CELEX: 31973L0046
Language: en
Date: 1972-12-05 00:00:00
Title: Fourth Commission Directive 73/46/EEC of 5 December 1972 establishing Community methods of analysis for the official control of feedingstuffs

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31973L0046

Fourth Commission Directive 73/46/EEC of 5 December 1972 establishing Community methods of analysis for the official control of feedingstuffs  

Official Journal L 083 , 30/03/1973 P. 0021 - 0034 Finnish special edition: Chapter 3 Volume 5 P. 0115  Greek special edition: Chapter 03 Volume 9 P. 0114  Swedish special edition: Chapter 3 Volume 5 P. 0115  Spanish special edition: Chapter 03 Volume 6 P. 0230  Portuguese special edition Chapter 03 Volume 6 P. 0230 

FOURTH COMMISSION DIRECTIVE  of 5 December 1972  establishing Community methods of analysis for the official control of feedingstuffs  (73/46/EEC)  THE COMMISSION OF THE EUROPEAN COMMUNITIES,  Having regard to the Treaty establishing the European Economic Community;  Having regard to the Council Directive of 20 July 1970 (1) on the introduction of Community methods of sampling and analysis for the official control of feedingstuffs, as amended by the Directive No 72/275/EEC of 20 July 1972 (2), and in particular Article 2 thereof;  Whereas that Directive requires that official controls of feedingstuffs be carried out using Community methods of sampling and analysis for the purpose of checking compliance with requirements arising under the provisions laid down by law, Regulation or administrative action concerning the quality and composition of feedingstuffs;  Whereas Directives No 71/250/EEC of 15 June 1971 (3), No 71/393/EEC of 18 November 1971 (4) and No 72/199/EEC of 27 April 1972 (5) have already established a number of Community methods of analysis ; whereas the progress of work since then makes it advisable to adopt a fourth set of methods;  Whereas the measures provided for in this Directive are in accordance with the Opinion of the Management Committee for Feedingstuffs;  HAS ADOPTED THIS DIRECTIVE:    Article 1 The Member States shall require that analyses for official controls of feedingstuffs as regards moisture contents of animal and vegetable fats and oils and magnesium and crude fibre contents of feedingstuffs be carried out according to the methods described in Annex I to this Directive.  The general provisions set out in Part 1 (Introduction) of the Annex to the First Commission Directive No 71/250/EEC of 15 June 1971 shall be applicable to the methods described in Annex I to this Directive.   Article 2 The Member States shall require that analyses for official controls of feedingstuffs as regards their contents of retinol (vitamin A), thiamine (aneurine, vitamin B1), ascorbic and dehydroascorbic acids (vitamin C be carried out according to the methods described in Annex II to this Directive.  The general provisions set out in Part 1 (Introduction) of the Annex to the First Commission Directive 71/250/EEC of 15 June 1971, with the exception of the part dealing with preparation of the sample to be analysed, shall be applicable to the methods described in Annex II to this Directive.   Article 3 The Member States shall, not later than 1 January 1974, bring into force the laws, regulations or administrative provisions necessary to comply with this Directive. They shall forthwith notify the Commission thereof.   Article 4 This Directive is addressed to the Member States.     Done at Brussels, 5 December 1972.  For the Commission  The President  S.L. MANSHOLT  (1)OJ No L 170, 3.8.1970, p. 2. (2)OJ No L 171, 29.7.1972, p. 39. (3)OJ No L 155, 12.7.1971, p. 13. (4)OJ No L 279, 20.12.1971, p. 7. (5)OJ No L 123, 29.5.1972, p. 6.     ANNEX I 1. DETERMINATION OF MOISTURE IN ANIMAL AND VEGETABLE FATS AND OILS   1. Purpose and Scope  This method makes it possible to determine the water and volatile substances content of animal and vegetable fats and oils.  2. Principle  The sample is dried to constant weight at 103 ºC. The loss in mass is determined by weighing.  3. Apparatus    3.1. Flat-bottomed dish, of a corrosion-resistant material, 8 to 9 cm in diameter and approximately 3 cm high.       3.2. Mercury thermometer with a strengthened bulb and expansion tube at the top end, graduated from approximately 80 ºC to at least 110 ºC, and approximately 10 cm in length.       3.3. Sand bath or electric hot-plate.       3.4. Desiccator, containing an efficient drying agent.       3.5. Analytical balance.         4. Procedure  Weigh out to the nearest mg approximately 20 g of the homogenized sample into the dry, weighed dish (3.1) containing the thermometer (3.2). Heat on the sand bath or hot-plate (3.3), stirring continuously with the thermometer, so that the temperature reaches 90 ºC in about 7 minutes.  Reduce the heat, watching the frequency with which bubbles rise from the bottom of the dish. The temperature must not exceed 105 ºC. Continue to stir, scraping the bottom of the dish, until bubbles stop forming.  