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Commission Regulation (EEC) No 4154/87 of 22 December 1987 laying down the methods of analysis and other technical provisions necessary for the implementation of Regulation (EEC) No 3033/80 laying down the trade arrangements applicable to certain goods resulting from the processing of agricultural products Official Journal L 392 , 31/12/1987 P. 0019 - 0028 Finnish special edition: Chapter 3 Volume 25 P. 0226 Swedish special edition: Chapter 3 Volume 25 P. 0226

COMMISSION REGULATION (EEC) No 4154/87 of 22 December 1987 laying down the methods of analysis and other technical provisions necessary for the implementation of Regulation (EEC) No 3033/80 laying down the trade arrangements applicable to certain goods resulting from the processing of agricultural products THE COMMISSION OF THE EUROPEANCOMMUNITIES, Having regard to the Treaty establishing the European Economic Community, Having regard to Council Regulation (EEC) No 2658/87 of 23 July 1987 on the tariff and statistical nomenclature and on the Common Customs Tariff (1) as amended by Regulation (EEC) No 3985/87 (2) and in particular Article 9 thereof, Whereas Commission Regulation (EEC) No 1061/69 (3), as last amended by Regulation (EEC) No 1822/86 (4), defined the analytical methods to be used for the application of Council Regulation (EEC) No 1059/69 (5); whereas Regulation (EEC) No 1059/69 was repealed and replaced by Council Regulation (EEC) No 3033/80 (6), as last amended by Regulation (EEC) No 3743/87 (7); Whereas the procedures for implementing Regulation (EEC) No 3033/80 for imports, were prescribed by Council Regulation (EEC) No 3034/80, of 11 November 1980, fixing the quantities of basic products considered to have been used in the manufacture of goods covered by Regulation (EEC) No 3033/80 (8) amending Regulation (EEC) No 950/68 on the Common Customs Tariff, as last amended by Regulation (EEC) No 4091/87 (9); Whereas in order to take account of the scientific and technological evolution of analytical methods and to continue to ensure the uniform treatment on import into the Community of goods covered by Regulation (EEC) No 3033/80, it is appropriate to repeal and replace Regulation (EEC) No 1061/69; Whereas the measures provided for in this Regulation are in accordance with the opinion of the Nomenclature Committee, HAS ADOPTED THIS REGULATION: Article 1 This Regulation lays down the methods of analysis necessary for the implementation of Regulations (EEC) No 3033/80 (as far as imports are concerned) and (EEC) No 3034/80, or, in the absence of a method of analysis, the nature of the analytical operations to be carried out or the principle of a method to be applied. Article 2 In accordance with the definitions set out in Annex III to Regulation (EEC) No 3034/80 concerning:- starch/glucose, content, and - sucrose/invert sugar/isoglucose contentand for the purpose of implementing Annexes II and III to that Regulation, the following formulae, procedures and methods shall be used: 1. Starch/glucose content:(expressed as 100 % anhydrous starch content of the goods as presented)(a) (Z F) × 0,9,if the glucose content is not less than the fructose content; or(b)(Z G) × 0,9,if the glucose content is less than the fructose contentwhere:Z is the glucose content determined by the method set out in Annex I of this Regulation; Fis the fructose content determined by HPLC (high performance liquid chromatography); Gis the glucose content determined by HPLC.In case 1 (a), where the presence of a lactose hydrolysate is declared and/or quantities of lactose and galactose are detected, a glucose content equivalent to the galactose content (determined by HPLC), shall be deducted from the glucose content (Z) before any other calculation is made. 2.Sucrose/invert sugar/isoglucose content:(expressed as sucrose content of the goods as presented)(a)S + (2F) × 0,95,if the glucose content is not less than the fructose content(b)S + (G + F) × 0,95,if the glucose content is less than the fructose contentwhere:Sis the sucrose content determined by HPLC; Fis the fructose content determined by HPLC; Gis the glucose content determined by HPLC.Where the presence of a lactose hydrolysate is declared and/or quantities of lactose and galactose are detected, a glucose content, equivalent to the galactose content (determined by HPLC), shall be deducted from the glucose (G) content before any other calculation is made. 3. Milk fat content:(a) Save as otherwise provided in paragraph (b) below, the milkfat content by weight shall be determined by extraction with light petroleum after hydrolysis with hydrochloric acid.(b)Where fats other than milkfats are also declared in the composition of the goods, the following procedure shall be applied: - the percentage of weight of the total fats in the goods shall be determined as mentioned in (a) above, -the percentage of weight of butyric acid in the total fats shall be obtained by IUPAC method No 23.10 (reference: Pure and Applied Chemistry, 1986, 58, No 10 pp. 1419-1428), -the percentage by weight of milk fats in the goods shall be calculated by multiplying the percentage butyric acid content of the total fats by a factor of 27, multiplying the product by the percentage by weight of total fats in the goods and dividing by 100. 4. Milk protein content:(a) Save as otherwise provided in (b) below, the milk protein content of the goods shall be calculated by multiplying the nitrogen content (determined by the Kjeldahl method) by the factor 6,38.