7) to the assembled cylinder and crucible and add a few drops of antifoaming agent (point 3.2). Bring the liquid to boiling point within 5 ± 2 minutes and boil vigorously for exactly 30 minutes. Filter and repeat the washing procedure used for the sulphuric acid step.

After the final washing and drying, disconnect the crucible and its contents and reconnect it to the cold extraction unit (point 4.6). Apply the vacuum and wash the residue in the crucible with three consecutive 25 ml portions of acetone (point 3.4) ensuring that the residue is filtered dry after each washing.

Dry the crucible to constant weight in the oven at 130 °C. After each drying cool in the desiccator and weigh rapidly. Place the crucible in a muffle furnace and ash to constant weight (loss in weight between two successive weighings must be less than or equal to 2 mg) at 475 °C to 500 °C for at least 30 minutes.

After each heating cool first in the furnace and then in the desiccator before weighing.

Carry out a blank test without the sample. Loss of weight resulting from ashing must not exceed 4 mg.

6. Calculation of results

The crude fibre content as a percentage of the sample is given by the expression:

Image omitted; see official source

where:

m	=	weight of sample in g;
---	---	---
m0	=	loss of weight after ashing during the determination, in g;
---	---	---
m1	=	loss of weight after ashing during the blank test, in g.

7. Repeatability

The difference between two parallel determinations carried out on the same sample must not exceed:

• — 0,6 % in absolute value for crude fibre contents lower than 10 %,

• — 6 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.

8. Reproducibility

The difference between the results of two determinations carried out on the same sample in different laboratories must not exceed:

• — 1,0 % in absolute value for crude fibre contents lower than 10 %,

• — 10 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.

9. Observations

• 9.1.Feed containing more than 10 % crude fat must be defatted prior to analysis with light petroleum (point 3.5). Connect the filter crucible (point 4.2) and its contents to the cold extraction unit (point 4.6) and apply vacuum and wash the residue with three consecutive 30 ml portions of light petroleum, ensuring that the residue is dry. Connect the crucible and its contents to the heating unit (point 4.1) and continue as described under point 5.

• 9.2.Feed containing fats which cannot be extracted directly with light petroleum (point 3.5) must be defatted as shown in point 8.1 and defatted once more after boiling with acid. After boiling with acid and the subsequent washing connect the crucible and its contents to the cold extraction unit (point 4.6) and wash three times with 30 ml acetone followed by three further washings with 30 ml portions of light petroleum. Filter under vacuum until dry and continue the analysis as described under point 5, beginning with potassium hydroxide treatment.

• 9.3.If the feed contain over 5 % of carbonates, expressed as calcium carbonate, connect the crucible (point 4.2) with the weighed sample to the heating unit (point 4.1). Wash the sample three times with 30 ml hydrochloric acid (point 3.6). After each addition let the sample stand for about one minute before filtering. Wash once with 30 ml water and then continue as described under point 5.

• 9.4.If an apparatus in the form of a stand is used (several crucibles attached to the same heating unit) no two individual determinations on the same sample for analysis may be carried out in the same series.

• 9.5.If after boiling it is difficult to filter the acidic and basic solutions, use compressed air through the discharge pipe of the heating unit and then continue filtering.

• 9.6.The temperature for ashing shall not be higher than 500 °C in order to extend the lifetime of the glass filter crucibles. Care must be taken to avoid excessive thermal shock during heating and cooling cycles.

I. DETERMINATION OF SUGAR

1. Purpose and scope

This method makes it possible to determine the amount of reducing sugars and total sugars after inversion, expressed as glucose or where appropriate as sucrose, converting by the factor 0,95. It is applicable to compound feed. Special methods are provided for other feed. Where necessary, lactose shall be measured separately and taken into account when calculating the results.

This method is to be used for the determination of the sugar content for use in energy value calculation of the feed.

In case the sugar content is to be determined for other purposes, other methods of analysis can be used.

2. Principle

The sugars are extracted in dilute ethanol; the solution is clarified with Carrez solutions I and II. After eliminating the ethanol, the quantities before and after inversion are determined by the Luff-Schoorl method.

3. Reagents

• 3.1.Ethanol solution 40 % (v/v) density: 0,948 g/ml at 20 °C, neutralised to phenolphthalein.

• 3.2.Carrez solution I: dissolve in water 21,9 g of zinc acetate Zn (CH3COO)2 2H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.

• 3.3.Carrez solution II: dissolve in water 10,6 g of potassium ferrocyanide K4Fe (CN)6 3H2O. Make up to 100 ml with water.

• 3.4.Methyl orange, solution 0,1 % (w/v).

• 3.5.Hydrochloric acid 4 mol/litre.

• 3.6.Hydrochloric acid 0,1 mol/litre.

• 3.7.Sodium hydroxide solution 0,1 mol/litre.

• 3.8.Luff-Schoorl reagent:

• Stirring carefully, pour the citric acid solution (point 3.8.2) into the sodium carbonate solution (point 3.8.3). Add the copper sulphate solution (point 3.8.1) and make up to 1 litre with water. Leave to settle overnight and filter.

• Check the concentration of the reagent thus obtained (Cu 0,05 mol/litre; Na2 CO3 1 mol/litre), see point 5.4, last paragraph. The solution’s pH shall be approximately 9,4.

• 3.8.1.Copper sulphate solution: dissolve 25 g of copper sulphate, Cu SO4 5H2O, free from iron, in 100 ml of water.

• 3.8.2.Citric acid solution: dissolve 50 g of citric acid, C6H8O7•H2O in 50 ml of water.

• 3.8.3.Sodium carbonate solution: dissolve 143,8 g of anhydrous sodium carbonate in approximately 300 ml of warm water. Leave to cool.

• 3.9.Sodium thiosulphate solution 0,1 mol/litre.

• 3.10.Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes, leave to cool and if necessary add 10 mg of mercuric iodide as a preservative.

• 3.11.Sulphuric acid 3 mol/litre.

• 3.12.Potassium iodide, solution 30 % (w/v).

• 3.13.Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.

• 3.14.3-methylbutan-1-ol.

4. Apparatus

Mixer (tumbler): approximately 35 to 40 rpm.

5. Procedure

5.1.   Extraction of sample

Weigh 2,5 g of the sample to the nearest mg and place in a 250 ml volumetric flask. Add 200 ml of ethanol (point 3.1) and mix in the tumbler for one hour. Add 5 ml of Carrez solution I (point 3.2) and stir for approximately 30 seconds. Add 5 ml of Carrez solution II (point 3.3) and again stir for one minute. Make up to volume with ethanol (point 3.1), homogenise and filter. Remove 200 ml of the filtrate and evaporate to approximately half volume in order to eliminate most of the ethanol. Transfer the evaporation residue quantitatively to a 200 ml volumetric flask using warm water, cool, bring up to volume with water, homogenise and filter if necessary. This solution will be used to determine the amount of reducing sugars and, after inversion, of total sugars.

5.2.   Determination of reducing sugars

Using a pipette, remove not more than 25 ml of the solution containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage content of glucose in the sample.

5.3.   Determination of total sugars after inversion

Using a pipette take 50 ml of the solution and transfer to a 100 ml volumetric flask. Add a few drops of methyl orange solution (point 3.4) then, carefully and stirring continuously, add hydrochloric acid (point 3.5) until the liquid turns a definite red. Add 15 ml of hydrochloric acid (point 3.6), immerse the flask in a fast boiling water bath and keep there for thirty minutes. Cool rapidly to approximately 20 °C and add 15 ml of sodium hydroxide solution (point 3.7). Make up to 100 ml with water and homogenise. Remove not more than 25 ml containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage of glucose or, where appropriate, sucrose, by multiplying by the factor 0,95.

