S.I. No. 250/1978 - European Communities (Feeding Stuffs) (Methods of Analysis) Regulations, 1978.

(1)In any prosecution for an offence under either of the Regulations mentioned in paragraph 2 of these Regulations, the production of a certificate in the form set out in the Schedule to these Regulations, purporting to be signed by the State Chemist or the Assistant State Chemist, shall be evidence of the facts stated in the certificate and of the analysis referred to in the certificate having been carried out, unless the defendant requires the person who made the analysis to be called as a witness.

(2)In this paragraph— "the State Chemist" means the head of the State Laboratory or a person authorised by him for the purposes of these Regulations. "the Assistant State Chemist" means the Assistant State Chemist of the State Laboratory. PART II. METHODS OF ANALYSIS OF THE COMPONENTS OF FEEDINGSTUFFS AND PREMIXTURES The main divisions in this Part of these Regulations are as follows:— 1. General Provisions 2. Determination of volatile nitrogenous bases 3. Detection and identification of antibiotics of the tetracycline group 4. Determination of: chlortetracycline oxytetracycline tetracycline 5. Determination of oleandomycin 6. Determination of tylosin 7. Determination of virginiamycin 8. Determination of amprolium 9. Determination of ethopabate 10. Determination of dinitolmide (DOT) 11. Determination of nicarbazin 12. Determination of vitamin A (retinol) 13. Determine of thiamine (vitamin B1, aneurine) 14. Determination of vitamin C 15. Determination of menadione (Vitamin K3) 16. Determination of hydrocyanic acid 17. Determination of free and total gossypol 18. Determination of theobromine 19. Determination of volatile mustard oil 20. Determination of buquinolate 21. Determination of sulphaquinoxaline 22. Determiation of furazolidone 23. Determination of aflatoxin B1 24. Determination of urea 1. GENERAL PROVISIONS. 1. Methods of analysis of the components of feeding stuffs are generally applicable to all feeding stuffs and premixtures. However, certain feeding stuffs, by reason of their inherent compositional characteristics, require certain modifications in analytical procedure. These have been provided for under the heading 'observations' in the description of methods. When two or more methods may be used to determine a component of a feeding stuff, the choice of method shall, except where otherwise indicated, be left to the State Laboratory; the method used must however be indicated in the certificate of analysis. 2. Preparation of the sample for analysis 2.1 The sample shall be divided into two parts. One part shall be taken as it is for macroscopic and microscopic determinations and also for the determination of moisture. The other part shall be prepared as follows for chemical analysis. 2.2 Mix this part carefully on a clean dry surface. (If the sample is very moist it must be pre-dried to bring the moisture content down to a level between 8—12%. To do this, dry the sample at a suitable temperature for an adequate length of time). Divide it by means of a divider or by hand using the quartering method (which consists of taking portions in turn from two opposite sections). Reduce it by either of these methods to approximately 100 g and, if necessary, crush the reduced portion so that it will pass through a 1 mm round mesh sieve. Transfer this portion immediately to a dry container with an air-tight fitting. 2.3 The general provisions set out in 2.1 and 2.2 are applicable to all methods of analysis with the exception of those methods dealing with antibiotics, coccidiostats and vitamins. 3. Reagents All reagents used should be of analytical quality. References to water mean distilled or de-ionised water. Where a solution of a reagent is mentioned without any other indication, this means a solution in distilled or de-ionised water. 4. Expression of Results The result given in the certificate of analysis shall be the average value obtained on the basis of at least two determinations. Subject to special provisions, the result shall be expressed as a proportion by weight of the original sample as it was when it reached the State Laboratory. The result must not be given to more significant figures than the accuracy of the method of analysis allows. 2. DETERMINATION OF VOLATILE NITROGENOUS BASES (

1. a)BY MICRODIFFUSION 1. Purpose and scope To determine the content of volatile nitrogenous bases, expressed as ammonia, in feeding stuffs. 2. Principle The sample is extracted with water and the solution clarified and filtered. The volatile nitrogenous bases are displaced by microdiffusion using a solution of potassium carbonate, collected in a solution of boric acid and titrated with sulphuric acid. 3. Reagents 3.1 Trichloroacetic acid solution, 20% (w/v). 3.2 Indicator: dissolve 33 mg of bromocresol green and 65 mg of methyl red in 100 ml of 95—96% (v/
2. v)ethanol. 3.3 Boric acid solution: in a 1 litre graduated flask dissolve 10 g of boric acid in 200 ml of 95—96% (v/
3. v)ethanol and 700 ml of water. Add 10 ml of indicator (3 2). Mix and, if necessary, adjust the colour of the solution to light red by adding a solution of sodium hydroxide. 1 ml of this solution will fix a maximum of 300µg of NH3. 3.4 Saturated potassium carbonate solution: dissolve 100 g of potassium carbonate in 100 ml of boiling water. Leave to cool, filter. 3.5 Sulphuric acid, 0.02 N. 4. Apparatus 4.1 Shaker: approximately 35—40 rpm. 4.2 Glass or plastic Conway cells (see diagram). 4.3 Microburettes graduated in 1/100 ml. 5. Procedure Weigh, to the nearest mg, 10 g of sample and place with 100 ml of water in a 200 ml graduated flask. Shake in the shaker (4.1) for 30 minutes. Add 50 ml of trichloroacetic acid solution (3.1), make up to volume with water, shake vigorously and filter through a pleated filter. Using a pipette, introduce 1 ml of boric acid solution (3.3) into the central part of the Conway cell (4.2) and 1 ml of the sample filtrate into the crown of the cell. Cover partially with the greased lid. Drop 1 ml of saturated potassium carbonate solution (3.4) quickly into the crown and close the lid so that the cell is airtight. Turn the cell carefully, rotating it in a horizontal plane so that the two reagents are mixed. Leave to incubate either for at least 4 hours at room temperature or for 1 hour at 40°C. Using a microburette (4.3), titrate the volatile bases in the boric acid solution with sulphuric acid 0.02 N (3.5). Carry out a blank test using the same procedure but without a sample to be analysed. 6. Calculation of results 1 ml of H2SO4 0.02 N corresponds to 0.34 mg of ammonia. Express the result as a percentage of the sample. Repeatability The difference between results of two parallel determinations carried out on the same sample should not exceed: 10%, relative to the higher result for ammonia contents of less than 1.0%; 0.1%, in absolute value, for ammonia contents of 1.0% or more. 7. Observation If the ammonia content of the sample exceeds 0.6%, dilute the initial filtrate. (
4. b)BY DISTILLATION 1. Purpose and scope To determine the content of volatile nitrogenous bases, expressed as ammonia, in fish-meal containing practically no urea. It is applicable only to ammonia contents of less than 0.25%. 2. Principle The sample is extracted with water and the solution clarified and filtered. The volatile nitrogenous bases are displaced at boiling point by adding magnesium oxide and collected in a specific quantity of sulphuric acid, the excess of which is back-titrated with a solution of sodium hydroxide. 3. Reagents 3.1 Trichloroacetic acid solution, 20% (w/v). 3.2 Magnesium oxide. 3.3 Antifoam (eg silicone). 3.4 Sulphuric acid, 0.1 N. 3.5 Sodium hydroxide solution, 0.1 N. 3.6 0.3% (w/