(b)Where components containing proteins other than milk proteins are also declared in the composition of the goods: (i) the total nitrogen content shall be determined by the Kjeldahl method; (ii)the milk protein content shall be calculated as mentioned in (a) above by subtracting from the total nitrogen content the nitrogen content corresponding to the non-milk proteins. Article 3 For the purposes of implementing Annex I to Regulation (EEC) No 3034/80, the following methods and/or procedures shall be used: 1. For the purposes of classifying goods falling within subheadings 0403 10 51 to 0403 10 59, 0403 10 91 to 0403 10 99, 0403 90 71 to 0403 90 79 and 0403 90 91 to 0403 90 99 of the combined nomenclature, the milk fat content shall be determined by the method referred to in Article 2 (3);2.For the purposes of classifying goods falling within subheadings 1704 10 11 to 1704 10 99 and 1905 20 10 to 1905 20 90 of the combined nomenclature, the sucrose content, including invert sugar expressed as sucrose, shall be determined by an HPLC method; (invert sugar expressed as sucrose means the sum of equal amounts of glucose and fructose multiplied by 0,95);3.For the purposes of classifying goods falling within subheadings 1806 10 10 to 1806 10 90 of the combined nomenclature, the sucrose/invert sugar/isoglucose content shall be determined in accordance with the formulae, method and procedures set out in Article 2 (2);4.For the purposes of classifying goods falling within subheadings 3505 20 10 to 3505 20 90 of the combined nomenclature, the starch, dextrin or other modified starch content shall be determined by the method set out in Annex II of this Regulation;5.For the purposes of classifying goods falling within subheadings 3809 10 10 to 3809 10 90 of the combined nomenclature, the amylaceous substances shall be determined by the method set out in Annex II to this Regulation;6.For the purposes of classifying goods falling within either subheading 1901 90 11 or subheading 1901 90 19 of the combined nomenclature, the distinction shall be drawn on the basis of the dry extract determined by drying at a temperature of 103 ± 2 °C to constant weight;7.For the purposes of classifying goods falling within subheadings 1902 19 10 and 1902 19 90 of the combined nomenclature, the method set out in Annex III to this Regulation shall be used to test for the presence of common wheat flours and semolinas in pasta; 8.The content of mannitol and D-glucitol (sorbitol) of the goods falling within subheadings 2905 44 11 to 2905 44 99 and 3823 60 11 to 3823 60 99 of the combined nomenclature, is determined using a method based on HPLC. Article 4 1. A test report shall be drawn up. 2. The test report shall include the following particulars:- all the information necessary for identifying the sample,-the method used and precise reference to the legal instrument in which it is laid down, or, where appropriate, detailed reference to a method, specifying the nature of the analytical operations to be carried out, or the principle of the method to be applied, as indicated in this Regulation,-any factors liable to have influenced the results,-the results of the analysis, with due regard to the way in which they are expressed in the method used and the means of expression dictated by the needs of the customs or administrative departments that requested the analysis. Article 5 Regulation (EEC) No 1061/69 is hereby repealed. Article 6 This Regulation shall enter into force on 1 January 1988. This Regulation shall be binding in its entirety and directly applicable in all Member States. Done at Brussels, 22 December 1987. For the Commission COCKFIELD Vice-President (1) OJ No L 256, 7. 9. 1987, p. 1. (2) OJ No L 376, 31. 12. 1987, p. 1. (3) OJ No L 141, 12. 6. 1969, p. 24. (4) OJ No L 158, 13. 6. 1986, p. 1. (5) OJ No L 141, 12. 6. 1969, p. 1. (6) OJ No L 323, 29. 11. 1980, p. 1. (7) OJ No L 352, 15. 12. 1987, p. 29. (8) OJ No L 323, 29. 11. 1980, p. 7. (9) OJ No L 382, 31. 12. 1987, p. 27. ANNEX I DETERMINATION OF STARCH CONTENT AND ITS DEGRADATION PRODUCTS INCLUDING GLUCOSE 1. Purpose and field of application(a) The method permits the determination of the starch content, its degradation products including glucose, hereafter referred to as 'starch'.(b)'Starch' content referred to in 1 (a) is equal to value E, as calculated in paragraph 6 of the present method. 2.PrincipleThe sample is broken down by means of sodium hydroxide and the starch divided into glucose units with amyloglucosidase. The glucose determination is performed by the enzymatic route. 3.Reagents(Use double-distilled water.)3.1.0,5 N Sodium hydroxide solution (0,5 mol/l).3.2.96 % (minimum) glacial acetic acid.3.3.Solution of amyloglucosidase: Immediately before use, dissolve approximately 10 mg of amyloglucosidase (EC 3.2.1.3) (6 U per mg) in one ml of water (1).3.4.Triethanolamine buffer solution: Dissolve 14,0 g of triethanolamine hydrochloride [tris(2-hydroxyethyl)ammonium chloride] and 0,25 g of magnesium sulphate MgSO47H2O) in 80 ml of water, add approximately 5 ml of 5 N sodium hydroxyde solution (5 mol/l) and adjust the pH to 7,6, using a 1 N sodium hydroxide solution (1 mol/l). Make up to 100 ml with water. This buffer solution can be kept for at least four weeks at 4 °C.3.5.NADP (nicotinamide adenine dinucleotide phosphate, disodium salt) solution: Dissolve 60 mg of NADP in 6 ml of water. This solution can be kept for at least four weeks at 4 °C.3.6.ATP (adenosine-5 -triphosphate, disodium salt) solution: Dissolve 300 mg of ATP.3H2O and 300 mg of sodium hydrogen carbonate (NaHCO3) in 6 ml of water. This solution can be kept for at least four weeks at 4 °C.3.7.Suspension HK/G6P-DH [Hexokinase (EC 2.7.1.1) and glucose-6-phosphate-dehydrogenase (EC 1.1.1.49)]: suspend 280 U HK and 140 U of G6P-DH in 1 ml of 3,2 M ammonium sulphate solution. This suspension can be kept for at least one year at 4 °C. 4.Apparatus4.1.Magnetic stirrer water bath at 60 °C.4.2.Magnetic rods.4.3.UV spectrophotometer with 1 cm optical cells.4.4.Pipettes for enzymatic analysis. 