In order to ensure complete elimination of moisture, reheat several times to 103 ºC ± 2 ºC, cooling to 93 ºC between successive heatings. Then leave to cool to room temperature in the desiccator (3.4) and weigh. Repeat this operation until the loss in mass between two successive weighings no longer exceeds 2 mg.  N.B. An increase in the mass of the sample after repeated heating indicates an oxidation of the fat, in which case calculate the result from the weighing carried out immediately before the mass began to increase.  5. Calculation of results  The moisture content, as a percentage of the sample, is given by the following formula: >PIC FILE= "T0006028">   where:  M0 = mass, in grammes, of the test sample;  M1 = mass, in grammes, of the dish with its contents before heating;  M2 = mass, in grammes, of the dish with its contents after heating.  Results lower than 0.05 % must be recorded as "lower than 0 705 %".  Repeatability  The difference in moisture between the results of two parallel determinations carried out on the same sample must not exceed 0 705 %, in absolute value.   2. DETERMINATION OF MAGNESIUM  - by atomic absorption spectrophotometry -    1. Purpose and Scope  This method makes it possible to determine the quantity of magnesium in feeding stuffs. It is particularly appropriate for determining magnesium contents lower than 5 %.       2. Principle  The sample is ashed and dissolved in dilute hydrochloric acid. If it contains no organic substances, it is dissolved directly in dilute hydrochloric acid. The solution is diluted and the magnesium content determined by atomic absorption spectrophotometry at 285 72 nm, by comparison with standard solutions.       3. Reagents      3.1. Hydrochloric acid a.p. d : 1 716.           3.2. Concentrated hydrochloric acid a.p. d : 1 719.           3.3. Magnesium ribbon or wire, or magnesium sulphate heptahydrate, dried at room temperature.           3.4. Strontium salt solution (chloride or nitrate) at 2 75 % (w/v) strontium (= 76 708 g SrC12  7 6H2O a.p., or 60 738 g Sr (NO3)2 a.p.).           3.5. Standard magnesium solution : weigh out to the nearest mg 1 g magnesium (3.3) which has previously had its oxide coating carefully removed, or the corresponding quantity (10 7143 g) of magnesium sulphate heptahydrate (3.3). Place in a 1 000 ml graduated flask, add 80 ml hydrochloric acid (3.1), leave to dissolve and make up to 1 000 ml with water. 1 ml of this solution contains 1.000 mg magnesium.                  4. Apparatus      4.1. Platinum, silica or porcelain ashing crucibles.           4.2. Thermostatically controlled electric muffle furnace.           4.3. Atomic absorption spectrophotometer.                  5. Procedure      5.1. Preparation of the sample solution        5.1.1. Feeding stuffs composed exclusively of mineral substances  Weigh out to the nearest mg 5 g of the sample into a 500 ml graduated flask with 250 to 300 ml water. Add 40 ml hydrochloric acid (3.1), bring to the boil and keep the liquid gently boiling for 30 minutes. Leave to cool, make up to volume with water, mix and filter into a dry beaker through a dry pleated filter. Discard the first 30 ml of the filtrate. In the presence of silica, treat 5 g of sample with a sufficient quantity (15-30 ml) of hydrochloric acid (3.2), evaporate to dryness on a water bath and transfer to an oven at 105 ºC for one hour. Proceed as from the third sentence of 5.1.2.               5.1.2. Feeding stuffs composed predominantly of mineral substances  Weigh out to the nearest mg 5 g of the sample into a crucible and ash at 550 ºC in the muffle furnace until an ash which is free from carbonaceous particles is obtained, and leave to cool. In order to eliminate silica, add to the ash a sufficient quantity (15-30 ml) of hydrochloric acid (3.2), evaporate to dryness on a water bath and transfer to an oven at 105 ºC for one hour. Treat the residue with 10 ml hydrochloric acid (3.1) and transfer to a 500 ml graduated flask using warm water. Leave to cool and make to volume with water. Mix and filter into a dry beaker through a dry pleated filter. Discard the first 30 ml of the filtrate.                5.1.3. Feedingstuffs composed predominantly of organic substances  Weigh out to the nearest mg 5 g of the sample into a crucible and ash at 550 ºC in the muffle furnace until an ash which is free from carbonaceous particles is obtained. Treat the ash with 5 ml hydrochloric acid (3.2), evaporate to dryness on a water bath and then dry for one hour in the oven at 105 ºC in order to render the silica insoluble. Treat the ash with 5 ml hydrochloric acid (3.1), transfer to a 250 ml graduated flask using warm water, bring to the boil, leave to cool and make up to volume with water. Mix and filter into a dry beaker through a dry pleated filter. Discard the first 30 ml of the filtrate.           