5.4.   Titration by the Luff-Schoorl method

Using a pipette, take 25 ml of Luff-Schoorl reagent (point 3.8) and transfer to a 300 ml Erlenmeyer flask; add exactly 25 ml of the clarified sugar solution. Add 2 granules of pumice stone (point 3.13), heat, stirring by hand, over a free flame of medium height and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly ten minutes. Cool immediately in cold water and after approximately five minutes titrate as follows:

Add 10 ml of potassium iodide solution (point 3.12) and immediately afterwards (carefully, because of the risk of abundant foaming), add 25 ml of sulphuric acid (point 3.11). Titrate with sodium thiosulphate solution (point 3.9) until a dull yellow colour appears, add the starch indicator (point 3.10) and complete titration.

Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (point 3.8) and 25 ml of water, after adding 10 ml of potassium iodide solution (point 3.12) and 25 ml of sulphuric acid (point 3.11) without boiling.

6. Calculation of results

Using the table establish the amount of glucose in mg which corresponds to the difference between the values of the two titrations, expressed in ml of sodium thiosulphate 0,1 mol/litre. Express the result as a percentage of the sample.

7. Special procedures

• 7.1.In the case of feed which are rich in molasses and other feed which are not particularly homogeneous, weigh out 20 g and place with 500 ml of water in a 1 litre volumetric flask. Mix for one hour in the tumbler. Clarify using Carrez solutions I (point 3.2) and II (point 3.3) reagents as described under point 5.1, this time however using four times the quantities of each reagent. Bring up to volume with 80 % ethanol (v/v).

• Homogenise and filter. Eliminate the ethanol as described under point 5.1. If there is no dextrinised starch, bring up to volume with distilled water.

• 7.2.In the case of molasses and feed materials which are rich in sugar and almost starch-free (carobs, dried beetroot cossettes, etc.), weigh out 5 g, place in a 250 ml volumetric flask, add 200 ml of distilled water and mix in the tumbler for one hour, or more if necessary. Clarify using Carrez solutions I (point 3.2) and II (point 3.3) reagents as described under point 5.1. Bring up to volume with cold water, homogenise and filter. In order to determine the amount of total sugars, continue as described under point 5.3.

8. Observations

• 8.1.In order to prevent foaming it is advisable to add (irrespective of the volume) approximately 1 ml of 3-methylbutan-1-ol (point 3.14) before boiling with Luff-Schoorl reagent.

• 8.2.The difference between the content of total sugars after inversion, expressed as glucose, and the content of reducing sugars, expressed as glucose, multiplied by 0,95, gives the percentage content of sucrose.

• 8.3.In order to determine the content of reducing sugars, excluding lactose, two methods may be adopted:

• 8.3.1.For an approximate calculation, multiply by 0,675 the lactose content established by a different method of analysis and subtract the result obtained from the content of reducing sugars.

• 8.3.2.For an accurate calculation of reducing sugars, excluding lactose, the same sample must be used for the two final determinations. One of the analyses is carried out on part of the solution obtained under point 5.1, the other on part of the solution obtained during the determination of lactose by the method laid down for that purpose (after fermenting the other types of sugar and clarifying).

• In both cases the amount of sugar present is determined by the Luff-Schoorl method and calculated in mg of glucose. One of the values is subtracted from the other and the difference is expressed as a percentage of the sample.

• Example

• The two volumes taken correspond, for each determination, to a sample of 250 mg.

• In the first case 17 ml of sodium thiosulphate solution 0,1 mol/litre corresponding to 44,2 mg of glucose is consumed; in the second, 11 ml, corresponding to 27,6 mg of glucose.

• The difference is 16,6 mg of glucose.

• The content of reducing sugars (excluding lactose), calculated as glucose, is therefore:

Image omitted; see official source

Table of values for 25 ml of Luff Schoorl reagent

ml of Na2 S2 O3 0,1 mol/litre, two minutes’ heating, ten minutes’ boiling

Na2 S2 O3 0,1 mol/litre	Glucose, fructose invert sugars C6 H12 O6	Glucose, fructose invert sugars C6 H12 O6	Lactose C12 H22 O11	Lactose C12 H22 O11	Na2 S2 O3 0,1 mol/litre
ml	mg	difference	mg	difference	ml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23	2,4 4,8 7,2 9,7 12,2 14,7 17,2 19,8 22,4 25,0 27,6 30,3 33,0 35,7 38,5 41,3 44,2 47,1 50,0 53,0 56,0 59,1 62,2	2,4 2,4 2,5 2,5 2,5 2,5 2,6 2,6 2,6 2,6 2,7 2,7 2,7 2,8 2,8 2,9 2,9 2,9 3,0 3,0 3,1 3,1	3,6 7,3 11,0 14,7 18,4 22,1 25,8 29,5 33,2 37,0 40,8 44,6 48,4 52,2 56,0 59,9 63,8 67,7 71,7 75,7 79,8 83,9 88,0	3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,8 3,8 3,8 3,8 3,8 3,8 3,9 3,9 3,9 4,0 4,0 4,1 4,1 4,1	1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

J. DETERMINATION OF LACTOSE

1. Purpose and scope

This method makes it possible to determine the level of lactose in feed containing more than 0,5 % of lactose.

2. Principle

The sugars are dissolved in water. The solution is subjected to fermentation by the yeast Saccharomyces cerevisiae which leaves the lactose intact. After clarification and filtration the lactose content of the filtrate is determined by the Luff-Schoorl method.

3. Reagents

• 3.1.Suspension of Saccharomyces cerevisiae: suspend 25 g of fresh yeast in 100 ml of water. The suspension will keep for a maximum period of one week in a refrigerator.

• 3.2.Carrez solution I: dissolve in water 21,9 g of zinc acetate, Zn (CH3 COO)2 2H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.

• 3.3.Carrez solution II: dissolve in water 10,6 g of potassium ferrocyanide K4Fe (CN)6 3H2O. Make up to 100 ml with water.

• 3.4.Luff-Schoorl reagent:

• Stirring carefully, pour the citric acid solution (point 3.4.2) into the sodium carbonate solution (point 3.4.3). Add the copper sulphate solution (point 3.4.1) and make up to 1 litre with water. Leave to settle overnight and filter. Check the concentration of the reagent thus obtained (Cu 0,05 mol/litre; Na2 CO3 1 mol/litre). The solution’s pH shall be approximately 9,4.

• 3.4.1.Copper sulphate solution: dissolve 25 g of copper sulphate Cu SO4 5H2O, free from iron, in 100 ml of water.

• 3.4.2.Citric acid solution: dissolve 50 g of citric acid C6H8O7 • H2O in 50 ml of water.

• 3.4.3.Sodium carbonate solution: dissolve 143,8 g of anhydrous sodium carbonate in approximately 300 ml of warm water. Leave to cool.

• 3.5.Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.

• 3.6.Potassium iodide, solution 30 % (w/v).

• 3.7.Sulphuric acid 3 mol/litre.

• 3.8.Solution of sodium thiosulphate 0,1 mol/litre.

• 3.9.Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes, leave to cool, and if necessary add 10 mg of mercuric iodide as a preservative.

4. Apparatus

Water bath with thermostat set at 38–40 °C.

5. Procedure

Weigh 1 g of the sample to the nearest mg and place this portion of the sample in a 100 ml volumetric flask. Add 25 to 30 ml of water. Place the flask in a boiling water bath for thirty minutes and then cool to approximately 35 °C. Add 5 ml of yeast suspension (point 3.1) and homogenise. Leave the flask to stand for two hours in a water bath, at a temperature of 38–40 °C. Cool to approximately 20 °C.