5.Method5.1.The sample is washed with ethanol, digested in sodium hydroxide and the 'starch' subject to enzymatic hydrolysis:5.1.1.Select the weight of sample as follows, according to the presumed 'starch' content (the 'starch' content must not exceed 0,4 g per sample) as follows: >TABLE> 5.2.6.Calculate the absorbence differences for reagent blank and sample (E2 - E1). Subtract absorbence difference of the reagent blank (AEAA reagent blank) from that of the sample (AEAA) sample): AEAA = AEAA sample - AEAA reagent blank This difference gives the glucose content of the test solution: Glucose content contained in test solution, g/l >TABLE> (3,22: volume of the solution to be measured; 1: cell thickness; 0,1: volume of the sample solution; The molecular weight of glucose is 180,16 (g/mol). 5.2.7.If, for any reason, a measurement cannot be made of 340 nm, the measurement may be made at a wavelength of 365 nm or 334 nm, the figure 6,3 in the formula for Gl above should be replaced by the figure 3,5 or 6,18 respectively. 6.Calculation and expression of results(a) E = 'Starch' content in g/100 g: >TABLE> (b) Z = 'Glucose' content in g/100 g: >TABLE> where: Gl = glucose in G/l (5.2.6); f= dilution factor (5.1.1); p= weight of sample in g; 0,9= glucose conversion factor for starch. Notes:(1) Where a test solution cannot be filtered according to 5.1.7 appropriate methods should be taken in order to obtain a clear solution. (2)Where an inhibition of enzymes occurs, it is advisable to apply a method which involves the addition of known amounts of pure starch. (1) U is the international unit of enzyme activity. ANNEX II DETERMINATION OF STARCHES OR DEXTRINS OR OTHER MODIFIED STARCHES CONTENT INTO GOODS OF SUBHEADINGS 3505 20 10 TO 3505 20 90 OF THE COMBINED NOMENCLATURE AND OF AMYLACEOUS SUBSTANCES CONTENT INTO GOODS OF SUBHEADINGS 3809 10 10 TO 3809 10 90 OF THE COMBINED NOMENCLATURE I. Principle Starch is converted by acid hydrolysis into reducing sugars which are determined by volume using Fehling's solution. II.Apparatus and reagents 1. 250 ml flask; 2. 200 ml graduated flask; 3. 25 ml graduated burette; 4. Hydrochloric acid at 1,19 density; 5. Potassium hydroxide solution; 6. Decolourizing charcoal; 7. Fehling's solution; 8. Methylene blue solution (1 %). III.Method Into a 250 ml flask place a sample containing about 1 g of starch. Add 100 ml of distilled water and 2 ml of hydrochloric acid. Bring to the boil and reflux for three hours.Transfer the contents of the flask and rinsings into a 200 ml graduated flask. Cool and nearly neutralize with potassium hydroxide solution. Add distilled water to 200 ml and filter through a little decolourizing charcoal.Then pour the solution into a graduated burette and reduce 10 ml of Fehling's solution by the following method:Into a flat-bottomed flask of about 250 ml pour 10 ml of Fehling's solution (5 ml of solution A and 5 ml of solution B). Shake until clear and add 40 ml of distilled water and a small quantity of quartz or pumice.Place the flask on a square asbestos plate with a round hole of about 6 cm diameter in the centre, the asbestos in turn resting on a piece of wire gauze. Heat the flask at such a rate that the liquid begins boiling after about two minutes.From the burette, add to the boiling liquid successive quantities of the sugar solution until the blue colour of the Fehling's solution becomes hardly discernible; then add 2 or 3 drops of methylene blue solution as indicator, and complete the titration by adding further quantities of the sugar solution, drop by drop, until the blue colour of the indicator disappears.For greater accuracy repeat the titration under the same conditions, but adding without a break almost all the sugar solution required to reduce the Fehling's solution. In this second titration, the reduction of the Fehling's solution should occur within three minutes.Keep boiling for exactly two further minutes, adding the reagent within one minute drop by drop to the boiling solution until the blue colour disappears.The percentage by weight of starch in the sample is determined by means of the following formula: >TABLE> where:T is the quantity in grammes of anhydrous dextrose corresponding to 10 ml of Fehling's solution (5 ml of solution A and 5 ml of solution B). This titer corresponds to 0,04945 g of anhydrous dextrose when solution A contains 17,636 g of copper per litre; nis the number of ml of the sugar solution used for titration; pis the weight of the sample amount; 0,95is the rate of conversion of anhydrous dextrose into starch. IV.Preparation of Fehling's solutions Solution A: In a graduated flask dissolve 69,278 g of pure crystallized copper sulphate - Analytical Reagent (CuSO4 7 5 H2O) - free from iron in distilled water and bring the volume to 1 litre with distilled water. The correct strength of this solution must be verified by a quantitative determination of the copper.Solution B:In a graduated flask dissolve 100 g of sodium hydroxide and 346 g of double sodium potassium tartrate (Rochelle salt) in distilled water and bring the volume to 1 litre with distilled water.The two solutions A and B must be mixed in equal quantities immediately before use. 10 ml of Fehling's solution (5 ml of solution A and 5 ml of solution B) is completely reduced, under the conditions described at III, by 0,04945 g of anhydrous dextrose.