5.2. Measurement by atomic absorption  By diluting the standard solution (3.5) with water, prepare at least 5 reference solutions of increasing concentration, corresponding to the optimal measuring range of the spectrophotometer. Add to each solution 10 ml strontium salt solution (3.4) and then make up the volume to 100 ml with water. Dilute with water one aliquot part of the filtrate obtained from 5.1.1, 5.1.2 or 5.1.3, so as to obtain a magnesium concentration which is within the limits of concentration of the reference solutions. The hydrochloric acid concentration of this solution must not exceed 0 74 N. Add 10 ml strontium salt solution (3.4) and then make up the volume to 100 ml with water. Measure the absorption of the solution to be determined and of the reference solutions at 285.2 nm.       6. Calculation of results  Calculate the quantity of magnesium in the sample by relation to the reference solutions. 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 must not exceed 5 %, in relative value.  3. DETERMINATION OF CRUDE FIBRE    1. Purpose and Scope  This method makes it possible to determine in feeding stuffs the quantity of fat-free organic substances which are insoluble in acid and alkaline media and are conventionally described as crude fibre.       2. Principle  The sample, defatted where necessary, is treated successively with boiling solutions of sulphuric acid and potassium hydroxide of specified concentrations. The residue is separated by filtration in the presence of asbestos, washed, dried, weighed and ashed at 900 ºC. The loss of weight resulting from ashing corresponds to the crude fibre present in the test sample.       3. Reagents      3.1. Sulphuric acid 0.26 N.           3.2. Treated asbestos : add to asbestos of the type used with a Gooch crucible approximately 5 times its weight of dilute hydrochloric acid (1 volume hydrochloric acid, d : 1 719 + 3 volumes of water). Boil the mixture for approximately 45 minutes, leave to cool and filter through a Buchner funnel. Wash the residue first with water until hydrochloric acid has disappeared from the washing water, and then with acetone (3.6). Dry the asbestos in the drying oven and then ash for 2 hours at 900 ºC. Leave to cool and keep in a stoppered flask. Asbestos treated in this way may be used several times. It must meet the specifications given in 5. regarding the blank test.            3.3. Antifoaming emulsion (eg silicone).           3.4. Potassium hydroxide solution 0 723 N.           3.5. Hydrochloric acid 0 75 N.           3.6. Acetone.           3.7. Diethyl ether.                  4. Apparatus      4.1. Beakers of at least 600 ml capacity, with measuring marks at the 200 ml level.           4.2. Porcelain discs approximately 80 mm in diameter and approximately 4 mm thick, perforated with approximately 32 holes, each approximately 4 mm in diameter.           4.3. Rubber-stoppered vacuum flasks of approximately 2 litre capacity, with measuring marks at the 800 ml level and fitted with glass funnels 120 mm in diameter.           4.4. Filter plates approximately 40 mm in diameter and approximately 4 mm thick, with slanting edges to fit the cone of the funnel (4.3), perforated with approximately 16 holes, each approximately 4 mm in diameter, and covered by a wire mesh, the mesh size being approximately 1 mm. Both plates and wire mesh must be resistant to acids and alkalis.           4.5. Platinum or silica ashing crucibles.           4.6. Thermostatically controlled electric muffle furnace.           4.7. Desiccator.           4.8. Asbestos filter : Suspend 2 70 g asbestos (3.2) in 100 ml water.  Filter under vacuum over a filter plate covered with a wire mesh (4.4) and placed in the funnel of a vacuum flask (4.3). Collect the filtrate and filter once more through the same filter. Discard the filtrate.       5. Procedure  Weigh out to the nearest mg 3 g of the sample and 2 g treated asbestos (3.2) into a beaker (4.1), add 200 ml sulphuric acid (3.1) and a few drops of antifoaming emulsion (3.3). Bring rapidly to the boil and leave to boil for exactly 30 minutes. To keep a constant volume, cover the beaker with a cooling device such as a 500 ml round-bottomed flask in which cold water is circulated. Stop boiling by adding approximately 50 ml cold water and filter immediately under vacuum through an asbestos filter previously prepared as shown in 4.8.  Wash the residue with 5 lots of approximately 100 ml of very hot water to obtain a final volume of filtrate of 800 ml. Transfer the residue quantitatively to the beaker (4.1) which has first been fitted with a porcelain disc (4.2) to regulate the boiling. Add 200 ml potassium hydroxide solution (3.4). Bring rapidly to the boil and leave to boil for exactly 30 minutes. Add approximately 50 ml cold water and filter immediately under vacuum through a fresh asbestos filter previously prepared as shown in 4.8. Wash the residue with very hot water until the washing water is neutral (test with litmus paper), then 3 times with acetone (3.6) (approximately 100 ml acetone in all).  Transfer the residue quantitatively to an ashing crucible (4.5), break up if necessary and dry to constant weight in the drying oven at 130 ºC.  Leave to cool in the desiccator (4.7) and weigh rapidly.  Place the crucible in the muffle furnace (4.6) and leave to ash for 30 minutes at 900 ºC. Leave to cool in the desiccator (4.7) and weigh rapidly,  Carry out a blank test applying the same procedure to the treated asbestos (3.2), but without the sample. Loss of weight resulting from ashing of the 6 g asbestos must not exceed 10 mg.       6. Calculation of results  The crude fibre content, as a percentage of the sample, is given by the ratio: >PIC FILE= "T0006029">    where:  a = loss of weight after ashing during the determination;  b = loss of weight after ashing during the blank test.  