Add 2,5 ml of Carrez solution I (point 3.2) and stir for thirty seconds, then add 2,5 ml of Carrez solution II (point 3.3) and again stir for thirty seconds. Make up to 100 ml with water, mix and filter. Using a pipette, remove an amount of filtrate which does not exceed 25 ml and which preferably contains from 40 to 80 mg of lactose and transfer it to a 300 ml Erlenmeyer flask. If necessary, make up to 25 ml with water.

Carry out a blank test in the same way with 5 ml of yeast suspension (point 3.1). Determine the lactose content according to Luff-Schoorl, as follows: add exactly 25 ml of Luff-Schoorl reagent (point 3.4) and two granules of pumice stone (point 3.5). Stir by hand while heating over a free flame of medium height and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly ten minutes. Cool immediately in cold water and after approximately five minutes titrate as follows:

Add 10 ml of potassium iodide solution (point 3.6) and immediately afterwards (carefully, because of the risk of abundant foaming) add 25 ml of sulphuric acid (point 3.7). Titrate with sodium thiosulphate solution (point 3.8) until a dull yellow colour appears, add the starch indicator (point 3.9) and complete titration.

Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (point 3.4) and 25 ml of water, after adding 10 ml of potassium iodide solution (point 3.6) and 25 ml of sulphuric acid (point 3.7) without boiling.

6. Calculation of results

Using the attached table, establish the amount of lactose in mg which corresponds to the difference between the results of the two titrations, expressed in ml of sodium thiosulphate 0,1 mol/litre.

Express the result of anhydrous lactose as a percentage of the sample.

7. Observation

• 1.For products containing more than 40 % of fermentable sugar, use more than 5 ml of yeast suspension (point 3.1).

• 2.In ‘lactose reduced’ feed (e.g. cat milk), lactose is converted into fructose, which is not completely fermented within 2 hours resulting in higher or false positive results (because residues of fructose remain in the extract).

Table of values for 25 ml of Luff-Schoorl reagent

ml of Na2 S2 O3 0,1 mol/litre, two minutes’ heating, ten minutes’ boiling

Na2 S2 O3 0,1 mol/litre	Glucose, fructose invert sugars C6 H12 O6	Glucose, fructose invert sugars C6 H12 O6	Lactose C12 H22 O11	Lactose C12 H22 O11	Na2 S2 O3 0,1 mol/litre
ml	mg	difference	mg	difference	ml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23	2,4 4,8 7,2 9,7 12,2 14,7 17,2 19,8 22,4 25,0 27,6 30,3 33,0 35,7 38,5 41,3 44,2 47,1 50,0 53,0 56,0 59,1 62,2	2,4 2,4 2,5 2,5 2,5 2,5 2,6 2,6 2,6 2,6 2,7 2,7 2,7 2,8 2,8 2,9 2,9 2,9 3,0 3,0 3,1 3,1	3,6 7,3 11,0 14,7 18,4 22,1 25,8 29,5 33,2 37,0 40,8 44,6 48,4 52,2 56,0 59,9 63,8 67,7 71,7 75,7 79,8 83,9 88,0	3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,8 3,8 3,8 3,8 3,8 3,8 3,9 3,9 3,9 4,0 4,0 4,1 4,1 4,1	1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

K. DETERMINATION OF STARCH

POLARIMETRIC METHOD

1. Purpose and scope

This method makes it possible to determine the levels of starch and of high molecular weight starch degradation products in feed for the purpose of checking compliance with the declared energy value (provisions in Annex VII) and Regulation (EC) No 767/2009.

This method is to be used for the determination of the starch content for use in energy value calculation of the feed.

In case the starch content is to be determined for other purposes, other methods of analysis can be used.

2. Principle

The method comprises of two determinations. In the first one, the sample is treated with dilute hydrochloric acid. After clarification and filtration, the optical rotation of the solution is measured by polarimetry.

In the second one, the sample is extracted with 40 % ethanol. After acidifying the filtrate with hydrochloric acid, clarifying and filtering, the optical rotation is measured as in the first determination.

The difference between the two measurements, multiplied by a known factor, gives the starch content of the sample.

3. Reagents

• 3.1.Hydrochloric acid, solution 25 % (w/w) density: 1,126 g/ml.

• 3.2.Hydrochloric acid. solution 1,13 % (w/v)

• The concentration must be checked by titration using a sodium hydroxide solution 0,1 mol/litre in the presence of 0,1 % (w/v) methyl red in 94 % (v/v) ethanol. For the neutralisation of 10 ml, 30,94 ml of NaOH 0,1 mol/litre is needed.

• 3.3.Carrez solution I: dissolve 21,9 g of zinc acetate Zn(CH3COO)2 2H2O and 3 g of glacial acetic acid in water. Make up to 100 ml with water.

• 3.4.Carrez solution II: dissolve 10,6 g of potassium ferrocyanide K4 Fe(CN)6 3H2O in water. Make up to 100 ml with water.

• 3.5.Ethanol, solution 40 % (v/v), density: 0,948 g/ml at 20 °C.

4. Apparatus

• 4.1.250 ml Erlenmeyer flask with standard ground-glass joint and with reflux condenser.

• 4.2.Polarimeter or saccharimeter.

5. Procedure

5.1.   Preparation of the sample

Crush the sample until it is fine enough for all of it to pass through a 0,5 mm round-meshed sieve.

5.2.   Determination of the total optical rotation (P or S) (see observation point 7.1)

Weigh 2,5 g of the crushed sample to the nearest mg and place in a 100 ml graduated flask. Add 25 ml of hydrochloric acid (point 3.2), shake to obtain even distribution of the test sample and add a further 25 ml of hydrochloric acid (point 3.2). Immerse the flask in a boiling water bath shaking vigorously and steadily for the first three minutes to prevent the formation of agglomerates. The quantity of water in the water bath must be sufficient for the bath to remain at boiling point when the flask is introduced into it. The flask must not be taken out of the bath whilst being shaken. After exactly 15 minutes, remove from the bath, add 30 ml of cold water and cool immediately to 20 °C.

Add 5 ml of Carrez solution I (point 3.3) and shake for approximately 30 seconds. Then add 5 ml of Carrez solution II (point 3.4) and shake again for approximately 30 seconds. Make up to volume with water, mix and filter. If the filtrate is not perfectly clear (which is rare), repeat the determination using a larger quantity of Carrez solutions I and II, for example 10 ml.

Measure the optical rotation of the solution in a 200 mm tube with the polarimeter or saccharimeter.

5.3.   Determination of the optical rotation (P' or S') of substances soluble in 40 % ethanol

Weigh 5 g of the sample to the nearest mg, place in a 100 ml graduated flask and add about 80 ml of ethanol (point 3.5) (see observation point 7.2). Leave the flask to stand for 1 hour at room temperature; during this time, shake vigorously on six occasions so that the test sample is thoroughly mixed with the ethanol. Make up to volume with ethanol (point 3.5), mix and filter.

Pipette 50 ml of the filtrate (corresponds to 2,5 g of the sample) into a 250 ml erlenmeyer flask, add 2,1 ml of hydrochloric acid (point 3.1) and shake vigorously. Fit a reflux condenser to the erlenmeyer flask and immerse the latter in a boiling water bath. After exactly 15 minutes, remove the erlenmeyer flask from the bath, transfer the contents to a 100 ml graduated flask, rinsing with a little cold water, and cool to 20 °C.

Clarify using Carrez solutions I (point 3.3) and II (point 3.4), make up to volume with water, mix, filter and measure the optical rotation as indicated in point 5.2, second and third paragraphs.