COMMISSION REGULATION (EEC) No 4154/87 of 22 December 1987 laying down the methods of analysis and other technical provisions necessary for the implementation of Regulation (EEC) No 3033/80 laying down the trade arrangements applicable to certain goods resulting from the processing of agricultural products

THE COMMISSION OF THE EUROPEANCOMMUNITIES, Having regard to the Treaty establishing the European Economic Community, Having regard to Council Regulation (EEC) No 2658/87 of 23 July 1987 on the tariff and statistical nomenclature and on the Common Customs Tariff (1) as amended by Regulation (EEC) No 3985/87 (2) and in particular Article 9 thereof, Whereas Commission Regulation (EEC) No 1061/69 (3), as last amended by Regulation (EEC) No 1822/86 (4), defined the analytical methods to be used for the application of Council Regulation (EEC) No 1059/69 (5); whereas Regulation (EEC) No 1059/69 was repealed and replaced by Council Regulation (EEC) No 3033/80 (6), as last amended by Regulation (EEC) No 3743/87 (7); Whereas the procedures for implementing Regulation (EEC) No 3033/80 for imports, were prescribed by Council Regulation (EEC) No 3034/80, of 11 November 1980, fixing the quantities of basic products considered to have been used in the manufacture of goods covered by Regulation (EEC) No 3033/80 (8) amending Regulation (EEC) No 950/68 on the Common Customs Tariff, as last amended by Regulation (EEC) No 4091/87 (9); Whereas in order to take account of the scientific and technological evolution of analytical methods and to continue to ensure the uniform treatment on import into the Community of goods covered by Regulation (EEC) No 3033/80, it is appropriate to repeal and replace Regulation (EEC) No 1061/69; Whereas the measures provided for in this Regulation are in accordance with the opinion of the Nomenclature Committee, HAS ADOPTED THIS REGULATION:

This Regulation lays down the methods of analysis necessary for the implementation of Regulations (EEC) No 3033/80 (as far as imports are concerned) and (EEC) No 3034/80, or, in the absence of a method of analysis, the nature of the analytical operations to be carried out or the principle of a method to be applied.

In accordance with the definitions set out in Annex III to Regulation (EEC) No 3034/80 concerning:- starch/glucose, content, and - sucrose/invert sugar/isoglucose contentand for the purpose of implementing Annexes II and III to that Regulation, the following formulae, procedures and methods shall be used: 1. Starch/glucose content:(expressed as 100 % anhydrous starch content of the goods as presented)(a) (Z F) × 0,9,if the glucose content is not less than the fructose content; or(b)(Z G) × 0,9,if the glucose content is less than the fructose contentwhere:Z is the glucose content determined by the method set out in Annex I of this Regulation; Fis the fructose content determined by HPLC (high performance liquid chromatography); Gis the glucose content determined by HPLC.In case 1 (a), where the presence of a lactose hydrolysate is declared and/or quantities of lactose and galactose are detected, a glucose content equivalent to the galactose content (determined by HPLC), shall be deducted from the glucose content (Z) before any other calculation is made. 2.Sucrose/invert sugar/isoglucose content:(expressed as sucrose content of the goods as presented)(a)S + (2F) × 0,95,if the glucose content is not less than the fructose content(b)S + (G + F) × 0,95,if the glucose content is less than the fructose contentwhere:Sis the sucrose content determined by HPLC; Fis the fructose content determined by HPLC; Gis the glucose content determined by HPLC.Where the presence of a lactose hydrolysate is declared and/or quantities of lactose and galactose are detected, a glucose content, equivalent to the galactose content (determined by HPLC), shall be deducted from the glucose (G) content before any other calculation is made. 3. Milk fat content:(a) Save as otherwise provided in paragraph (b) below, the milkfat content by weight shall be determined by extraction with light petroleum after hydrolysis with hydrochloric acid.(b)Where fats other than milkfats are also declared in the composition of the goods, the following procedure shall be applied: - the percentage of weight of the total fats in the goods shall be determined as mentioned in (a) above, -the percentage of weight of butyric acid in the total fats shall be obtained by IUPAC method No 23.10 (reference: Pure and Applied Chemistry, 1986, 58, No 10 pp. 1419-1428), -the percentage by weight of milk fats in the goods shall be calculated by multiplying the percentage butyric acid content of the total fats by a factor of 27, multiplying the product by the percentage by weight of total fats in the goods and dividing by 100. 4. Milk protein content:(a) Save as otherwise provided in (b) below, the milk protein content of the goods shall be calculated by multiplying the nitrogen content (determined by the Kjeldahl method) by the factor 6,38.(b)Where components containing proteins other than milk proteins are also declared in the composition of the goods: (i) the total nitrogen content shall be determined by the Kjeldahl method; (ii)the milk protein content shall be calculated as mentioned in (a) above by subtracting from the total nitrogen content the nitrogen content corresponding to the non-milk proteins.