Repeatability  The difference between the results of two parallel determinations carried out on the same sample must not exceed:  0 73, in absolute value, for crude fibre contents lower than 10 %;  3 %, in relative value, for crude fibre contents equal to or greater than 10 %.       7. Observations     7.1. Feedingstuffs containing more than 10 % oils and fats must be defatted prior to analysis with diethyl ether (3.7). To do this, place the test sample (3 g weighed to the nearest mg) on an asbestos filter (4.8). Cover 3 times with approximately 50 ml diethyl ether (3.7) and each time filter carefully under vacuum. Transfer the defatted test sample and the asbestos quantitatively to a beaker (4.1) and continue the analysis as shown in 5.       7.2. Feedingstuffs containing oils and fats which cannot be extracted directly must be defatted as shown in 7.1 and defatted a further time after the acid attack has been washed from the residue.  To do this, wash this attack from the residue 3 times with acetone (3.6) (100 ml in all), then 3 times with 50 ml diethyl ether (3.7). Then transfer the residue quantitatively to a beaker (4.1) and continue the analysis as shown in the second paragraph of 5 (treatment with potassium hydroxide solution).       7.3. If the feedingstuffs are rich in calcium (more than 2 % calcium), place the test sample (3 g weighed to the nearest mg) in a beaker (4.1) with 100 ml hydrochloric acid 0 75 N (3.5) and leave to stand in a cool temperature for 5 minutes. Filter immediately and wash in cold water. Use as a filtration aid the 2 70 g asbestos specified for boiling with sulphuric acid. If filtration proves difficult, dilute the suspension with acetone (3.6). Then proceed as shown in 5.               ANNEX II 1. DETERMINATION OF RETINOL (VITAMIN A)    1. 1. Purpose and Scope  This method makes it possible to determine the quantity of retinol (Vitamin A) in feedingstuffs, concentrates and premixes. The lower limit of the determination is 10 000 IU/kg for highly pigmented feeds and 4 000 IU/kg for others (1). Products are classified in two groups, according to their presumed retinol content:  Group A : contents lower than 200 000 IU/kg;  Group B : contents equal to or greater than 200 000 IU/kg.  2. Principle  The sample is hydrolised when hot with a potassium hydroxide solution in an ethanol medium and in the presence of an antioxidant or in a nitrogen atmosphere. The mixture is extracted with 1,2-dichlorethane. The extract is evaporated to dryness and treated with light petroleum. The solution is chromatographed on a column of aluminium oxide (for  (1)1 IU = 0.3 ¶g of retinol.   Group B products, chromatography is only required in certain cases). For Group A products the retinol is determined by spectrophotometry at 610 nm after development of a coloured complex according to the Carr-Price reaction ; for Group B products by spectrophotometry in the UV at 325 nm.  3. Reagents    (a) used for analysing products of Groups A and B  3.1. 96 % (v/v) ethanol.  3.2. 10 % (w/v) sodium ascorbate solution a.p., or  3.3. Purified nitrogen.  3.4. 50 % (w/v) potassium hydroxide solution a.p.  3.5. Potassium hydroxide solution 1 N a.p.  3.6. Potassium hydroxide solution 0 75 N a.p.  3.7. 1,2-dichlorethane a.p.  3.8. Light petroleum, boiling range : 30-50 ºC. If necessary, purify as follows : stir 1 000 ml light petroleum with 20 ml lots of concentrated sulphuric acid until the acid remains colourless. Remove the acid and wash the light petroleum successively with 500 ml water, twice with 250 ml of 10 % (w/v) sodium hydroxide solution and three times with 500 ml water. Remove the aqueous layer, dry the light petroleum for 1 hour over active carbon and anhydrous sodium sulphate, filter and distil.  3.9. Aluminium oxide, standardized according to Brockmann : ash for 8 hours at 750 ºC, cool in a desiccator and keep in a brown glass bottle fitted with a ground-glass stopper. Before use in chromatography moisten as follows : place in a brown glass bottle 10 g aluminium oxide and 0 77 ml water, seal with a stopper, reheat for 5 minutes in a boiling water bath while shaking. Leave to cool. Verify the activity of the aluminium thus prepared by subjecting a known quantity of retinol (3.17) (ca. 500 IU) to the procedure of 5.3 and 5.4 and checking recovery.  3.10. Basic aluminium oxide, degree of activity 1 (Woelm, Merck or equivalent).  3.11. Pure diethyl ether. Remove peroxides and traces of water by chromatography on a column of basic aluminium oxide (3.10). (25 g aluminium oxide per 250 ml diethyl ether.)  3.12. Light petroleum solutions (3.8) at 4, 8, 12, 16 and 20 % (v/v) diethyl ether (3.11).  3.13. Sodium sulphide solution 0 75 molar in 70 % (v/v) glycerine, prepared from sodium sulphide a.p.       (b) used exclusively for analysing Group A products  3.14. Crystallizable benzene a.p.  3.15. Chloroform a.p. Remove the ethanol, phosgene and traces of water by chromatography on a column of basic aluminium oxide (3.10) (50 g aluminium oxide per 200 ml chloroform ; it is advisable to chromatograph the first 50 ml of the eluate a second time).  3.16. Carr-Price reagent : stir approximately 25 g antimony trichloride a.p. (kept in a desiccator) with 100 ml chloroform (3.15) until the solution is saturated. A slight deposit of antimony trichloride causes no problem. Add 2 ml acetic anhydride a.p. Keep in a refrigerator in a brown glass bottle with ground-glass stopper. The solution keeps for several weeks.  3.17. Retinol - standardized spectrophotometrically.       (c) used exclusively for analysing Group B products  3.18. Isopropanol, for chromatography.   4. Apparatus    4.1. Water bath.       4.2. Vacuum evaporation apparatus with round flasks of different capacities.       4.3. Glass chromatography tubes (length : 300 mm ; internal diameter : about 13 mm).       4.4. Spectrophotometer with 10 mm cells. Measurements in the UV require silica cells.       4.5. UV lamps suitable for 365 nm.         5. Procedure  N.B. All operations must be carried out away from direct light, if necessary in brown glass equipment.    5.1. Test Sample  From the finely divided sample, take a test sample proportional to the presumed retinol content, thus:  0 71-1 70 g for concentrates (contents greater than 20 000 IU/g);   3 70-5 70 g for premixes (contents of between 400 and 20 000 IU/g);  10-20 g for mineral mixtures;  30 g for Group A products.  Immediately place the test sample in a 500 ml flask with a ground-glass stopper.         5.2. Hydrolysis and extraction (1)  Add successively to the test sample 40 ml ethanol (3.1), 2 ml sodium ascorbate solution (3.2) (2), 10 ml potassium hydroxide solution (3.4) and 2 ml sodium sulphide solution (3.13).  Heat for 30 minutes at 70-80 ºC under a reflux condenser and then leave to cool under a stream of water. Add 50 ml ethanol (3.1) and 100 ml 1,2-dichlorethane (3.7) (taken with a pipette). Shake vigorously and then decant the supernatant liquid into a decanting container. Add to the container 150 ml potassium hydroxide solution (3.5), shake for 30 seconds and leave to stand until the layers are separated. Collect the dichlorethane layer (lower layer) in a decanting container, add 40 ml potassium hydroxide solution (3.6), shake for 10 seconds and leave to stand until the layers are separated. Collect the dichlorethane layer in a decanting container and wash 6-8 times with 40 ml lots of water until free of alkali (phenolphthalein test). Collect the dichlorethane layer and remove the last traces of water using strips of filter paper.  Evaporate to dryness an aliquot part of the solution under vacuum and on the water bath at 40 ºC. Rapidly treat the residue with 5 ml light petroleum (3.8).  For Group A products, chromatograph as shown in 5.3.1.  For Group B products, transfer the solution to a 50 ml graduated flask, make up to volume with light petroleum (3.8), mix and measure the optical density as shown in 5.4.2.       5.3. Chromatography      5.3.1. Group A products  Fill a chromatography tube (4.3) to a height of 200 mm with 10 g aluminium oxide (3.9) previously slurried with light petroleum (3.8). Place in the tube the solution obtained in 5.2 and immediately add 20 ml light petroleum (3.8). Elute successively with 10 ml lots of the light petroleum solutions at 4, 8, 12, 16 and 20 % diethyl ether (3.12) under pressure or partial vacuum, the rate of flow being 2 to 3 drops per second.  (1)For milk feeds and products with a tendency to agglomerate or swell, double the quantity of the reagents shown in the first and second paragraphs of 5.2. (2)Sodium ascorbate need not be added when hydrolysis is carried out in a nitrogen atmosphere.   The carotene is eluted first (1). The retinol is generally eluted with the light petroleum solution at 20 % diethyl ether (3.12). The elution is followed under UV light (brief irradiation of the column with the mercury lamp). The fluorescent zone of the retinol is clearly separated from the yellow xanthophyll zones following it. Collect the eluate fraction containing the retinol in an Erlenmeyer flask.           5.3.2. Group B products  Chromatography must only be carried out if the optical density measurements obtained in 5.4.2 do not conform to the requirements given in 5.4.2.  If chromatography proves necessary, place in the chromatography column an aliquot part of the solution in the light petroleum obtained in 5.2, containing approximately 500 IU of retinol, and chromatograph as shown in 5.3.1.       5.4. Measurement of the optical density    5.4.1. Group A products  Evaporate to dryness under vacuum the eluate containing the retinol obtained in 5.3.1. Treat the residue with 2 ml benzene (3.14). Take 0.3 ml of this solution and add 3 ml of the Carr-Price reagent (3.16). A blue colouring develops. Measure the optical density with the spectrophotometer at 610 nm exactly 30 seconds after the reaction has begun. Determine the retinol content by reference to a standard curve obtained from benzene solutions of increasing retinol-standard concentrations treated with Carr-Price reagent (2 to 16 IU retinol-standard (3.17) per 0 73 ml benzene (3.14) +3 ml Carr-Price reagent (3.16)). The standard curve must be checked regularly and frequently using the standard and a freshly prepared Carr-Price reagent solution.       