6. Calculation of results

The starch content (%) is calculated as follows:

6.1.   Measurement by polarimeter

Image omitted; see official source

P	=	Total optical rotation in angle degrees
---	---	---
P'	=	Optical rotation in angle degrees of the substances soluble in 40 % (V/V) ethanol
---	---	---
Image omitted; see official source	=	Specific optical rotation of pure starch. The numerical values conventionally accepted for this factor are the following:
---	---	---
+ 185,9°	:	rice starch
---	---	---
+ 185,7°	:	potato starch
---	---	---
+ 184,6°	:	maize starch
---	---	---
+ 182,7°	:	wheat starch
---	---	---
+ 181,5°	:	barley starch
---	---	---
+ 181,3°	:	oat starch
---	---	---
+ 184,0°	:	other types of starch and starch mixtures in compound feed.

6.2.   Measurement by saccharimeter

Image omitted; see official source

S	=	Total optical rotation in saccharimeter degrees
---	---	---
S'	=	Optical rotation in saccharimeter degrees of the substances soluble in 40 % (v/v) ethanol
---	---	---
N	=	weight (g) of saccharose in 100 ml of water yielding an optical rotation of 100 saccharimeter degrees when measured using a 200 mm tube 16,29 g for the French saccharimeters 26,00 g for the German saccharimeters 20,00 g for mixed saccharimeters.
---	---	---
Image omitted; see official source	=	Specific optical rotation of pure starch (see point 6.1).

6.3.   Repeatability

The difference between the results of two parallel determinations carried out on the same sample must not exceed 0,4 in absolute value for a starch content lower than 40 % and 1 % relative for starch contents equal to or greater than 40 %.

7. Observations

• 7.1.If the sample contains more than 6 % of carbonates, calculated in terms of calcium carbonate, they must be destroyed by treatment with an exactly appropriate quantity of dilute sulphuric acid before determination of the total optical rotation.

• 7.2.In the case of products with a high lactose content, such as powdered milk serum or skimmed milk powder, proceed as follows after adding 80 ml of ethanol (point 3.5). Fit a reflux condenser to the flask and immerse the latter in a water bath at 50 °C for 30 minutes. Leave to cool and continue the analysis as indicated in point 5.3.

• 7.3.The following feed materials, where they are present in significant amounts in feed, are known to give rise to interferences when determining the starch content by the polarimetric method and thereby incorrect results could be yielded:

• — (sugar) beet products such as (sugar)beet pulp, (sugar) beet molasses, (sugar) beet pulp – molassed, (sugar) beet vinasse, (beet) sugar,

• — citrus pulp,

• — linseed; linseed expeller; linseed extracted,

• — rape seed; rape seed expeller; rape seed extracted; rape seed hulls,

• — sunflower seed; sunflower seed extracted; sunflower seed, partially decorticated, extracted,

• — copra expeller; copra extracted,

• — potato pulp,

• — dehydrated yeast,

• — products rich in inulin (e.g. Chips and meal of Jerusalem artichokes),

• — greaves,

• — soybean products. In these cases the method of analysis as provided by Commission Regulation (EC) No 121/2008 ([^E0033]) can be applied. This method can also be used for feed containing less than 1 % starch.

L. DETERMINATION OF CRUDE ASH

1. Purpose and scope

This method makes it possible to determine the crude ash content of feed.

2. Principle

The sample is ashed at 550 °C; the residue is weighed.

3. Reagents

Ammonium nitrate, solution 20 % (w/v).

4. Apparatus

• 4.1.Hot-plate.

• 4.2.Electric muffle-furnace with thermostat.

• 4.3.Crucibles for ashing made of silica, porcelain or platinum either rectangular (approx. 60 × 40 × 25 mm) or circular (diameter: 60 to 75 mm, height: 20 to 40 mm).

5. Procedure

Weigh out to the nearest mg approximately 5 g of the sample (2,5 in the case of products which have a tendency to swell) and place in a crucible for ashing which has first been heated at 550 °C, cooled down and tared. Place the crucible on the hot-plate and heat gradually until the substance carbonises. Ash according to point 5.1 or 5.2.

• 5.1.Put the crucible into the calibrated muffle furnace set at 550 °C. Keep at this temperature until white, light grey or reddish ash is obtained which appears to be free from carbonaceous particles. Place the crucible in a desiccator, leave to cool and weigh immediately.

• 5.2.Put the crucible into the calibrated muffle-furnace set at 550 °C. Ash for 3 hours. Place the crucible in a desiccator, leave to cool and weigh immediately. Ash again for 30 minutes to ensure that the weight of the ash remains constant (loss in weight between two successive weighings must be less than or equal to 1 mg).

6. Calculation of results

Calculate the weight of the residue by deducting the tare.

Express the result as a percentage of the sample.

7. Observations

• 7.1.The ash of substances which are difficult to ash must be subjected to an initial ashing of at least three hours, cooled and then a few drops of 20 % solution of ammonium nitrate or water added to it (carefully, to avoid dispersal of the ash or the formation of lumps). Continue calcining after drying in the oven. Repeat the operation as necessary until ashing is complete.

• 7.2.In the case of substances resistant to the treatment described under point 7.1, proceed as follows: after ashing for three hours, place the ash in warm water and filter through a small, ash-free filter. Ash the filter and its contents in the original crucible. Place the filtrate in the cooled crucible, evaporate until dry, ash and weigh.

• 7.3.In the case of oils and fats, weigh accurately a sample of 25 g in a suitably sized crucible. Carbonise by setting light to the substance with a strip of ash-free filter paper. After combustion, moisten with as little water as possible. Dry and ash as described under point 5.

M. DETERMINATION OF ASH WHICH IS INSOLUBLE IN HYDROCHLORIC ACID

1. Purpose and scope

This method makes it possible to determine the level in feed of mineral substances which are insoluble in hydrochloric acid. Two methods can be used, depending on the nature of the sample.

• 1.1. Method A: applicable to organic feed materials and to most compound feed;

• 1.2. Method B: applicable to mineral compounds and mixtures and to compound feed, whose content in substances insoluble in hydrochloric acid, as determined by Method A, is greater than 1 %.

2. Principle

• 2.1. Method A: the sample is ashed, the ash boiled in hydrochloric acid and the insoluble residue filtered and weighed.

• 2.2. Method B: the sample is treated with hydrochloric acid. The solution is filtered, the residue ashed and the ash thus obtained treated in accordance with Method A.

3. Reagents

• 3.1.Hydrochloric acid 3 mol/litre.

• 3.2.Trichloroacetic acid, solution 20 % (w/v).

• 3.3.Trichloroacetic acid, solution 1 % (w/v).

4. Apparatus

• 4.1.Hot plate.

• 4.2.Electric muffle-furnace with thermostat.

• 4.3.Crucibles for ashing made of silica, porcelain or platinum, either rectangular (approx. 60 × 40 × 25 mm) or circular (diameter: 60 to 75 mm, height: 20 to 40 mm).

• 4.4.Ash free filters

5. Procedure

5.1.   Method A

Ash the sample using the method described for the determination of crude ash. Ash obtained from that analysis may also be used.

Place the ash in a 250 to 400 ml beaker using 75 ml of hydrochloric acid (point 3.1). Bring slowly to the boil and boil gently for fifteen minutes. Filter the warm solution through an ash-free filter paper and wash the residue with warm water until the acid reaction is no longer visible. Dry the filter containing the residue and ash in a tared crucible at a temperature of not less than 550 °C and not more than 700 °C. Cool in a desiccator and weigh.