Article 3

For the purposes of implementing Annex I to Regulation (EEC) No 3034/80, the following methods and/or procedures shall be used: 1. For the purposes of classifying goods falling within subheadings 0403 10 51 to 0403 10 59, 0403 10 91 to 0403 10 99, 0403 90 71 to 0403 90 79 and 0403 90 91 to 0403 90 99 of the combined nomenclature, the milk fat content shall be determined by the method referred to in Article 2 (3);2.For the purposes of classifying goods falling within subheadings 1704 10 11 to 1704 10 99 and 1905 20 10 to 1905 20 90 of the combined nomenclature, the sucrose content, including invert sugar expressed as sucrose, shall be determined by an HPLC method; (invert sugar expressed as sucrose means the sum of equal amounts of glucose and fructose multiplied by 0,95);3.For the purposes of classifying goods falling within subheadings 1806 10 10 to 1806 10 90 of the combined nomenclature, the sucrose/invert sugar/isoglucose content shall be determined in accordance with the formulae, method and procedures set out in Article 2 (2);4.For the purposes of classifying goods falling within subheadings 3505 20 10 to 3505 20 90 of the combined nomenclature, the starch, dextrin or other modified starch content shall be determined by the method set out in Annex II of this Regulation;5.For the purposes of classifying goods falling within subheadings 3809 10 10 to 3809 10 90 of the combined nomenclature, the amylaceous substances shall be determined by the method set out in Annex II to this Regulation;6.For the purposes of classifying goods falling within either subheading 1901 90 11 or subheading 1901 90 19 of the combined nomenclature, the distinction shall be drawn on the basis of the dry extract determined by drying at a temperature of 103 ± 2 °C to constant weight;7.For the purposes of classifying goods falling within subheadings 1902 19 10 and 1902 19 90 of the combined nomenclature, the method set out in Annex III to this Regulation shall be used to test for the presence of common wheat flours and semolinas in pasta; 8.The content of mannitol and D-glucitol (sorbitol) of the goods falling within subheadings 2905 44 11 to 2905 44 99 and 3823 60 11 to 3823 60 99 of the combined nomenclature, is determined using a method based on HPLC.

Article 4

1. A test report shall be drawn up. 2. The test report shall include the following particulars:- all the information necessary for identifying the sample,-the method used and precise reference to the legal instrument in which it is laid down, or, where appropriate, detailed reference to a method, specifying the nature of the analytical operations to be carried out, or the principle of the method to be applied, as indicated in this Regulation,-any factors liable to have influenced the results,-the results of the analysis, with due regard to the way in which they are expressed in the method used and the means of expression dictated by the needs of the customs or administrative departments that requested the analysis.

Article 5

Regulation (EEC) No 1061/69 is hereby repealed.

Article 6

This Regulation shall enter into force on 1 January 1988.

This Regulation shall be binding in its entirety and directly applicable in all Member States. Done at Brussels, 22 December 1987. For the Commission COCKFIELD Vice-President

(1) OJ No L 256, 7. 9. 1987, p. 1.

(2) OJ No L 376, 31. 12. 1987, p. 1.