5.4.2. Group B products  Take an aliquot part of the solution in light petroleum obtained in 5.2 containing approximately 200 IU retinol. Evaporate to dryness under vacuum and treat the residue with 25 ml isopropanol (3.18). Measure the optical density in the spectrophotometer at 325, 310 and 334 nm. The absorption maximum is located at 325 nm. The retinol content of the solution is calculated as follows:  E325  7 18 730 = IU of retinol/ml  However, the ratio of the optical densities  E310 : E325 and E334 : E325  must be 6 : 7 = 0 7857.  If one of these ratios differs appreciably from this value ( 0 7880), the measurement of the optical densities must be preceded by chromatography in accordance with the method given in 5.3.2. If the measurement of the optical densities carried out after chromatography shows that the abovementioned ratios still differ appreciably from the value of 0 7857 ( 0 7880), the determination must be carried out in accordance with the method given for Group A products.  6. Calculation of results  Calculate the retinol content of the sample taking into account the weight of the test sample and the dilutions carried out in the course of analysis. Express the results in IU of retinol per kg of feedingstuff or per kg of concentrate or premix. (1) >PIC FILE= "T0006030">    Repeatability  The difference between the results of two parallel determinations carried out on the same sample must not exceed:      - 20 %, in relative value, for retinol contents lower than 75 000 IU/kg;           - 15 000 IU for contents between 75 000 and 150 000 IU/kg;           - 10 %, in relative value, for contents between 150 000 and 250 000 IU/kg;           - 25 000 IU for contents between 25 000 and 500 000 IU/kg;           - 5 %, in relative value, for contents greater than 500 000 IU/kg.             2. DETERMINATION OF THIAMINE (VITAMIN B1, ANEURINE)      1. Purpose and Scope  This method makes it possible to determine the quantity of thiamine (aneurine, Vitamin B1) in feeding stuffs, concentrates and premixes. The lower limit of the determination is 5 ppm.           2. Principle  The solution is treated when hot with dilute sulphuric acid and then hydrolysed enzymatically. The solution obtained is subjected to alkaline oxidation. The thiochrome formed is extracted with isobutanol and determined by fluorimetry.           3. Reagents  3.1. 100 ¶g/ml standard thiamine solution : dissolve 112 73 mg thiamine hydrochloride, previoulsly dried under vacuum to constant weight, in 1000 ml sulphuric acid 0 72 N (3.2). If stored in a cool, dark place, this solution keeps for one month.  3.2. Sulphuric acid 0 7.2 N.  3.3. Pure sodium bisulphite.  3.4. 20 % (w/v) potassium ferricyanide solution a.p.  3.5. 25 % (w/v) potassium hydroxide solution a.p.  3.6. Oxidizing mixture : mix 2 ml potassium ferricyanide solution (3.4) with 48 ml potassium hydroxide solution (3.5). This mixture does not keep for more than 4 hours.  3.7. Isobutanol a.p.  3.8. Sodium acetate solution 2 75 N.  3.9. Multienzymatic preparation containing protease, phosphatase and amylase (eg, Clarase).  3.10. 96 % (v/v) ethanol.           4. Apparatus        4.1. Water bath.               4.2. Centrifuge (3 500 rpm), with 30 to 50 ml tubes fitted with ground-glass stoppers.               4.3. Fluorimeter.                          5. Procedure        5.1. Enzymatic hydrolysis  Place in each of two 250 ml graduated flasks, A and B, identical amounts of the finely divided sample containing approximately 100 ¶g thiamine and 125 ml sulphuric acid (3.2). Also add, to flask A only, 1 70 ml standard solution (3.1) (internal standard).   Shake the flasks vigorously, place on a boiling water bath and keep there for 15 minutes, shaking occasionally. Leave to cool to approximately 45 ºC. Add to each flask 20 ml sodium acetate solution (3.8) and 0 75 g multienzymatic preparation (3.9), then leave to stand for 20 minutes at room temperature. Add 20 ml sodium acetate solution (3.8), make up to volume with water, homogenize and filter. Collect filtrates A and B after having discarded the first 15 ml. Prepare the following solutions:          5.1.1. Reference solution T Place in a centrifuge tube (4.2) 5 ml filtrate A and approximately 10 mg sodium bisulphite (3.3). Immerse the tube in a boiling water bath for 15 minutes and then leave to cool to room temperature.                   5.1.2. Solutions A (internal standard) and B (sample)  Place 5 ml filtrate A in a centrifuge tube (4.2) and 5 ml filtrate B in another centrifuge tube (4.2).               5.2. Oxidation  Add to solutions T, A and B 5 ml of the oxidising mixture (3.6) and, one minute later, 10 ml isobutanol (3.7). Stopper the tubes and shake vigorously for 5 seconds. Leave to stand for one minute and centrifuge so as to separate the layers. From each tube transfer 5 ml of the supernatant isobutanol layer to each of the 25 ml graduated flasks, make up to volume with ethanol (3.10) and homogenize (= extracts T, A and B).               