5.2.   Method B

Weigh 5 g of the sample to the nearest mg and place in a 250 to 400 ml beaker. Add 25 ml of water and 25 ml of hydrochloric acid (point 3.1) successively, mix and wait for effervescence to cease. Add a further 50 ml of hydrochloric acid (point 3.1). Wait for any release of gas to cease then place the beaker in a boiling water bath and keep it there for thirty minutes or longer, if necessary, in order to hydrolyse thoroughly any starch which may be present. Filter while warm through an ash-free filter and wash the filter in 50 ml of warm water (see observation point 7). Place the filter containing the residue in a crucible for ashing, dry and ash at a temperature of not less than 550 °C and not more than 700 °C. Place the ash in a 250 to 400 ml beaker using 75 ml of hydrochloric acid (point 3.1); continue as described in the second subparagraph of point 5.1.

6. Calculation of results

Calculate the weight of the residue by deducting the tare. Express the result as a percentage of the sample.

7. Observation

If filtration proves difficult recommence the analysis, replacing the 50 ml of hydrochloric acid (point 3.1) by 50 ml of trichloroacetic acid, solution 20 % (w/v) (point 3.2) and washing the filter in a warm solution of 1 % trichloroacetic acid (point 3.3).

N. DETERMINATION OF TOTAL PHOSPHORUS

The total phosphorus is to be determined by

• — the method of analysis provided for by EN 15510 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt, molybdenum and lead by ICP-AES, or

• — the method of analysis provided for by EN 15621 Animal feeding stuffs: Methods of sampling and analysis – Determination of calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt after pressure digestion by ICP-AES, or

• — the photometric method, as described hereafter.

PHOTOMETRIC METHOD

1. Purpose and scope

This method makes it possible to determine the content of total phosphorus in feed. It is particularly appropriate for the analysis of products low in phosphorus. In certain cases (product rich in phosphorus), a gravimetric method may be used.

2. Principle

The sample is mineralised, either by dry combustion (in the case of organic feed) or by acid digestion (in the case of mineral compounds and liquid feed), and placed in an acid solution. The solution is treated with molybdovanadate reagent. The optical density of the yellow solution thus formed is measured in a spectrophotometer at 430 nm.

3. Reagents

• 3.1.Calcium carbonate.

• 3.2.Hydrochloric acid, ρ20 = 1,10 g/ml (approx 6 mol/litre).

• 3.3.Nitric acid, ρ20 = 1,045 g/ml.

• 3.4.Nitric acid, ρ20 = 1,38 to 1,42 g/ml.

• 3.5.Sulphuric acid, ρ20 = 1,84 g/ml.

• 3.6.Molybdovanadate reagent: mix 200 ml of ammonium heptamolybdate solution (point 3.6.1), 200 ml of ammonium monovanadate solution (point 3.6.2) and 134 ml of nitric acid (point 3.4) in a 1 litre graduated flask. Make up to volume with water.

• 3.6.1.Ammonium heptamolybdate solution: dissolve in hot water 100 g of ammonium heptamolybdate (NH4) 6Mo7O24.4H2O. Add 10 ml of ammonia (density 0,91 g/ml) and make up to 1 litre with water.

• 3.6.2.Ammonium monovanadate solution: dissolve 2,35 g of ammonium monovanadate NH4VO3 in 400 ml of hot water. Stirring constantly, slowly add 20 ml of dilute nitric acid (7 ml of HNO3 (point 3.4) + 13 ml of H2O and make up to 1 litre with water.

• 3.7.Standard solution of 1 mg phosphorus per ml: dissolve 4,387 g of potassium dihydrogen phosphate KH2PO4 in water. Make up to 1 litre with water.

4. Apparatus

• 4.1.Silica, porcelain or platinum ashing crucibles.

• 4.2.Electric muffle-furnace with thermostat set at 550 °C.

• 4.3.250 ml Kjeldahl flask.

• 4.4.Graduated flasks and precision pipettes.

• 4.5.Spectrophotometer.

• 4.6.Test tubes about 16 mm in diameter, with stoppers graded to a diameter of 14,5 mm; capacity: 25 to 30 ml.

5. Procedure

5.1.   Preparation of the solution

According to the nature of the sample, prepare a solution as indicated in point 5.1.1 or 5.1.2.

5.1.1.   Usual procedure

Weigh 1 g or more of the sample to the nearest 1 mg. Place the test sample in a Kjeldahl flask, add 20 ml of sulphuric acid (point 3.5), shake to impregnate the substance completely with acid and to prevent it from sticking to the sides of the flask, heat and keep at boiling point for 10 minutes. Leave to cool slightly, add 2 ml of nitric acid (point 3.4), heat gently, leave to cool slightly, add a little more nitric acid (point 3.4) and bring back to boiling point. Repeat this procedure until a colourless solution is obtained. Cool, add a little water, decant the liquid into a 500 ml graduated flask, rinsing the Kjeldahl flask with hot water. Leave to cool, make up to volume with water, homogenise and filter.

5.1.2.   Samples containing organic substances and free from calcium and magnesium dihydrogen phosphates

Weigh about 2,5 g of the sample to the nearest 1 mg in an ashing crucible. Mix the test sample until completely merged with 1 g of calcium carbonate (point 3.1). Ash in the oven at 550 °C until white or grey ash is obtained (a little charcoal does not matter). Transfer the ash into a 250 ml beaker. Add 20 ml of water and hydrochloric acid (point 3.2) until effervescence ceases. Add a further 10 ml of hydrochloric acid (point 3.2). Place the beaker on a sand bath and evaporate until dry to make the silica insoluble. Redissolve the residue in 10 ml of nitric acid (point 3.3) and boil on the sand bath or hot plate for 5 minutes without evaporating until dry. Decant the liquid into a 500 ml graduated flask, rinsing the beaker several times with hot water. Leave to cool, make up to volume with water, homogenise and filter.

5.2.   Development of coloration and measurement of optical density

Dilute an aliquot part of the filtrate obtained by point 5.1.1 or 5.1.2 to obtain a phosphorus concentration of not more than 40 μg/ml. Place 10 ml of this solution in a test tube (point 4.6) and add 10 ml of molybdovanadate reagent (point 3.6). Homogenise and leave to stand for at least 10 minutes at 20 °C. Measure the optical density in a spectrophotometer at 430 nm against a solution obtained by adding 10 ml of the molybdovanadate reagent (point 3.6) to 10 ml of water.

5.3.   Calibration curve

From the standard solution (point 3.7) prepare solutions containing respectively 5, 10, 20, 30 and 40 μg of phosphorus per ml. Take 10 ml of each of these solutions and add thereto 10 ml of molybdovanadate reagent (point 3.6). Homogenise and leave to stand for at least 10 minutes at 20 °C. Measure the optical density as indicated in point 5.2. Trace the calibration curve by plotting the optical densities against the corresponding quantities of phosphorus. For concentrations between 0 and 40 μg/ml, the curve will be linear.

6. Calculation of results

Determine the amount of phosphorus in the test sample by using the calibration curve.

Express the result as a percentage of the sample.

Repeatability

The difference between the results of two parallel determinations carried out on the same sample shall not exceed:

• — 3 %, relative to the higher result, for phosphorus contents of less than 5 %,

• — 0,15 % in absolute value, for phosphorus contents of 5 % or more.

O. DETERMINATION OF CHLORINE FROM CHLORIDES

1. Purpose and scope

This method makes it possible to determine the amount of chlorine in chlorides which are soluble in water, conventionally expressed as sodium chloride. It is applicable to all feed.

2. Principle

The chlorides are dissolved in water. If the product contains organic matter it is clarified. The solution is slightly acidified with nitric acid and the chlorides precipitated in the form of silver chloride by means of a solution of silver nitrate. The excess silver nitrate is titrated with a solution of ammonium thiocyanate, by Volhard’s method.