5. v)solution of methyl red in 95—96% (v/
6. v)ethanol. Conway Cell Scale 1/1 4. Apparatus 4.1 Shaker: approximately 35—40 rpm. 4.2 Distilling apparatus of the Kjeldahl type. 5. Procedure Weigh, to the nearest mg, approximately 10 g of the sample, and place with 100 ml of water in a 200 ml graduated flask. Shake in the shaker (4.1) for 30 minutes. Add 50 ml of trichloroacetic acid solution (3.1), make up to volume with water, shake vigorously and filter through a pleated filter. Take a quantity of clear filtrate appropriate for the presumed content of volatile nitrogenous bases (100 ml is usually suitable). Dilute to 200 ml and add 2 g of magnesium oxide (3.2) and a few drops of antifoam (3.3). The solution should be alkaline to litmus paper; otherwise add some magnesium oxide (3.2). Distil about 150 ml of the solution in the Kjeldahl apparatus (4.2) and collect the distillate in an Erlenmeyer flask containing an accurately measured volume (25—50
7. ml)of sulphuric acid 0.1 N (3.4). While distilling, avoid over-heating the sides of the flask. Boil the sulphuric acid solution for 2 minutes, cool it and back-titrate the excess sulphuric acid with the sodium hydroxide solution 0.1 N (3.5) in the presence of the methyl red indicator (3.6). Carry out a blank test using the same procedure but without a sample to be analysed. 6. Calculation of results 1 ml of H2SO4 0.1 N corresponds to 1.7 mg of ammonia. 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 should not exceed 10% relative to the higher result. 3. DETECTION AND IDENTIFICATION OF ANTIBIOTICS OF THE TETRACYCLINE GROUP 1. Purpose and scope To detect and identify antibiotics of the tetracycline group in feeding stuffs containing at least 0.1 mg/kg of antibiotics, in concentrates and in premixes. 2. Principle The sample is extracted with a mixture of methanol and hydrochloric acid. The extract and reference solutions for comparison are subjected to ascending paper chromatography. The antibiotics are detected and identified by comparing their Rf-values with those of the standard substances, either by fluorescence in UV light (high antibiotic contents) or by bioautography on an agar medium inoculated with B. cereus. 3. Reagents and culture medium 3.1 Buffer solution, pH 3.5 Citric acid monohydrate 10.256 g di Sodium hydrogen phosphate Na2 HPO4. 2H2O 7.45 g Acetone 300 ml Water to 1000 ml 3.2 Phosphate buffer solution, pH 5.5 Potassium dihydrogen phosphate KH2PO4 130.86 g di Sodium hydrogen phosphate Na2HPO4. 2H2O 6.947 g Water to 1000 ml 3.3 Eluent I: mixture of pure nitromethane/pure chloroform/1.3-dichloropropan-2-ol: 20/10/1.5 by volume, prepare immediately before use. 3.4 Eluent II: mixture of pure nitromethane/pure chloroform/2-picoline: 20/10/3 by volume. Prepare immediately before use. 3.5 Mixture of pure methanol/hydrochloric acid (d: 1.19): 98/2 by volume. 3.6 Hydrochloric acid, 0.1 N. 3.7 Ammonia, d: 0.91. 3.8 Standard substances: chlortetracycline, oxytetracycline, tetracycline, the activity of which is expressed in terms of hydrochloride. 3.9 Micro-organism: B. cereus ATCC No 11.778 (NCIB 8849: NCTC 10320) Maintenance of the parent strain, preparation of the spore suspension and inoculation of the culture medium: follow the directions given in 3.1 and 3.2 of the method for the determination of chlortetracycline, oxytetracycline and tetracycline contents by diffusion on agar; these instructions are the subject of Method 4 (
8. a)paragraphs 3.1 and 3.2. 3.10 Culture medium1 Glucose 1 g Tryptic peptone 10 g Meat extract 1.5 g Yeast extract 3 g Agar 20 g Water to 1000 ml Adjust the pH to 5.8 immediately before use. 1 Any commercial culture medium of similar composition and giving the same results may be used. 3.11 0.1% (w/
9. v)2, 3, 5 triphenyltetrazolium chloride solution and 5% (w/
10. v)glucose solution. 4. Apparatus 4.1 Apparatus for ascending paper chromatography (height of paper: 25 cm). Schleicher and Schull paper (2040b or 2043b) or equivalent. 4.2 Centrifuge 4.3 Incubator set at 30°C. 4.4 U.V. lamp for the detection of fluorescence. 4.5 Glass plates approximately 20 x 30 cm for bioautography. 5. Standard solutions 5.1 Stock solutions Using hydrochloric acid (3.6). prepare from the standard substances (3.8) solutions with concentrations corresponding to 500 µg per ml of chlortetracycline-HC1, of oxytetracycline-HC1, and of tetracycline-HC1. 5.2 Reference solutions for detections by UV light Dilute the solutions (5.1) with the phosphate buffer solution (3.2) to obtain solutions with concentrations corresponding to 100 µg per ml of chlortetracycline-HC1, of oxytetracycline-HC1 and of tetracycline-HC1. 5.3 Reference solutions for detection by bioautograph Dilute the solutions (5.1) with the phosphate buffer solution (3.2) to obtain solutions with concentrations corresponding to 5 µg per ml of chlortetracycline-HC1, of oxytetracycline-HC1 and of tetracycline-HC1. 6. Extraction When the presumed antibiotic content is less than 10 mg/kg either the mixed sample or the finest fraction separated by sieving may be used, since the antibiotics are to be found mainly in this fraction. Suspend the sample in the mixture (3.5) and centrifuge. Collect the supernatant liquid and use it directly or dilute, if necessary, with the mixture (3.5) to obtain antibiotic concentrations of approximately 100 µg per mg (6.1) and 5 µg per ml (6.2). 7. Detection and Mentification 7.1 Chromatography Immerse the paper in the buffer solution, pH 3.5 (3.1). Remove the excess liquid by pressing the paper between sheets of dry filter paper. Then place volumes of 0.01 ml of the reference solutions (5.2 and 5.3) and of the extract (6.1 and 6.2) on the paper. To give a good separation, the paper must have the correct moisture content; if necessary, leave to dry a little. Develop by ascending chromatography. Use eluent 1 (3.3) for detection by bioautography and eluent II (3.4) for detection by UV light. When the solvent front has reached 15 to 20 cm (approx. 1 hour 30 minutes), stop chromatography and dry the paper. 7.2 Detection by UV light If the antibiotic level is greater than 1 µg per cm2, after the chromatogram has been treated with ammonia vapours (3.7) golden yellow fluorescent spots will be seen on irradiation under the UV lamp (4.4). 7.3 Detection by bioautography Pour the culture medium (3.10); previously inoculated with B. cereus (3.9), into glass plates (4.5) and place the paper on the culture medium. After 5 minutes' contact, detach the paper and place it on another spot in the culture medium, where it will remain during the incubation period. Incubate overnight at 30°C. If an antibiotic of the tetracycline group is present, light inhibition zones will appear in the cloudy culture medium. To fix the chromatogram, the solution (3.11) is vaporized on the paper, after incubation. 7.4 Identification The relative Rf values of antibiotics of the tetracycline group are given below. These values may vary slightly according to the quality of the paper and its moisture content: Chlortetracycline (CTC) 0.60 Tetracycline (TC) 0.40 Oxytetracycline (OTC) 0.20 4-epi-CTC 0.15 4-epi-TC 0.13 4-epi-OTC 0.10 The antibiotic activity of the 'epi' compounds is less than that of the normal compounds. 4. DETERMINATION OF CHLORTETRACYCLINE, OXYTETRACYCLINE AND TETRACYCLINE (