(3) OJ No L 141, 12. 6. 1969, p. 24.

(4) OJ No L 158, 13. 6. 1986, p. 1.

(5) OJ No L 141, 12. 6. 1969, p. 1.

(6) OJ No L 323, 29. 11. 1980, p. 1.

(7) OJ No L 352, 15. 12. 1987, p. 29.

(8) OJ No L 323, 29. 11. 1980, p. 7.

(9) OJ No L 382, 31. 12. 1987, p. 27.

ANNEX I

DETERMINATION OF STARCH CONTENT AND ITS DEGRADATION PRODUCTS INCLUDING GLUCOSE

1. Purpose and field of application(a) The method permits the determination of the starch content, its degradation products including glucose, hereafter referred to as 'starch'.(b)'Starch' content referred to in 1 (a) is equal to value E, as calculated in paragraph 6 of the present method. 2.PrincipleThe sample is broken down by means of sodium hydroxide and the starch divided into glucose units with amyloglucosidase. The glucose determination is performed by the enzymatic route. 3.Reagents(Use double-distilled water.)3.1.0,5 N Sodium hydroxide solution (0,5 mol/l).3.2.96 % (minimum) glacial acetic acid.3.3.Solution of amyloglucosidase: Immediately before use, dissolve approximately 10 mg of amyloglucosidase (EC 3.2.1.3) (6 U per mg) in one ml of water (1).3.4.Triethanolamine buffer solution: Dissolve 14,0 g of triethanolamine hydrochloride [tris(2-hydroxyethyl)ammonium chloride] and 0,25 g of magnesium sulphate MgSO47H2O) in 80 ml of water, add approximately 5 ml of 5 N sodium hydroxyde solution (5 mol/l) and adjust the pH to 7,6, using a 1 N sodium hydroxide solution (1 mol/l). Make up to 100 ml with water. This buffer solution can be kept for at least four weeks at 4 °C.3.5.NADP (nicotinamide adenine dinucleotide phosphate, disodium salt) solution: Dissolve 60 mg of NADP in 6 ml of water. This solution can be kept for at least four weeks at 4 °C.3.6.ATP (adenosine-5 -triphosphate, disodium salt) solution: Dissolve 300 mg of ATP.3H2O and 300 mg of sodium hydrogen carbonate (NaHCO3) in 6 ml of water. This solution can be kept for at least four weeks at 4 °C.3.7.Suspension HK/G6P-DH [Hexokinase (EC 2.7.1.1) and glucose-6-phosphate-dehydrogenase (EC 1.1.1.49)]: suspend 280 U HK and 140 U of G6P-DH in 1 ml of 3,2 M ammonium sulphate solution. This suspension can be kept for at least one year at 4 °C. 4.Apparatus4.1.Magnetic stirrer water bath at 60 °C.4.2.Magnetic rods.4.3.UV spectrophotometer with 1 cm optical cells.4.4.Pipettes for enzymatic analysis. 5.Method5.1.The sample is washed with ethanol, digested in sodium hydroxide and the 'starch' subject to enzymatic hydrolysis:5.1.1.Select the weight of sample as follows, according to the presumed 'starch' content (the 'starch' content must not exceed 0,4 g per sample) as follows: >TABLE>

5.2.6.Calculate the absorbence differences for reagent blank and sample (E2 - E1). Subtract absorbence difference of the reagent blank (AEAA reagent blank) from that of the sample (AEAA) sample): AEAA = AEAA sample - AEAA reagent blank This difference gives the glucose content of the test solution: Glucose content contained in test solution, g/l >TABLE>

(3,22: volume of the solution to be measured; 1: cell thickness; 0,1: volume of the sample solution; The molecular weight of glucose is 180,16 (g/mol). 5.2.7.If, for any reason, a measurement cannot be made of 340 nm, the measurement may be made at a wavelength of 365 nm or 334 nm, the figure 6,3 in the formula for Gl above should be replaced by the figure 3,5 or 6,18 respectively. 6.Calculation and expression of results(a) E = 'Starch' content in g/100 g: >TABLE>

(b) Z = 'Glucose' content in g/100 g: >TABLE>

where: Gl = glucose in G/l (5.2.6); f= dilution factor (5.1.1); p= weight of sample in g; 0,9= glucose conversion factor for starch. Notes:(1) Where a test solution cannot be filtered according to 5.1.7 appropriate methods should be taken in order to obtain a clear solution. (2)Where an inhibition of enzymes occurs, it is advisable to apply a method which involves the addition of known amounts of pure starch. (1) U is the international unit of enzyme activity.