5.3. Measurement of fluorescence  Carry out the measurements at the wavelength for which the fluorimeter gives an optimal response to the fluorescence of the thiochrome. Irradiate at approximately 365 nm.  Adjust the instrument to zero using extract T. Measure the intensity of fluorescence of extracts A and B.           6. Calculation of results  The thiamine content in mg/kg of the sample is given by the ratio: >PIC FILE= "T0006031">   where:  a = intensity of fluorescence of extract A (internal standard);  b = intensity of fluorescence of extract B (sample);  c = weight of the test sample in g;  d = amount of thiamine in ¶g added to the test sample (internal standard).  Repeatability  The difference between the results of two parallel determinations carried out on the same sample must not exceed:  10 %, in relative value, for contents lower than 500 mg/kg, and  5 %, in relative value, for contents equal to or greater than 500 mg/kg.  3. DETERMINATION OF ASCORBIC ACID AND DEHYDROASCORBIC ACID (VITAMIN C)      1. Purpose and Scope  This method makes it possible to determine the total quantity of ascorbic and dehydroascorbic acids (Vitamin C) in feedingstuffs, concentrates and premixes. The lower limit of the determination is 5 ppm. Products are classified in two groups, according to their presumed Vitamin C content:  Group A : contents lower than 10 g/kg;  Group B : contents equal to or greater than 10 g/kg.            2. Principle  The sample is suspended in a dilute solution of metaphosphoric acid and extracted with chloroform. The aqueous phase is treated with a solution of 2,6-dichlorophenol-indophenol in order to transform the ascorbic acid into dehydroascorbic acid, and then with a solution of 2,4-dinitrophenylhydrazine. The hydrazone formed is extracted with a mixture of ethyl acetate, glacial acetic acid and acetone. The solution is chromatographed on a column of silica gel, the eluate evaporated to dryness and the residue dissolved in dilute sulphuric acid. The optical density of the solution is measured by a spectrophotometer at 509 nm.  For Group A products the eluate resulting from chromatography on the column is further subjected to thin layer chromatography to isolate the hydrazone.           3. Reagents        3.1. Standard solution of 0 705 % L-ascorbic acid : dissolve 50 mg L-ascorbic acid a.p. in approximately 20 ml metaphosphoric acid solution (3.2) and make up to 100 ml with water. Prepare immediately before use.  3.2. 10 % (w/v) metaphosphoric acid solution : after grinding it in a mortar, dissolve in water 200 g metaphosphoric acid a.p. and make up to 2 000 ml with water. Keep at 4 ºC. This is stable for one week.  3.3. Chloroform a.p.  3.4. Solution of 0 75 % (w/v) 2,6-dichlorophenol-indophenol a.p. Prepare immediately before use.  3.5. Filtration aid (S. and S. No 121 or equivalent).  3.6. Acid solution of 2 % (w/v) 2,4-dinitrophenylhydrazine : dissolve 2 g 2,4-dinitrophenylhydrazine in 100 ml dilute sulphuric acid (25 ml sulphuric acid a.p., d : 1.84, diluted by making up to 100 ml with water). Stored at a cool temperature this solution keeps for one week.  3.7. Nitrogen, or  3.8. Carbon dioxide.  3.9. Mixture of ethyl acetate a.p. /glacial acetic acid/acetone a.p. : 96/2/2 in volume.  3.10. Mixture of dichloromethane a.p. /glacial acetic acid : 97/3 in volume.  3.11. Silica gel, particle size : 0 705 to 0 72 mm.  3.12. Stahl grade silica gel H, for thin layer chromatography.  3.13. Dilute sulphuric acid : place 105 ml water in a 200 ml graduated flask, make up to volume with sulphuric acid a.p., d : 1 784.  3.14. Eluting solvent for thin layer chromatography : mix 75 ml diethyl ether a.p., 25 ml ethyl acetate a.p. and 4 70 ml 96 % (w/v) acetic acid a.p. Renew after 2 to 3 chromatographies.           4. Apparatus        4.1. Water bath fitted with a thermostat set at 20 ºC.               4.2. Centrifuge (3 500 rpm) with 40 to 50 ml tubes fitted with ground-glass stoppers.               4.3. Rotary vacuum evaporator with 250 ml flasks.               4.4. Glass chromatography tubes (length : 100 mm, internal diameter : 20 mm), with a sintered disc (eg Allihn tubes).               4.5. Spectrophotometer or colorimeter with filters with 10 mm cells.               4.6. Apparatus for thin layer chromatography, with silica gel plates (3.12) coated to a depth of 0 75 to 0 76 mm. (Ready-made plates are appropriate.) Dry the plates for 2 1/2 to 3 hours in the drying oven at 120 to 130 ºC. Leave to cool and then keep in a desiccator for at least 24 hours before use.               4.7. Drying oven set at 120 to 130 ºC.                           5. Procedure        5.1. Extraction  Place in each of two 250 ml graduated flasks (with ground-glass stoppers), A and B, identical quantities of the finely divided sample containing about 200 ¶g vitamin C. Add to flask A only 0 74 ml standard solution (3.1) and mix, shaking gently (internal standard).  