11. a)BY DIFFUSION ON AGAR 1. Purpose and scope To determine the levels of chlortetracycline (CTC), oxytetracycline (OTC) and tetracycline (TC) in feedingstuffs. concentrates and premixes where more than 5 ppm are present. Contents of less than 5 mg/kg may be estimated by graphic interpolation. 2. Principle For contents of 50 ppm or less, the sample is extracted with dilute formamide. For contents greater than 50 ppm, it is extracted with a mixture of acetone, water and hydrochloric acid, for the determination of CTC, and with a mixture of methanol and hydrochloric acid for the determination of OTC and TC. The extracts are then diluted and their antibiotic activity determined by measuring the diffusion of the CTC, OTC or TC on an agar medium seeded with B. cerus. The diffusion is made evident by the formation of inhibition zones in the presence of the micro-organism. The diameter of these zones is directly proportional to the logarithm of the antibiotic concentration. 3. Micro-organism: B. cereus, ATCC No. 11.778 (NCIB 8849; NCTC 10320) 3.1 Maintenance of the parent strain Inoculate with B. cereus a tube of sloped agar taken from culture medium (4.1) free from methylene blue and boric acid. Incubate overnight at approximately 30°C. Keep the culture in a refrigerator and re-inoculate sloped agar with it every 14 days. 3.2 Preparation of the spore suspension Collect the bacteria from a tube of sloped agar (3.1) using 2—3 ml of physiological saline (4.5). With this suspension, seed a Roux flask containing 300 ml of culture medium (4.1), free from methylene blue and boric acid, with 3—4% agar concentration. Incubate for 3—5 days at 28—30°C, then collect the spores in 15 ml of ethanol (4.6) after checking sporulation under a microscope, and mix. This suspension will keep in a refrigerator for 5 months or more. By preliminary tests on plates with the basic medium for the determination (4.1) establish the quantity of inoculum which, for the different concentrations of antiobiotic used, will give the largest possible inhibition zones that are still clear. This quantity is usually between 0.2—0.3 ml per 1000 ml. The culture medium is inoculated at between 50—60°C. 4. Culture media and reagents 4.1 Basic medium for the determination1 1 Any commercial culture medium of similar composition and giving the same results may be used. Glucose 1 g Tryptic peptone 10 g Meat extract 1.5 g Yeast extract 3 g Agar, according to quality 10—20 g 'Tween 80' 1 ml Phosphate buffer solution, pH 5.5 (4.2) 10 ml Boric acid solution 5% (w/
12. v)15 ml Methylene blue, ethanol solution, 0.5% (w/
13. v)4 ml Water to 1000 ml Adjust to pH 5.8 before use. 4.2 Phosphate buffer solution, pH 5.5 Potassium dihydrogen phosphate KH2PO4 130.86 g di Sodium hydrogen phosphate Na2HPO4. 2H2O 6.947 g Water to 1000 ml 4.3 Phosphate buffer solution, pH 5.5, diluted to 1/10. 4.4 Phosphate buffer solution, pH 8 Potassium dihyrogen phosphate KH2PO4 1.407 g di Sodium hydrogen phosphate Na2HPO4. 2H2O 57.539 g Water to 1000 ml 4.5 Sterile physiological saline. 4.6 Ethanol solution, 20% (v/v). 4.7 Hydrochloric acid, 0.1 N. 4.8 Formamide solution, 70% (v/v): prepare fresh before use and adjust the pH to 4.5 using sulphuric acid approximately 2N. 4.9 Mixture of pure acetone/water/hydrochloric acid (d: 1.19): 65/33/2 by volume. 4.10 Mixture of pure methanol/hydrochloric acid (d:1.19): 98/2 by volume. 4.11 Standard substances: CTC, OTC, TC, the activity of which is expressed in terms of hydrochloride. 5. Standard solutions 5.1 Chlortetracycline Using hydrochloric acid (4.7), prepare from the standard solution (4.11) a stock solution with a concentration corresponding to 500 µg per ml of chlortetracycline-HC1. This solution will keep for one week in a refrigerator. From this stock solution, prepare a standard working solution S8 with a concentration corresponding to 0.2 µg per ml of chlortetracycline-HC1. Dilution is carried out using the phosphate buffer solution, pH 5.5, diluted to 1/10 (4.3), to which 0.01% of amido black has been added1. 1 Amido black is used to make evident the inhibition zones of the standard solutions (blue rings). Then prepare by successive dilutions (1 + 1), using the buffer solution (4.3), the following concentrations: S4 0.1 µg/ml S2 0.05 µ/ml S1 0.025 µg/ml 5.2 Oxytetracycline Proceeding as indicated in 5.1, prepare, from a stock solution with a concentration corresponding to 400 µg per ml of oxytetracycline-HC1, a standard working solution S8 containing 1.6 µg per ml of oxytetracycline-HC1, and the following concentrations: S4 0.8 µg/ml S2 0.4 µg/ml S1 0.2 µg/ml 5.3 Tetracycline Proceeding as indicated in 5.1, prepare, from a stock solution with a concentration corresponding to 500 µg per ml of tetracycline-HCl, a standard working solution S8 containing 1.0 µg per ml of tetracycline-HC1 and the following concentrations: S4 0.5 µg/ml S2 0.25 µg/ml S1 0.125 µg/ml 6. Extraction 6.1 Contents of 50 mg/kg or less To test sample add formamide (4.8) in the quantities indicated in the table below. Shake for 30 minutes on a shaking platform. Then dilute immediately with the phosphate buffer solution (4.3) according to the indications given in the table below to obtain the concentration U8. The formamide concentration of this solution must not exceed 40%. Centrifuge or decant to obtain a clear solution. Then prepare the concentrations U4, U2 and U1 by successive dilutions (1 + 1) using the phosphate buffer solution (4.3). Antibiotic CTC OTC TC Presumed content in ppm 10 50 10 50 10 50 Test sample in g 10 10 24 9.6 20 10 ml of formamide(4.8) 100 100 80 100 80 100 ml of phosphate buffer solution (4.3) dilution 1:5 (
14. a)dilution 1:25 (
15. b)70 200 120 dilution 1:5 (
16. a)U8 concentration in µg/ml 0.2 0.2 1.6 1.6 1.0 1.0 (
17. a)Take 20 ml of extract and make up to 100 ml in a graduated flask with the buffer solution. (
18. b)Take 4 ml of extract and make up to 100 ml in a graduated flask with the buffer solution. 6.2 Contents greater than 50 mg/kg 6.2.1 Chlortetracycline To a test sample of 2—10 g, depending on the presumed antibiotic content of the sample or its manufacturer's guarantee, add 20 times its volume of mixture (4.9). Shake for 30 minutes on a shaking platform. The pH must remain below 3 during extraction; if necessary, readjust to pH 3 (using 10% acetic acid for mineral compounds). Take an aliquot part of the extract and adjust the pH to 5.5 using the phosphate buffer solution, pH 8 (4.4) in the presence of bromocresol green (turning from yellow to blue). Dilute, using the phosphate buffer solution, pH 5.5, diluted to 1/10 (4.3), to obtain the concentration U8 (see 6.1). Then prepare the concentrations U4, U2 and U1, by successive dilutions (1 + 1) using the phosphate buffer solution (4.3). 6.2.2 Oxytetracycline and tetracycline Proceed as indicated in 6.2.1, using the mixture (4.10) instead of the mixture (4.9). 7. Determination method 7.1 Inoculation of the culture medium Inoculate at 50—60°C the basic medium for the determination (4.1) with the spore suspension (3.2). 7.2 Preparation of the trays Diffusion on agar is carried out in trays using 4 concentrations of the standard solution (S8, S4, S2, S1) and 4 concentrations of the extract (U8, U4, U2, U1). The 4 concentrations of extract and of standard solution must be placed in each tray. Choose trays, therefore, which are large enough to allow at least 8 holes 10—13 mm in diameter to be made in the agar medium. Calculate the quantity of inoculated culture medium (7.1) needed to provide a uniform covering approximately 2 mm thick. The test should preferably be carried out on trays consisting of flat glass plates fitted with a perfectly level aluminium or plastic ring, 200 mm in diameter and 20 mm high. Pipette into the holes accurately measured quantities of between 0.10—0.15 ml of antibiotic solution, depending on the diameter of the holes. For each sample, repeat the diffusion at least 4 times with each concentration so that each determination comprises an evaluation of 32 inhibition zones. 7.3 Incubation Incubate the trays for approximately 18 hours at 28—30°C. 8. Evaluation Measure the diameter of the inhibition zones, preferably by projection. Record the measurements on semi-logarithmic paper, plotting the logarithm of the concentrations against the diameter of the inhibition zones. Trace the lines of the standard solution and of the extract. Provided there is no interference, the two lines will be parallel. The logarithm of the relative activity is calculated by using the following formula:— Real activity = presumed activity x relative activity. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed 10%, relative to the higher result. (