ANNEX II

DETERMINATION OF STARCHES OR DEXTRINS OR OTHER MODIFIED STARCHES CONTENT INTO GOODS OF SUBHEADINGS 3505 20 10 TO 3505 20 90 OF THE COMBINED NOMENCLATURE AND OF AMYLACEOUS SUBSTANCES CONTENT INTO GOODS OF SUBHEADINGS 3809 10 10 TO 3809 10 90 OF THE COMBINED NOMENCLATURE

I. Principle Starch is converted by acid hydrolysis into reducing sugars which are determined by volume using Fehling's solution. II.Apparatus and reagents 1. 250 ml flask; 2. 200 ml graduated flask; 3. 25 ml graduated burette; 4. Hydrochloric acid at 1,19 density; 5. Potassium hydroxide solution; 6. Decolourizing charcoal; 7. Fehling's solution; 8. Methylene blue solution (1 %). III.Method Into a 250 ml flask place a sample containing about 1 g of starch. Add 100 ml of distilled water and 2 ml of hydrochloric acid. Bring to the boil and reflux for three hours.Transfer the contents of the flask and rinsings into a 200 ml graduated flask. Cool and nearly neutralize with potassium hydroxide solution. Add distilled water to 200 ml and filter through a little decolourizing charcoal.Then pour the solution into a graduated burette and reduce 10 ml of Fehling's solution by the following method:Into a flat-bottomed flask of about 250 ml pour 10 ml of Fehling's solution (5 ml of solution A and 5 ml of solution B). Shake until clear and add 40 ml of distilled water and a small quantity of quartz or pumice.Place the flask on a square asbestos plate with a round hole of about 6 cm diameter in the centre, the asbestos in turn resting on a piece of wire gauze. Heat the flask at such a rate that the liquid begins boiling after about two minutes.From the burette, add to the boiling liquid successive quantities of the sugar solution until the blue colour of the Fehling's solution becomes hardly discernible; then add 2 or 3 drops of methylene blue solution as indicator, and complete the titration by adding further quantities of the sugar solution, drop by drop, until the blue colour of the indicator disappears.For greater accuracy repeat the titration under the same conditions, but adding without a break almost all the sugar solution required to reduce the Fehling's solution. In this second titration, the reduction of the Fehling's solution should occur within three minutes.Keep boiling for exactly two further minutes, adding the reagent within one minute drop by drop to the boiling solution until the blue colour disappears.The percentage by weight of starch in the sample is determined by means of the following formula: >TABLE>

where:T is the quantity in grammes of anhydrous dextrose corresponding to 10 ml of Fehling's solution (5 ml of solution A and 5 ml of solution B). This titer corresponds to 0,04945 g of anhydrous dextrose when solution A contains 17,636 g of copper per litre; nis the number of ml of the sugar solution used for titration; pis the weight of the sample amount; 0,95is the rate of conversion of anhydrous dextrose into starch. IV.Preparation of Fehling's solutions Solution A: In a graduated flask dissolve 69,278 g of pure crystallized copper sulphate - Analytical Reagent (CuSO4 7 5 H2O) - free from iron in distilled water and bring the volume to 1 litre with distilled water. The correct strength of this solution must be verified by a quantitative determination of the copper.Solution B:In a graduated flask dissolve 100 g of sodium hydroxide and 346 g of double sodium potassium tartrate (Rochelle salt) in distilled water and bring the volume to 1 litre with distilled water.The two solutions A and B must be mixed in equal quantities immediately before use. 10 ml of Fehling's solution (5 ml of solution A and 5 ml of solution B) is completely reduced, under the conditions described at III, by 0,04945 g of anhydrous dextrose.