Add to each flask 30 ml chloroform (3.3) and 25 ml metaphosphoric acid solution (3.2) at 4 ºC. Shake briefly and then leave to stand for 10 to 15 minutes. Add 25 ml water, stopper the flasks, shake vigorously for 10 seconds and leave to stand for 10 to 15 minutes in the water bath (4.1). Centrifuge to separate the aqueous phase from the chloroform phase. Carry out the operations simultaneously, as described below, on the aqueous extracts A (internal standard) and B.               5.2. Oxidation  Using a pipette, transfer 40 ml of the supernatant aqueous solution (slightly cloudy) obtained in 5.1 to a reaction tube fitted with a ground-glass stopper, add 0 75 to 1 ml 2,6-dichlorophenol-indophenol solution (3.4) and mix. A red colouring develops which should remain for at least 15 minutes. Then add approximately 300 mg filtration aid (3.5), shake and filter through a dry pleated filter. The filtrate need not necessarily be clear.               5.3. Reaction with 2,4-dinitrophenylhydrazine and hydrazone extraction  Using a pipette, transfer 10 ml of the filtrate obtained in 5.2 to a centrifuge tube (4.2), add 2 ml 2,4-dinitrophenylhydrazine solution (3.6) and mix. Pass a stream of nitrogen (3.7) or carbon dioxide (3.8) rapidly into the tube, stopper the tube and immerse it for approximately 15 hours (overnight) in the water bath (4.1).  Then add 3 ml water, 20 ml of the ethyl acetate/glacial acetic acid/acetone mixture (3.9) and approximately 800 mg filtration aid (3.5). Stopper the tube, shake vigorously for 30 seconds and centrifuge. Place 15 ml of the supernatant phase in an evaporation flask and evaporate under reduced pressure in the rotary evaporator (4.3) until an oily residue is obtained. Dissolve the residue in 2 ml of the ethyl acetate/glacial acetic acid/acetone mixture (3.9) by re-heating at 50 ºC, leave to cool, add 10 ml of the dichloromethane/glacial acetic acid mixture (3.10) and mix.               5.4. Chromatography on a column  Fill a chromatography tube (4.4) up to a level of 30 mm with the dichloromethane/ glacial acetic acid mixture (3.10). Suspend (shaking vigorously) 5 g silica gel (3.11) in 30 ml of the dichloromethane/glacial acetic acid mixture (3.10) ; pour the suspension into the tube. Leave to stand and then compress under nitrogen (3.7) at low pressure. Decant into the tube the solution obtained in 5.3, rinse the flask with a small quantity of the dichloromethane/glacial acetic acid mixture (3.10) and decant into the tube, then fill the latter with the mixture (3.10) and proceed to wash the column with the same mixture (3 to 4 lots of approximately 5 ml) until a colourless eluate is obtained. Discard the part of the eluate which is coloured yellow.  Elute the reddish zone at the top of the column with the ethyl acetate/glacial acetic acid/acetone mixture (3.9), collect the eluate and evaporate to dryness.          5.4.1. For Group A products (contents in vitamin C lower than 10 g/kg), dissolve the residue in 2.0 ml of the ethyl acetate/glacial acetic acid/acetone mixture (3.9) and chromatograph immediately on a thin layer as shown in 5.5.                   5.4.2. For Group B products (contents in vitamin C equal to or greater than 10 g/kg), treat the oily residue with 4.0 ml dilute sulphuric acid (3.13), shake vigorously to dissolve the residue completely and measure the optical density as shown in 5.6.                                   5.5. Thin layer chromatography  Carry out in duplicate the operations described as follows. Place in a thin line on the plate (4.6) 0 75 ml of the solution obtained in 5.4.1. Using the eluting solvent (3.14) develop for at least 20 minutes in a tank saturated with solvent vapour, until the pinkcoloured hydrazone zone is clearly separated. Leave to dry in the open. Mark out the limit of the pink zone, scrape away the zone with a spatula and quantitatively transfer the powder into a chromatography tube (4.4).  Elute successively once with 2 ml and twice with 1 75 ml of the ethyl acetate/glacial acetic acid/acetone mixture (3.9). Collect the eluate in a small flask (the last part must be colourless). Evaporate to dryness, treat the oily residue with 4 70 ml dilute sulphuric acid (3.13), shake vigorously to dissolve the residue completely and measure the optical density.               5.6. Measurement of the optical density  Measure the optical density with a spectrophotometer at 509 nm 20 to 30 minutes after dissolving the residue in sulphuric acid. Carry out the measurements by comparison with dilute sulphuric acid (3.13).               5.7. Blank test  Carry out a blank test applying the same procedure but without the sample.           6. Calculation of results  The vitamin C content of the sample in g per kg is given by the ratio: >PIC FILE= "T0006032">   where:  a = optical density of the blank;  b = optical density of the internal standard solution;  c = optical density of the sample solution;  d = weight, in grammes, of the test sample.  Repeatability  The difference between the results of two parallel determinations carried out on the same sample must not exceed:  10 %, in relative value, for vitamin C contents lower than 10 g/kg and  5 %, in relative value, for contents equal to or greater than 10 g/kg.