19. b)BY TURBIDIMETRY 1. Purpose and scope To determine the levels of chlortetracycline (CTC), oxytetracycline (OTC) and tetracycline (TC) where concentrations are greater than 1 g per kg, provided there is no interference from other substances clouding the extracts. This method is quicker than diffusion on agar. 2. Principle For the determination of CTC, the sample is extracted with a mixture of acetone, water and hydrochloric acid, and with a mixture of methanol and hydrochloric acid for the determination of OTC and TC. The extracts are then diluted and their antibiotic effect determined by measuring the light transmission of a culture medium which has been seeded with Staphylococcus aureus and to which the antibiotic has been added. The light transmission depends on the antibiotic concentration. 3. Micro-organism: Staphylococcus aureus K 1411 (NCIB 11182; NCTC 10988) 1 This strain, isolated by the LUFA at Kiel, grows more rapidly than S. aureus ATCC 6538P. 3.1 Maintenance of the parent strain Inoculate with S. aureus a tube of sloped agar taken from the culture medium (4.1), to which 1.5—3% of agar has been added (depending on the quality). Incubate overnight at 37°C. Keep the culture in a refrigerator and re-inoculate sloped agar with it every 4 weeks. At the same time prepare sub-cultures for laboratory use. 3.2 Preparation of the inoculum 24 hours before use, re-inoculate sloped agar with a sub-culture and incubate overnight at 37°C. Suspend all the culture contained in a tube of agar in approximately 2 ml of the basic medium (4.1), then transfer the suspension under sterile conditions into approximately 100 ml of the same basic medium (4.1). Incubate in a water bath at 37°C until the growth of the strain enters its logarithmic phase (1 hour 30 minutes to 2 hours). 4. Culture media and reagents 4.1 Basic medium for the determination2 Peptone 2Any commercial culture medium of similar composition and giving the same results may be used. 5 g Yeast extract 1.5 g Meat extract 1.5 g Sodium chloride 3.5 g Glucose 1.0 g Potassium dihydrogen-phospate KH2PO4 1.32 g di Potassium hydrogen phosphate K2HPO4 3.68 g Water to pH after sterilisation: 6.8—7.0 1000 ml 4.2. Phosphate buffer solution, pH 4.5 Potassium dihydrogen phosphate KH2PO4 13.6 g Water to 1000 ml 4.3. Hydrochloric acid, 0.1 N. 4.4. Mixture of pure acetone/water/hydrochloric acid (d: 1.19): 65/33/2 by volume. 4.5. Mixture of pure methanol/hydrochloric acid (d: 1.19): 98/2 by volume. 4.6. Formaldehyde solution, approximately 10% (w/v). 4.7. Standard substances: CTC, OTC, TC, the activity of which is expressed in terms of hydrochloride. 5. Standard solution Using hydrochloric acid (4.3), prepare from the standard substance (4.7) a stock solution with a concentration corresponding to 400—500 µg per ml of CTC-HC1, OTC-HC1 or TC-HC1. This solution will keep for one week in a refrigerator. 6. Extraction 6.1. Chlortetracycline Place a 1—2 g test sample in a 200 or 250 ml graduated flask. Add approximately 100 ml of the mixture (4.4) and shake for 30 minutes on a shaking platform. Make up to volume with the phosphate buffer solution, pH 4.5 (4.2). Mix and leave to settle. 6.2. Oxytetracycline and tetracycline Place a 1—2 g test sample in a 200 or 250 ml graduated flask. Add approximately 100 ml of the mixture (4.5) and shake for 30 minutes on a shaking platform. Make up to volume with the phosphate buffer solution, pH 4.5 (4.2). Mix and leave to settle. 7. Determination method 7.1. Preparation of the standard series and of the extract Dilute the standard solution

(5)and the extract

(6)with the phosphate buffer solution, pH 4.5 (4.2), to obtain a series of concentrations. For each determination, a calibration curve is drawn from the respective concentration, permitting the interpolation of at least two values relating to the extract. The dilutions should be chosen according to the conditions under which the strain is grown, which may vary from one laboratory to another. The procedure is generally the following: 7.1.1.Chlortetracycline Dilute the standard solution

(5)with the phosphate buffer solution (4.2) to obtain a standard working solution with a concentration corresponding to 0.2 µg per ml of CTC-HC1. Then, using the phosphate buffer solution (4.2), prepare in test tubes, as indicated below, 6 dilutions, each dilution in duplicate. ml of standard working solution ml of phosphate buffer solution (4.2) Concentration of CTC-HC1 (µ/ml) 0.7 0.3 0.14 0.6 0.4 0.12 0.55 0.45 0.11 0.45 0.55 0.09 0.4 0.6 0.08 0.3 0.7 0.06 Dilute the extract (6.1) with the phosphate buffer solution (4.2) to obtain a presumed CTC-HC1 concentration of 0.12 µg per ml. Place 1 ml of this solution in each of 2 tubes; and 0.75 ml (=0.09 µg) in each of 2 other tubes. Make the volume of the latter 2 tubes up to 1 ml with the phosphate buffer solution (4.2). 7.1.2 Oxytetracycline and tetracycline Dilute the standard solution

(5)with the phosphate buffer solution (4.2) to obtain a standard working solution with a concentration corresponding to 0.6 µg per ml of OTC-HC1 or of TC-HC1. Then, using the phosphate buffer solution (4.2), prepare in test tubes, as indicated below, 7 dilutions, each dilution in duplicate. ml of standard working solution ml of phosphate buffer solution(4.2) Concentration of OTC-HC1 or TC-HC1 (µ/

1. ml)0.9 0.1 0.54 0.8 0.2 0.48 0.7 0.3 0.42 0.6 0.4 0.36 0.4 0.6 0.24 0.3 0.7 0.18 0.2 0.8 0.12 Dilute the extract (6.2) with the phosphate buffer solution (4.2) to obtain a presumed OTC-HC1 or TC-HC1 concentration of 0.48 µg per ml. Place 1 ml of this solution in each of 2 tubes, and 0.5 ml (=0.24 µg) in each of 2 other tubes. Make the volume of the latter 2 tubes up to 1 ml with the phosphate buffer solution (4.2). 7.2. Inoculation of the culture medium Inoculate the basic medium for the determination (4.1) with the inoculum (3.2) to obtain with the photometer at 590 nm 85% light transmission in a 5 cm cell or 92% transmission in a 2 cm cell, the apparatus being set at 100% transmission on the non-inoculated basic medium (4.1). 7.3. Seeding Place 9 ml of the inoculated culture medium (7.2) in each tube (7.1.1 or 7.1.2). The tubes must be filled under clean but not necessarily sterile conditions. 7.4. Incubation Incubation must be carried out in a water bath whose temperature is kept uniform at 37°C±0.1°C by stirring. The incubation period chosen (generally 2 hours 30 minutes to 3 hours) must be such that it will be possible to trace transmission curves with gradients suitable for accurate measurement. Then block further growth by rapidly injecting 1 ml of formaldehyde solution (4.6) into each tube. 7.5. Measurement of growth Measure the transmissions with the photometer at 590 nm, setting the apparatus at 100% transmission on the clearest standard solution (corresponding to the highest antibiotic content). Since the different tubes will show slight differences of turbidity, at least 2 cm, and preferably 5 cm, cells should be used. 8. Calculation of results Trace the calibration curve on millimetre graph paper by plotting the photometric transmissions against the antibiotic concentrations. Interpolate on the curve the transmission values of the extract. Calculate the antibiotic content of the sample. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed 10%, relative to the higher result. 5. DETERMINATION OF OLEANDOMYCIN by diffusion on agar 1. Purpose and scope To determine, even in the presence of tetracyclines, the oleandomycin content of feedingstuffs, concentrates and premixes, where more than 0.5 ppm mg/kg are present. 2. Principle The sample is extracted with a dilute methanol solution of tri (hydroxymethylamino) methane. After centrifuging, the extract is diluted and its antibiotic activity determined by measuring the diffusion of the oleandomycin on an agar medium seeded with B. cereus. The diffusion is made evident by the formation of inhibition zones in the presence of the micro-organism. The diameter of these zones is directly proportional to the logarithm of the antibiotic concentration. 3. Micro-organism: B. cereus K 250 TR1 (resistant to tetracyclines) (NCIB 11183; NCTC 10989) 1 Strain isolated by the LUFA at Kiel. 3.1 Maintenance of the parent strain Inoculate with B. cereus a tube of sloped agar taken from the culture medium (4.1) to which 100 µg per 5 ml oxytetracycline has been added. Incubate overnight at approximately 30°C. Keep the culture in a refrigerator and re-inoculate sloped agar with it every 4 weeks. 3.2 Preparation of the spore suspension Collect the bacteria from a tube of sloped agar (3.1) using approximately 3 ml of physiological saline (4.3). With this suspension, seed a Roux flask containing 300 ml of culture medium (4.1) which has a 3—4% agar concentration. Incubate for 3—5 days at 28—30°C, then collect the spores in 15 ml of ethanol (4.4) after checking sporulation under a microscope, and mix. This suspension will keep in a refrigerator for 5 months or more. By preliminary tests on plates with the basic medium for the determination (4.2), establish the quantity of inoculum which, for the different concentrations of oleandomycin used will give the largest possible inhibition zones that are still clear. This quantity is usually between 0.1—0.2 ml per 1000 ml. The culture medium is inoculated at 60°C. 4. Culture media and reagents 4.1 Medium for maintenance of the parent strain1 1 Any commercial culture medium of similar composition and giving the same results may be used. Glucose 1 g Tryptic peptone 10 g Meat extract 1.5 g Yeast extract 3 g Agar, according to quality 10—20 g Water to 1000 ml Adjust the pH to 6.5 immediately before use. 4.2 Basic medium for the determination1 Medium (4.1) adjusted to pH 8.8. 4.3 Sterile physiological saline. 4.4 Ethanol solution, 20% (v/v). 4.5 Methanol. 4.6 Tri (hydroxymethylamino) methane solution, 0.5% (w/v). 4.7 Extraction solution Pure methanol 50 ml Water 50 ml Tri (hydroxymethylamino)methane 0.5 g 4.8 Standard substance: oleandomycin of known activity. 5. Standard solution Dissolve some of the standard substance (4.8) in 5 ml of methanol (4.5) and dilute with the solution (4.6) to obtain an oleandomycin concentration of 100 µg per ml. From this stock solution, prepare a standard working solution S8 containing 0.1 µg per ml of oleandomycin by diluting with the solution (4.6). Then prepare by successive dilutions (1 + 1), using the solution (4.6), the following concentrations: S4 0.05 µg/ml S2 0.025 µg/ml S1 0.0125 µg/ml 6. Extraction Take a test sample of 2—10 g, depending on the presumed oleandomycin content of the sample, add 100 ml of the solution (4.7) and shake for 30 minutes on a shaking platform. Centrifuge, take an aliquot part of the extract and dilute with the solution (4.6) to obtain a presumed oleandomycin concentration of 0.1 µg per ml (= U8). Then prepare the concentrations U4, U2 and U1 by successive dilutions (1 + 1) using the solution (4.6). 7. Determination method 7.1 Inoculation of the culture medium Inoculate at 60°C the basic medium for the determination (4.2) with the spore suspension (3.2). 7.2 Preparation of the trays Diffusion on agar is carried out in trays using 4 concentrations of the standard solution (S8, S4, S2, S1 ) and four concentrations of the extract (U8, U4, U2, U1,). The 4 concentrations of standard solution and of extract must be placed in each tray. Choose trays, therefore, which are large enough to allow at least 8 holes 10—13 mm in diameter to be made in the agar medium. Calculate the quantity of inoculated culture medium (7.1) needed to provide a uniform covering approximately 2 mm thick. The test should preferably be carried out on trays consisting of flat glass plates fitted with a perfectly level aluminium or plastic ring 200 mm in diameter and 200 mm high. Pipette into the holes accurately measured quantities of between 0.10—0.15 ml of antibiotic solution, depending on the diameter of the holes. For each sample repeat the diffusion at least 4 times with each concentration so that each determination comprises an evaluation of 32 inhibition zones. 7.3 Incubation Incubate the trays for approximately 18 hours at 28—30°C. 8. Evaluation Measure the diameter of the inhibition zones, preferably by projection. Record the measurements on semi-logarithmic paper, plotting the logarithm of the concentrations against the diameter of the inhibition zones. Trace the lines of the standard solution and of the extract. Provided there is no interference, the two lines will be parallel. The logarithm of the relative activity is calculated by using the following formula: Real activity = presumed activity x relative activity. Repeatability The difference between the results of two parrallel determinations carried out on the same sample must not exceed 10% relative to the higher result. 6. DETERMINATION OF TYLOSIN by diffusion on agar 1. Purpose and scope To determine the tylosin content of feedingstuffs, concentrates and premixes where more than 2 mg/kg are present. 2. Principle The sample is treated with a pH 8 phosphate buffer solution, previously heated to 80°C, and then extracted with methanol. After centrifuging, the extract is diluted and its antibiotic activity determined by measuring the diffusion of the tylosin on an agar medium seeded with Sarcina lutea. The diffusion is made evident by the formation of inhibition zones in the presence of the micro-organism. The diameter of these zones is directly proportional to the logarithm of the antibiotic concentration. 3. Micro-organism: Sarcina lutea ATCC No. 9341 (NCIB 8553; NCTC 8340) 3.1 Maintenance of the parent strain Inoculate with Sarcina lutea a tube of sloped agar taken from the culture medium (4.1), adjust to pH 7.0. Incubate overnight at approximately 35°C. Keep the culture in a refrigerator and reinoculate sloped agar with it every month. 3.2 Preparation of the bacteria suspension Collect the bacteria from a recently prepared tube of sloped agar (3.1) using 2—3 ml of physiological saline (4.4). With this suspension seed a Roux flask containing 250 ml of the culture medium (4.1), adjusted to pH 7.0. Incubate for 24 hours at 35°C, then collect the bacteria in 25 ml of physiological saline (4.4). Mix, and dilute this suspension to obtain approximately 75% light transmission at 650 nm. If kept in a refrigerator this suspension may be used for one week. By preliminary tests on plates with the basic medium for the determination (4.1), establish the quantity of inoculum which, for the different concentrations of tylosin used, will give the largest possible inhibition zones that are still clear. The culture medium is inoculated at 48—50°C. 4. Culture media and reagents 4.1 Basic medium for the determination1 Glucose 1 g Tryptic peptone 10 g Meat extract 1.5 g Yeast extract 3 g Agar, according to quality 10—20 g Water to 1000 ml Adjust immediately before use to pH 7.0 for maintenance of the parent strain and preparation of the bacteria suspension, and to pH 8.0 for the determination. 