3. Reagents

• 3.1.Solution of ammonium thiocyanate 0,1 mol/litre.

• 3.2.Solution of silver nitrate 0,1 mol/litre.

• 3.3.Saturated solution of ammonium ferric sulphate (NH4)Fe(SO4)2.

• 3.4.Nitric acid, density: 1,38 g/ml.

• 3.5.Diethyl ether.

• 3.6.Acetone.

• 3.7.Carrez solution I: dissolve in water 21,9 g of zinc acetate, Zn (CH3COO)2·2H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.

• 3.8.Carrez solution II: dissolve in water 10,6 g of potassium ferrocyanide K4Fe(CN)6·3H2O. Make up to 100 ml with water.

• 3.9.Active carbon, free from chlorides and not absorbing them.

4. Apparatus

Mixer (tumbler): approximately 35 to 40 rpm.

5. Procedure

5.1.   Preparation of the solution

According to the nature of the sample, prepare a solution as shown under point 5.1.1, 5.1.2 or 5.1.3.

At the same time carry out a blank test omitting the sample to be analysed.

5.1.1.   Samples free from organic matter

Weigh to the nearest mg a sample of not more than 10 g and containing not more than 3 g of chlorine in the form of chlorides. Place with 400 ml of water in a 500 ml volumetric flask at approximately 20 °C. Mix for thirty minutes in the tumbler, bring up to volume, homogenise and filter.

5.1.2.   Samples containing organic matter, excluding the products listed under point 5.1.3

Weigh approximately 5 g of the sample to the nearest mg and place with 1 g of active carbon in a 500 ml volumetric flask. Add 400 ml of water at approximately 20 °C and 5 ml of Carrez solution I (point 3.7), stir for 30 seconds then add 5 ml of Carrez solution II (point 3.8). Mix for thirty minutes in the tumbler, bring up to volume, homogenise and filter.

5.1.3.   Cooked feed, flax cakes and flour, products rich in flax flour and other products rich in mucilage or in colloidal substances (for example, dextrinated starch)

Prepare the solution as described under point 5.1.2 but do not filter. Decant (if necessary centrifuge), remove 100 ml of the supernatant liquid and transfer to a 200 ml measuring flask. Mix with acetone (point 3.6) and bring up to volume with this solvent, homogenise and filter.

5.2.   Titration

Using a pipette, transfer to an Erlenmeyer flask from 25 ml to 100 ml of the filtrate (according to the assumed chlorine content) obtained as described under point 5.1.1, 5.1.2 or 5.1.3. The aliquot portion must not contain more than 150 mg of chlorine (Cl). Dilute if necessary to not less than 50 ml with water, add 5 ml of nitric acid (point 3.4), 2 ml of saturated solution of ammonium ferric sulphate (point 3.3) and two drops of ammonium thiocyanate solution (point 3.1) transferred by means of a burette filled up to the zero mark. Using a burette, transfer the silver nitrate solution (point 3.2) in such a way that an excess of 5 ml is obtained. Add 5 ml of diethyl ether (point 3.5) and shake hard to coagulate the precipitate. Titrate the excess silver nitrate with the ammonium thiocyanate solution (point 3.1) until the reddish-brown tint has lasted for one minute.

6. Calculation of results

The amount of chlorine (X), expressed as % sodium chloride is calculated by using the following formula:

Image omitted; see official source

where:

V1	=	ml of silver nitrate solution 0,1 mol/l added
---	---	---
V2	=	ml of ammonium thiocyanate solution 0,1 mol/l used for titration
---	---	---
m	=	g of weight of the sample in the aliquot.

If the blank test indicates that silver nitrate solution 0,1 mol/l has been consumed deduct this value from the volume (V1 – V2).

7. Observations

• 7.1.Titration may also be carried out by potentiometry or amperometry.

• 7.2.In the case of products which are very rich in oils and fats, first de-fat with diethyl ether or light petroleum.

• 7.3.In the case of fish-meal, titration may be carried out by Mohr’s method.

P. DETERMINATION OF CARBONATES

1. Purpose and Scope

This method makes it possible to determine the amount of carbonates, conventionally expressed as calcium carbonate, in feed with the exception of feed where iron carbnate is present.

2. Principle

The carbonates are decomposed in hydrochloric acid; the carbon dioxide released is collected in a graduated tube, and its volume compared with that released under the same conditions by a known quantity of calcium carbonate.

3. Reagents

• 3.1.Hydrochloric acid, density 1,10 g/ml.

• 3.2.Pure calcium carbonate.

• 3.3.Sulphuric acid, approximately 0,05 mol/litre, coloured with methyl red.

4. Apparatus

Scheibler-Dietrich apparatus (see diagram in the Appendix) or equivalent apparatus (calcimeter).

5. Procedure

Depending on the sample’s carbonate content, weigh a portion of the sample as shown below:

• (a) 0,5 g for products containing from 50 % to 100 % of carbonates, expressed as calcium carbonate;

• (b) 1 g for products containing from 40 % to 50 % of carbonates, expressed as calcium carbonate;

• (c) 2 g to 3 g for other products.

Hydrochloric acid (point 3.1, above) is added to the portion of the sample to decompose any carbonates present. The volume of carbon dioxide is measured by using a Scheibler-Dietrich apparatus or equivalent apparatus (calcimeter) and is compared with the volume of carbon dioxide produced by 0,5 g pure calcium carbonate (point 3.2, above).

All determinations shall be carried out under the same conditions in order to avoid making corrections for differences in temperature and pressure. The determination should be preferably carried out in a temperature-controlled room.

The procedure making use of the Scheibler-Dietrich apparatus is described in detail in the Appendix.

6. Calculation

The content of carbonates, expressed as pure calcium carbonate, is calculated by using the formula:

X =	V ×100
X =	V1 × 2m

where:

X	=	% (w/w) of carbonates in the sample, expressed as calcium carbonate
---	---	---
V	=	ml of CO2 released by the portion of the sample
---	---	---
V1	=	ml of CO2 released by 0,5 g of CaCO3
---	---	---
m	=	weight, in grammes, of the portion of the sample.

7. Observations

• 7.1.If the Scheibler-Dietrich apparatus is used and the sample weighs more than 2 g, first place 15 ml of distilled water in the flask (item 4 in the diagram in the Appendix, below) and mix before beginning the test by adding hydrochloric acid (point 3.1, above). Use the same volume of distilled water for the control test.

• 7.2.If the apparatus used has a different volume from that of the Scheibler-Dietrich apparatus, the portions taken from the sample and from the control substance and the calculation must be adapted accordingly.

Appendix

Detailed procedure making use of the Scheibler-Dietrich apparatus

Place the portion of the sample in the special flask (item 4 in the diagram) of the apparatus, fitted with a small tube of unbreakable material containing 10 ml of hydrochloric acid (point 3.1, above), and connect the flask to the apparatus. Turn the three-way cock (item 5 in the diagram) so that the graduated tube (item 1 in the diagram) connects with the outside. Using the mobile tube (item 2 in the diagram), which is filled with coloured sulphuric acid (point 3.3, above) and is connected to the graduated tube (item 1 in the diagram), bring the level of the liquid up to the zero mark. Turn the three-way cock (item 5 in the diagram) in order to connect up the tubes (items 1 and 3 in the diagram) and check that the level is at zero.

Run the hydrochloric acid (point 3.1, above) slowly over the portion of the sample, tilting the special flask (item 4 in the diagram). Make the pressure equal by lowering the mobile tube (item 2 in the diagram). Shake the special flask (item 4 in the diagram) until the release of carbon dioxide has stopped completely.

Restore the pressure by bringing the liquid back to the same level in the tubes (items 1 and 2 in the diagram). After a few minutes, when the volume of gas has become constant, take the reading.