4.2 Phosphate buffer solution, pH 8 Potassium dihydrogen phosphate KH2PO4 0.523 g di Potassium hydrogen phosphate K2HPO4 16.730 g Water to 1000 ml 4.3 Phosphate buffer solution, pH 7 Potassium dihydrogen phosphate KH2PO4 5.5 g di Potassium hydrogen phosphate K2HPO4, 13.6 g Water to 1000 ml 4.4 Sterile physiological saline. 4.5 Methanol. 4.6 Methanol solution, 10% (v/v). 4.7 Mixture of phosphate buffer solution (4.2)/pure methanol: 60/40 by volume. 4.8 Standard substance: tylosin of known activity. 5. Standard solutions Dry the standard substance (4.8) for 3 hours at 60°C in a vacuum oven (5 mm of mercury). Weigh 10—50 mg into a graduated flask, dissolve in 5 ml of methanol (4.5) and dilute the solution with the phosphate buffer solution, pH 7 (4.3), to obtain a tylosin-base concentration of 1000 µg per ml. Prepare a standard working solution S8 containing 2 µg per ml of tylosin base from this stock solution by diluting with the mixture (4.7). 1 Any commercial culture medium of similar composition and giving the same results may be used. Then prepare by successive dilutions (1 + 1), using the mixture (4.7), the following concentrations: S4 1 µg/ml S2 0.5 µg/ml S1 0.25 µg/ml 6. Extraction For concentrates, take a 10 g test sample; for premixes and feedingstuffs, a 20 g test sample. Add 60 ml of phosphate buffer solution, pH8 (4.2), previously heated to 80°C, and mix for 2 minutes (domestic mixer, Ultra-turrax, etc.). Leave to stand for 10 minutes, add 40 ml of methanol (4.5) and mix for 5 minutes. Centrifuge the extract and dilute an aliquot part with the mixture (4.7) to obtain a presumed tylosin concentration of 2 µg per ml (=U8). Then prepare the concentrations U4, U2 and U1, by successive dilutions (1 + 1) using the mixture (4.7). For contents of less than 10 ppm, evaporate the extract until dry in a rotary evaporator at 35°C and dissolve the residue in 10% methanol (4.6). 7. Determination method 7.1 Inoculation of the culture medium Inoculate at 48—50°C the basic medium for the determination (4.1), adjust to pH 8.0, with the bacteria suspension (3.2). 7.2 Preparation of the trays Diffusion on agar is carried out in trays using 4 concentrations of the standard solution (S8, S4, S2, S1) and 4 concentrations of the extract (U8, U4, U2, U1). The 4 concentrations of standard solution and of extract must be placed in each tray. Choose trays, therefore, which are large enough to allow at least 8 holes 10—13 mm in diameter to be made in the agar medium. Calculate the quantity of inoculated culture medium (7.1) needed to provide a uniform covering approximately 2 mm thick. The test should preferably be carried out on flat trays consisting of glass plates fitted with a perfectly level aluminium or plastic ring, 200 mm in diameter and 20 mm high. Pipette into the holes acurately measured quantities of between 0.10—0.15 ml of antibiotic solution, depending on the diameter of the holes. For each sample repeat the diffusion at least 4 times with each concentration so that each determination comprises an evaluation of 32 inhibition zones. 7.3 Incubation Incubate the trays overnight at 35—37°C. 8. Evaluation Measure the diameter of the inhibition zones, preferably by projection. Record the measurements on semi-logarithmic paper, plotting the logarithm of the concentrations against the diameter of the inhibition zones. Trace the lines of the standard solution and of the extract. Provided there is no interference the two lines will be parallel. The logarithm of the relative activity is calculated by using the following formula: Real activity = presumed activity x relative activity. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed 10% relative to the higher result. 7. DETERMINATION OF VIRGINIAMYCIN by diffusion on agar. 1. Purpose and scope To determine the virginiamycin content of feeding stuffs, concentrates and premixes where more than 2 mg/kg are present. 2. Principle The sample is extracted with a "Tween 80" methanol solution. After centrifuging or filtering, the extract is diluted and its antibiotic activity determined by measuring the diffusion of the virginiamycin on an agar medium seeded with Sarcina lutea. The diffusion is made evident by the formation of inhibition zones in the presence of the micro-organism. The diameter of these zones is directly proportional to the logarithm of the antibiotic concentration. 3. Micro-organism: Sarcina lutea ATCC No 9341 (NCIB 8553, NCTC 8340) 3.1 Maintenance of the parent strain Inoculate with S. Lutea a tube of sloped agar taken from the culture medium (4.1). Incubate overnight at approximately 35°C. Keep the culture in a refrigerator and re-inoculate sloped agar with it every 14 days. 3.2 Preparation of the bacteria suspension Collect the bacteria from a recently prepared tube of sloped agar (3.1) using 2—3 ml of physiological saline (4.3). With this suspension, seed a Roux flask containing 250 ml of the culture medium (4.1). Incubate for 24 hours at 35°C. then collect the bacteria in 25 ml of physiological saline (4.3). Mix, and dilute this suspension to obtain approximately 75% light transmission at 650 nm. If kept in a refrigerator this suspension may be used for 1 week. By preliminary tests on plates with the basic medium for the determination (4.1). establish the quantity of inoculum which, for the different concentrations of virginiamycin used, will give the largest possible inhibition zones that are still clear. The culture medium is inoculated at 48-50°C. 4. Culture media and reagents 4.1 Basic medium for the determination1 Glucose 1 g Tryptic peptone 10 g Meat extract 1.5 g Yeast extract 3 g Agar, according to quality 10-20 g Water to 1000 ml Adjust the pH to 6.5 before use. 4.2 Phosphate buffer solution, pH 6 Potassium dihydrogen phosphate KH2PO4 8.0 g di Potassium hydrogen phosphate K2HPO4 2.0 g Water to 1000 ml 4.3 Sterile physiological saline 4.4 Methanol 4.5 Mixture of phosphate buffer solution (4.2)/pure methanol: 80/20 by volume. 4.6 "Tween 80" methanol solution, 0.5% (w/v). 4.7 Standard substance: virginiamycin of known activity. 1 Any commercial culture medium of similar composition and giving the same results may be used. 5. Standard solutions Prepare a methanol solution of the standard substance (4.7) containing 800 µg per ml of virginiamycin. From this stock solution prepare a standard working solution S8 containing 1µg per ml of virginiamycin by diluting with the mixture (4.5). Then prepare by successive dilutions (1 + 1), using the mixture (4.5), the following concentrations: S4 0.5 µg/ml S2 0.25 µg/ml S1 0.125 µg/ml 6. Extraction 6.1 Products with a virginiamycin content of 50 mg/kg or less Take a test sample of 10—20 g, add 100 ml of the solution (4.6) and shake for 30 minutes on a shaking platform. Centrifuge or filter, take 20 ml of the clear solution and evaporate until dry in a rotary evaporator. Dissolve the residue in 20 ml or more of the mixture (4.5) to obtain a presumed virginiamycin concentration of 1 µg per ml (= U8). Then prepare the concentrations U4, U2 and U1 by successive dilutions (1 + 1) using the mixture (4.5). 6.2 Products with a virginiamycin content greater than 50 mg/kg Take a test sample of 1—10 g, add 100 ml of solution (4.6) and shake for 30 minutes on a shaking platform. Centrifuge or filter, then dilute with the mixture (4.5) to obtain a presumed virginiamycin concentration of 1 µg per ml (= U8). Then prepare the concentrations U4 U2 and U1 as indicated in 6.1. 7. Determination method 7.1 Inoculation of the culture medium Inoculate the basic medium for the determination (4.1) at 48—50°C with the suspension of bacteria (3.2). 7.2 Preparation of the trays Diffusion on agar is carried out in trays using 4 concentrations of the standard solution (S8, S4, S2, S1) and 4 concentrations of the extract (U8, U4, U2, U1). The 4 concentrations of the standard solution and of extract must be placed in each tray. Choose trays, therefore, which are large enough to allow at least 8 holes 10—13 mm in diameter to be made in the agar medium. Calculate the quantity of inoculated culture medium (7.1) needed to provide a uniform covering approximately 2 mm thick. The test should preferably be carried out on flat trays consisting of glass plates fitted with a perfectly level aluminium or plastic ring, 200 mm in diameter and 20 mm high. Pipette into the holes accurately measured quantities of between 0.10—0.15 ml of antibiotic solution, depending on the diameter of the holes. For each sample, repeat the diffusion at least 4 times with each concentration so that each determination comprises an evaluation of 32 inhibition zones. 7.3 Incubation Incubate the trays for approximately 18 hours at 28—30°C. 8 Evaluation Measure the diameter of the inhibition zones, preferably by projection. Record the measurements on semi-logarithmic paper, plotting the logarithm of the concentrations against the diameter of the inhibition zones. Trace the lines of the standard solution and of the extract. Provided there is no interference, the two lines will be parallel. The logarithm of the relative activity is calculated by using the following formula: Real activity = presumed activity x relative activity. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed 10% relative to the higher result. 8. DETERMINATION OF AMPROLIUM (Chloride hydrochloride of 1-(4-amino-2-propyl-5-pyrimidylmethyl)-2-picolinium) 1. Purpose and scope To determine the content of amprolium in feeding stuffs, concentrates and premixtures. The lower limit of the determination is 40 mg/kg. 2. Principle The sample is extracted with diluted methanol. The extract is purified on a column of aluminium oxide and treated with a methanol solution of 2, 7-dihydroxyn aphthalene, potassium ferricyanide, potassium cyanide and sodium hydroxide, forming a purple colour complex. Amprolium is determined by spectrophotometry at 530 nm. 3. Reagents 3.1 Methanol. 3.2 Diluted methanol: mix two volumes of methanol (3.1) with one volume of water. 3.3 Potassium ferricyanide (K3Fe (CN)6) solution, 0.2% (w/v). This solution is stable for 2 weeks. 3.4 Potassium cyanide solution, 1% (w/v). This solution is stable for two weeks. 3.5 Sodium hydroxide solution, 1.125% (w/v). 3.6 Methanolic sodium hydroxide solution: dilute 15 ml of the solution (3.5) to 200 ml with methanol (3.1). 3.7 2, 7—dihydroxynaphthalene solution, 0.0025% (w/v): dissolve 25 mg of 2, 7-dihydroxynaphthalene in methanol (3.1) and make it up to 1000 ml with methanol (3.1). 3.8 Colour reagent: transfer 90 ml of 2, 7-dihydroxynaphthalene solution (3.7) to a conical flask (4.1), add 5 ml of potassium ferricyanide solution (3.3) and mix well. Then add 5 ml of potassium cyanide solution (3.4), stopper the flask and mix well. Leave to stand for 30—35 minutes, add 100 ml of methanolic sodium hydroxide solution (3.6), mix and filter through a filter crucible (4.3). Use this reagent in the 75 minutes following filtration. 3.9 Aluminium oxide for column chromatography: before use, stir 100 g of aluminium oxide with 500 ml of water for 30 minutes, filter the slurry, wash the aluminium oxide on the filter 3 times with 50 ml of methanol (3.1), drying each time by suction, leave to stand overnight and then dry for 2 hours at 100°C in a vacuum drier. Put in a desiccator to cool. Check strength by subjecting a specified quantity of standard solution (3.11) to analysis, starting from point 5.2. The recovery rate of the amprolium must be 100%±4%. 3.10 Standard substance: pure amprolium complying with the characteristics below: Melting point (decomposition): 248°C. Molecular extinction co-efficient at both 265 and 235 nm in water: 11.0 x 103. 3.11 Standard solution: weigh out to within 0.1 mg, 50 mg of standard substance (3.10). Dissolve in diluted methanol (3.2) in a 500 ml volumetric flask, make up the volume with the same solvent and mix. Dilute 10.0 ml to 50 ml with diluted methanol (3.2) in a volumetric flask and mix well. 1 ml of this solution contains 20 µg of amprolium. 4. Apparatus 4.1 Conical flasks with ground glass stoppers. 4.2 Stirrer. 4.3 Filtering crucible, porosity G3, diameter: 60 mm. 4.4 Glass tube for chromatography (interior diameter: approximately 9 mm, length: 400—500 mm). 4.5 Centrifuge. 4.6 Spectrophotometer with suitable cells (10 mm). 5. Procedure 5.1 Extraction and purification 5.1.1 Feeding stuffs and premixtures Weigh, to the nearest mg, approximately 10 g of the finely divided and mixed sample. For premixtures weigh 3—6 g, to the nearest mg. Place the test portion in a conical flask (4.1) and add exactly 100 ml of diluted methanol (3.2). Shake for 60 minutes and filter. Dilute with diluted methanol (3.2) if necessary to obtain a solution containing 5—15 µg of amprolium per ml. Insert a cotton wool plug into the lower end of a chromatographic tube (4.4), and tamp in 5 g of alumium oxide (3.9) and then run in 25.0 ml of the extract. Let the liquid run through the column, discard the first 5 ml and collect the next 12 ml in a graduated test tube. 5.1.2 Concentrates Weigh, to the nearest mg, approximately 0.5 g of the finely divided and mixed sample, place it in a conical flask (4.1), add 250 ml of diluted methanol (3.2) shake or stir for 60 minutes and filter. Dilute 5.0 ml of the Filtrate to 200 ml with diluted methanol (3.2) in a volumetric flask. 5.2 Development of colour and measurement of the optical density Transfer 5.0 ml of the solution obtained in 5.1.1 or 5.1.2 into centrifuge tube A (4.5). Place 5.0 ml of diluted methanol (3.2) in centrifuge tube B (4.5). Add to each tube 10.0 ml of colouring reagent (3.8), stopper the tubes, mix and leave to stand for 18 minutes. Then centrifuge for 3 minutes in order to obtain a clear solution and decant solutions A and B in conical flasks (4.1). Immediately measure the optical density of solution A at 530 nm in the spectrophotometer (4.6) using solution B as a control. Determine the quantity of amprolium by referring to the calibration curve (5.3). 5.3 Calibration curve Pipette into centrifuge tubes (4.5) volumes of 1.0, 2.0, 3.0, 4.0 and 5.0 ml respectively of the standard solution (3.11). Make the volumes of the first four tubes up to 5.0 ml with diluted methanol (3.2). Add to all five tubes 10.0 ml of coloring reagent (3.8), stopper the tubes, mix and leave to stand for 18 minutes. Then centrifuge for 3 minutes and decant the solutions into conical flasks (4.1). Immediately measure the optical density of the solutions at 530 nm in the spectrophotometer (4.6), using a mixture of 5 ml diluted methanol (3.2) and 10 ml of colour reagent (3.8) as a control. Plot the calibration curve, using the optical density values as the ordinates and the corresponding quantities of amprolium in mg as the abscissae. 