Carry out a control test in the same conditions on 0,5 g of calcium carbonate (point 3.2, above).

SCHEIBLER-DIETRICH APPARATUS FOR THE DETERMINATION OF CO2

Image omitted; see official source

(measured in mm)

ANNEX IV

METHODS OF ANALYSIS TO CONTROL THE LEVEL OF AUTHORISED ADDITIVES IN FEED

A. DETERMINATION OF VITAMIN A

The vitamin A is to be determined by:

• — the method of analysis provided for by EN 17547 Animal feeding stuffs: Methods of sampling and analysis – Determination of vitamin A, E and D ([^E0034]) content – Method using solid phase extraction (SPE) clean-up and high-performance liquid chromatography (HPLC), or

• — by reversed phase high performance liquid chromatography (RP-HPLC) using a UV or a fluorescence detector, as described in the points 1 to 9 hereafter.

1. Purpose and scope

This method makes it possible to determine the level of vitamin A (retinol) in feed. Vitamin A includes all-trans-retinyl alcohol and its cis-isomers which are determined by this method. The content of vitamin A is expressed in International Units (IU) per kg. One IU corresponds to the activity of 0,300 μg all-trans-vitamin A alcohol or 0,344 μg all-trans-vitamin A acetate or 0,550 μg all-trans-vitamin A palmitate.

The limit of quantification is 2 000  IU vitamin A/kg.

2. Principle

The sample is hydrolysed with ethanolic potassium hydroxide solution and the vitamin A is extracted into light petroleum. The solvent is removed by evaporation and the residue is dissolved in methanol and, if necessary, diluted to the required concentration. The content of vitamin A is determined by reversed phase high performance liquid chromatography (RP-HPLC) using a UV or a fluorescence detector. The chromatographic parameters are chosen so that there is no separation between the all-trans- vitamin A alcohol and its cis isomers.

3. Reagents

• 3.1.Ethanol, σ = 96 %.

• 3.2.Light petroleum, boiling range 40 °C-60 °C.

• 3.3.Methanol.

• 3.4.Potassium hydroxide solution, c = 50 g/100 ml.

• 3.5.Sodium ascorbate solution, c = 10 g/100 ml (see point 7.7 observations).

• 3.6.Sodium sulphide, Na2S · x H2O (x = 7 – 9).

• 3.6.1.Sodium sulphide solution, c = 0,5 mol/l in glycerol, ß = 120 g/l (for x = 9) (see point 7.8 observations).

• 3.7.Phenolphthalein solution, c = 2 g/100 ml in ethanol (point 3.1).

• 3.8.2-Propanol.

• 3.9.Mobile phase for HPLC: mixture of methanol (point 3.3) and water, e.g. 980 + 20 (v + v). The exact ratio will be determined by the characteristics of the column employed.

• 3.10.Nitrogen, oxygen free.

• 3.11.All-trans-vitamin A acetate, extra pure, of certified activity, e.g. 2,80 × 10⁶ IU/g.

• 3.11.1.Stock solution of all-trans-vitamin A acetate: Weigh to the nearest 0,1 mg, 50 mg of vitamin A acetate (point 3.11) into a 100 ml graduated flask. Dissolve in 2-propanol (point 3.8) and make up to the mark with the same solvent. The nominal concentration of this solution is 1 400  IU vitamin A per ml. The exact content shall be determined according to point 5.6.3.1.

• 3.12.All-trans-vitamin A palmitate, extra pure, of certified activity, e.g. 1,80 × 10⁶ IU/g.

• 3.12.1.Stock solution of all-trans-vitamin A palmitate: Weigh to the nearest 0,1 mg, 80 mg of vitamin A palmitate (point 3.12) into a 100 ml graduated flask. Dissolve in 2-propanol (point 3.8) and make up to the mark with the same solvent. The nominal concentration of this solution is 1 400  IU vitamin A per ml. The exact content shall be determined according to point 5.6.3.2.

• 3.13.2,6-Di-tert-butyl-4-methylphenol (BHT) (see point 7.5 observations).

4. Apparatus

• 4.1.Vacuum rotary evaporator.

• 4.2.Amber glassware.

• 4.2.1.Flat bottom or conical flasks, 500 ml, with ground-glass socket.

• 4.2.2.Graduated flasks with ground-glass stoppers, narrow-necked, 10, 25, 100 and 500 ml.

• 4.2.3.Separating funnels, conical, 1 000  ml, with ground-glass stoppers.

• 4.2.4.Pear-shaped flasks, 250 ml, with ground-glass sockets.

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

• 4.4.Pleated filter paper for phase separation, diameter 185 mm (e.g. Schleicher & Schuell 597 HY 1/2).

• 4.5.HPLC equipment with injection system.

• 4.5.1.Liquid chromatographic column, 250 mm × 4 mm, C18, 5 or 10 μm packing, or equivalent (performance criterion: only a single peak for all retinol isomers under the HPLC-conditions).

• 4.5.2.UV or fluorescence detector, with variable wavelength adjustment.

• 4.6.Spectrophotometer with 10 mm quartz cells.

• 4.7.Water-bath with magnetic stirrer.

• 4.8.Extraction apparatus (see Figure 1) consisting of:

• 4.8.1.Glass cylinder of 1 l capacity fitted with a ground glass neck and stopper.

• 4.8.2.Ground glass insert equipped with a side-arm and an adjustable tube passing through the centre. The adjustable tube shall 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 into a separating funnel.

5. Procedure

• Note: Vitamin A 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

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

5.2.   Saponification

Depending on the vitamin A content weigh, to the nearest 1 mg, 2 g to 25 g of the sample into a 500 ml flat bottom or conical flask (point 4.2.1). In case of low concentrations, the sample weight can be increased in order to have enough particles in the test portion. Add successively with swirling 130 ml ethanol (point 3.1), approximately 100 mg BHT (point 3.13), 2 ml sodium ascorbate solution (point 3.5) and 2 ml sodium sulphide solution (point 3.6). Fit a condenser (point 4.3) to the flask and immerse the flask in a water-bath with magnetic stirrer (point 4.7). Heat to boiling and allow to reflux for 5 minutes. Then add 25 ml potassium hydroxide solution (point 3.4) through the condenser (point 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

Transfer by decantation the saponification solution quantitatively by rinsing with a total volume of 250 ml water to a 1 000  ml separating funnel (point 4.2.3) or to the extraction apparatus (point 4.8). Rinse the saponification flask successively with 25 ml ethanol (point 3.1) and 100 ml light petroleum (point 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 (point 4.2.3)

When the layers have separated (see observation point 7.3) transfer the light petroleum layer to another separating funnel (point 4.2.3). Repeat this extraction twice, with 100 ml light petroleum (point 3.2) and twice, with 50 ml light petroleum (point 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 (point 3.7) (washing four times is usually sufficient). Filter the washed extract through a dry pleated filter for phase separation (point 4.4) to remove any suspended water into a 500 ml graduated flask (point 4.2.2). Rinse the separating funnel and the filter with 50 ml light petroleum (point 3.2), make up to the mark with light petroleum (point 3.2) and mix well.

5.3.2.   Extraction using an extraction apparatus (point 4.8)

When the layers have separated (see observation point 7.3) replace the stopper of the glass cylinder (point 4.8.1) by the ground glass insert (point 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 (point 4.2.3). Add 100 ml light petroleum (point 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 (point 3.2), then twice with 50 ml portions of light petroleum (point 3.2) and add the light petroleum layers to the separating funnel.

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

5.4.   Preparation of the sample solution for HPLC

Pipette an aliquot portion of the light petroleum solution (from point 5.3.1 or 5.3.2) into a 250 ml pear-shaped flask (point 4.2.4). Evaporate the solvent nearly to dryness on the rotary evaporator (point 4.1) with reduced pressure at a bath temperature not exceeding 40 °C. Restore atmospheric pressure by admitting nitrogen (point 3.10) and remove the flask from the rotary evaporator. Remove the remaining solvent with a stream of nitrogen (point 3.10) and dissolve the residue immediately in a known volume (10–100 ml) of methanol (point 3.3) (the concentration of vitamin A must be in the range of 5 IU/ml to 30 IU/ml).

5.5.   Determination by HPLC

Vitamin A is separated on a C18 reversed phase column (point 4.5.1) and the concentration is measured by means of a UV detector (325 nm) or a fluorescence detector (excitation: 325 nm, emission: 475 nm) (point 4.5.2).

Inject an aliquot portion (e.g. 20 μl) of the methanolic solution obtained in point 5.4 and elute with the mobile phase (point 3.9). Calculate the mean peak height (area) of several injections of the same sample solution and the mean peak heights (areas) of several injections of the calibration solutions (point 5.6.2).

HPLC conditions

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

Liquid chromatographic column (point 4.5.1):	250 mm × 4 mm, C18, 5 or 10 μm packing, or equivalent
Mobile phase (point 3.9):	Mixture of methanol (point 3.3) and water, e.g. 980 + 20 (v + v)
Flow rate:	1–2 ml/min
Detector (point 4.5.2):	UV detector (325 nm) or fluorescence detector (excitation: 325 nm/emission: 475 nm).

5.6.   Calibration

5.6.1.   Preparation of the working standard solutions

Pipette 20 ml of the vitamin A acetate stock solution (point 3.11.1) or 20 ml of the vitamin A palmitate stock solution (point 3.12.1) into a 500 ml flat bottom or conical flask (point 4.2.1) and hydrolyse as described under point 5.2, but without addition of BHT. Subsequently extract with light petroleum (point 3.2) according to point 5.3 and make up to 500 ml with light petroleum (point 3.2). Evaporate 100 ml of this extract on the rotary evaporator (see point 5.4) nearly to dryness, remove the remaining solvent with a stream of nitrogen (point 3.10) and redissolve the residue in 10,0 ml of methanol (point 3.3). The nominal concentration of this solution is 560 IU vitamin A per ml. The exact content has to be determined according to point 5.6.3.3. The working standard solution has to be freshly prepared before use.

Pipette 2,0 ml of this working standard solution into a 20 ml graduated flask, make up to the mark with methanol (point 3.3) and mix. The nominal concentration of this diluted working standard solution is 56 IU vitamin A per ml.

5.6.2.   Preparation of the calibration solutions and calibration graph

Transfer 1,0, 2,0, 5,0 and 10,0 ml of the diluted working standard solution into a series of 20 ml graduated flasks, make up to the mark with methanol (point 3.3) and mix. The nominal concentrations of these solutions are 2,8, 5,6, 14,0 and 28,0 IU vitamin A per ml.

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 considering the results of the UV control (point 5.6.3.3).

5.6.3.   UV standardisation of the standard solutions

5.6.3.1.   Vitamin A acetate stock solution

Pipette 2,0 ml of the vitamin A acetate stock solution (point 3.11.1) into a 50 ml graduated flask (point 4.2.2) and make up to the mark with 2-propanol (point 3.8). The nominal concentration of this solution is 56 IU vitamin A per ml. Pipette 3,0 ml of this diluted vitamin A acetate solution into a 25 ml graduated flask and make up to the mark with 2-propanol (point 3.8). The nominal concentration of this solution is 6,72 IU vitamin A per ml. Measure the UV spectrum of this solution against 2-propanol (point 3.8) in the spectrophotometer (point 4.6) between 300 nm and 400 nm. The extinction maximum must be between 325 nm and 327 nm.

Calculation of the vitamin A content:

Image omitted; see official source

Image omitted; see official source

5.6.3.2.   Vitamin A palmitate stock solution

Pipette 2,0 ml of the vitamin A palmitate stock solution (point 3.12.1) into a 50 ml graduated flask (point 4.2.2) and make up to the mark with 2-propanol (point 3.8). The nominal concentration of this solution is 56 IU vitamin A per ml. Pipette 3,0 ml of this diluted vitamin A palmitate solution into a 25 ml graduated flask and make up to the mark with 2-propanol (point 3.8). The nominal concentration of this solution is 6,72 IU vitamin A per ml. Measure the UV spectrum of this solution against 2-propanol (point 3.8) in the spectrophotometer (point 4.6) between 300 nm and 400 nm. The extinction maximum must be between 325 nm and 327 nm.

Calculation of the vitamin A content:

Image omitted; see official source

Image omitted; see official source

5.6.3.3.   Vitamin A working standard solution

Pipette 3,0 ml of the undiluted vitamin A working standard solution, prepared according to point 5.6.1 into a 50 ml graduated flask (point 4.2.2) and make up to the mark with 2-propanol (point 3.8). Pipette 5,0 ml of this solution into a 25 ml graduated flask and make up to the mark with 2-propanol (point 3.8). The nominal concentration of this solution is 6,72 IU vitamin A per ml. Measure the UV spectrum of this solution against 2-propanol (point 3.8) in the spectrophotometer (point 4.6) between 300 nm and 400 nm. The extinction maximum must be between 325 nm and 327 nm.

Calculation of the vitamin A content:

Image omitted; see official source

Image omitted; see official source

6. Calculation of the results

From the mean height (area) of the vitamin A peaks of the sample solution determine the concentration of the sample solution in IU/ml by reference to the calibration graph (point 5.6.2).

The vitamin A content w in IU/kg of the sample is given by the following formula:

Image omitted; see official source

in which:

c	=	vitamin A concentration of the sample solution (point 5.4) in IU/ml
---	---	---
V1	=	volume of sample solution (point 5.4) in ml
---	---	---
V2	=	volume of aliquot taken in point 5.4 in ml
---	---	---
m	=	weight of the test portion in g.

7. Observations

• 7.1.For samples with low vitamin A concentration it may be useful to combine the light petroleum-extracts of two saponification-charges (amount weighed: 25 g) to one sample solution for HPLC-determination.

• 7.2.The weight of the sample taken for the analysis shall not contain more than 2 g fat.

• 7.3.If phase separation does not occur add approximately 10 ml ethanol (point 3.1) to break the emulsion.

• 7.4.With cod-liver oil and other pure fats the saponification time shall be extended to 45-60 minutes.

• 7.5.Hydroquinone can be used instead of BHT.

• 7.6.Using a normal phase-column the separation of retinol isomers is possible. But in that case, the heights (areas) of all cis and trans isomers peaks shall be summed for calculations.

• 7.7.Approximately 150 mg ascorbic acid can be used instead of sodium ascorbate solution.

• 7.8.Approximately 50 mg EDTA can be used instead of sodium sulphide solution.

• 7.9.In cases of analysis of vitamin A in milk replacers, specific attention has to be paid:

• — at saponification (point 5.2): due to the amount of fat present in the sample, increasing of potassium hydroxide solution amount (point 3.4) may be necessary,

• — at extraction (point 5.3): due to the presence of emulsions, adaptation of the water/ethanol 2:1 ratio may be necessary.

• To check if the applied method of analysis generates reliable results on this specific matrix (milk replacer), a recovery test shall be applied on an additional test portion. If the recovery rate is lower than 80 %, the analytical result has to be corrected for recovery.

8. Repeatability

The difference between the results of two parallel determinations carried out on the same sample must not exceed 15 % relative to the higher result.

9. Results of a collaborative study ([^E0035])