6. Calculation of results 6.1 Feeding stuffs and premixtures The amprolium content in mg per kg of sample is given by the formula in which A = quantity of amprolium in mg as determined by photometric measurement; W = weight of the test portion in grams; F = coefficient of dilution (possibly worked out in 5.1.1). 6.2 Concentrates The amprolium content per cent of sample is given by the formula in which A = quantity of amprolium in mg as determined by photometric measurement; W = weight of the test portion in grams. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed: 10 mg/kg in absolute value, for amprolium contents below 100 mg/kg; 10%, relative to the higher result, for contents between 100 and 5000 mg/kg; 500 mg/kg in absolute value, for contents between 5000 and 10000 mg/kg; 5%, relative to the higher result, for contents above 10000 mg/kg. 9. DETERMINATION OF ETHOPABATE (methyl-4-acetamido-2-ethoxybenzoate) 1. Purpose and scope To determine the content of ethopabate in feeding stuffs, concentrates and premixtures. The lower limit of the determination is 2 mg/kg. 2. Principle The sample is extracted with diluted methanol. The solution is acidified and extracted with chloroform. The chloroform extract is washed first with an alkaline solution and then with water. The purified extract is concentrated, the ethopabate is hydrolysed with diluted hydrochloric acid. The animo derivative thus formed is diazotised and coupled with 2-aminoethyl-1-naphthylamine dihydrochloride. The colored complex is extracted with butanol and the optical density of the solution is measured at 555 nm. 3. Reagents 3.1 Methanol. 3.2 Methanol solution, 50% (v/v): mix equal volumes of methanol (3.1) and water. 3.3 Hydrochloric acid, d: 1.18. 3.4 Hydrochloric acid solution, 10% (v/v): dilute 10.0 ml of hydrochloric acid (3.3) to 100 ml with water. 3.5 Hydrochloric acid, approximately 0.3 N: dilute 25 ml of hydrochloric acid (3.3) to 1,000 ml with water. 3.6 Chloroform. 3.7 Sodium carbonate solution, 4% (w/v): dissolve 40.0 g of anhydrous sodium carbonate in water and make up to 1,000 ml with water. 3.8 Sodium nitrate solution 0.2% (w/v): dissolve 100 mg of sodium nitrate in water and make up to 50 ml with water in a volumetric flask. Prepare immediately before use. 3.9 Ammonium sulphamate solution 1.0% (w/v): dissolve 500 mg of ammonium sulphamate in water and make up to 50 ml with water in a volumetric flask. Prepare immediately before use. 3.10 2-aminoethyl-1-naphthylamine dihydrochloride solution, 0.2% (w/v): dissolve 100 mg of 2-aminoethyl-1-naphthylamine dihydrochloride in water and make up to 50 ml with water in a volumetric flask. Prepare immediately before use. 3.11 Anhydrous sodium chloride. 3.12 n-butanol. 3.13 Standard substance: ethopabate. 3.14 Standard solutions: 3.14.1 Solution of 0.040 mg of ethopabate per ml: weigh out 40 mg to within 0.1 mg of standard substance (3.13). Dissolve in methanol (3.2) in a 100 ml volumetric flask; make up the volume with the same solvent and mix. Dilute 10.0—100 ml with methanol (3.2) in a volumetric flask and mix. This solution is stable for a month. 3.14.2 Solution of 0.016 mg of ethopabate per 20 ml: dilute 5.0 ml of the solution (3.14.1) to 250 ml with methanol (3.2) in a volumetric flask and mix well. Prepare before use. 4. Apparatus 4.1 Conical flasks, with ground glass stoppers. 4.2 Separating funnels, with ground glass stoppers. 4.3 Shaker. 4.4 Rotary vacuum evaporator. 4.5 Water bath. 4.6 Centrifuge. 4.7 Spectrophotometer with suitable cells (10 mm). 5. Procedure 5.1 Extraction Weigh, to the nearest mg, a quantity of the finely divided and mixed sample, containing about 80 µg of ethopabate. Place the test portion in a conical flask (4.1) and add 100.0 ml of diluted methanol (3.2). Mix, stopper the flask and shake for 1 hour with the aid of a shaker (4.3). Decant, filter and discard the first few ml of the filtrate. 5.2 Purification NB All operations under this point must be carried out rapidly. Transfer 20.0 ml of the clear extract into a separating funnel (4.2), add 5.0 ml of hydrochloric acid (3.4) and 20.0 ml of chloroform (3.6). Shake, first carefully and then vigorously for 3 minutes. Leave to stand until the zones separate and collect the chloroform phase in a second separating funnel (4.2). Extract the acid phase twice more with 20.0 ml of chloroform (3.6). Collect the chloroform extracts in the second separating funnel and discard the acid phase. Add to the combined chloroform solution 10 ml of sodium carbonate solution (3.7), shake for 3 minutes and leave to stand until the phases separate. Collect the chloroform phase in a third separating funnel (4.2) and discard the aqueous phase. Add to the chloroform solution 10 ml of sodium carbonate solution (3.7), shake for 3 minutes and leave to stand until the phases separate. Collect the chloroform phase in a fourth separating funnel (4.2) wash twice consecutively with 25.0 ml of water each time, separate the aqueous phases and quantitatively collect the chloroform extract in a round bottomed flask. Combine the aqueous phases together, in one of the separating funnels; rinse each empty funnel with a few ml chloroform; shake the aqueous phase with the same few ml chloroform, allow phases to separate, and transfer the chloroform phase to the chloroform extract collected in the flask. 5.3 Hydrolysis Evaporate the chloroform extract down to about 2 ml on a 50°C water bath with the aid of the rotary vacuum evaporator (4.4). Dissolve the residue in 2-3 ml of methanol (3.1), and transfer quantitatively the solution in a centrifuge tube (4.6) with the aid of two 10 ml portions and one 5 ml portion of 0.3 N hydrochloric acid (3.5). Add a few glass beads, shake well, plunge the tube in a bath of boiling water and keep it there for 45 minutes. Then cool under a stream of cold running water. 5.4 Development of colour and measurement of the optical density Add 1.0 ml of sodium nitrate solution (3.8), stir and leave to stand for 2 minutes. Add 1.0 ml of ammonium sulphamate solution (3.9), shake and leave to stand for 2 minutes. Add 1.0 ml of 2-aminoethyl-1-naphthylamine dihydrochloride solution (3.10), stir and leave to stand for 10 minutes. Add 5.0 g of sodium chloride (3.11) and 10.0 ml of n-butanol (3.12), shake vigorously until the sodium chloride has completely dissolved. Draw off the supernatant butanolic solution with the aid of a pipette, and transfer it to a centrifuge tube (4.6) and centrifuge. Then measure the optical density EA with a spectrophotometer at 555 nm using n-butanol (3.12) as blank. 5.5 Control test Carry out a control test, using the same procedure, starting from point 5.2, on 20.0 ml of diluted methanol (3.2). Measure the optical density EB at 555 nm using n-butanol (3.12) as blank. 5.6 Standard test Carry out a standard test, using the same procedure, starting from point 5.2, on 20.0 ml of standard solution (3.14.2). Measure the optical density EC at 555 nm using n-butanol (3.12) as blank. 6. Calculation of results The ethopabate content in mg per kg of sample is given by the formula (EA - EB) x 80 ___________ (EC - EB) x W in which —EA = optical density of the solution from the sample; —EB = optical density of the solution resulting from the control test; —EC = optical density of the solution resulting from the standard test; —W = weight of test portion in grams. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed: 20%, relative to the higher result, for contents of ethopabate below 7.5 mg/kg; 1.5 mg/kg in absolute value, for contents between 7.5—10 mg/kg; 15%, relative to the higher result, for contents above 10 mg/kg. 10. DETERMINATION OF DINITOLMIDE (DOT) (3, 5-dinitro-o-toluamide) 1. Purpose and scope To determine the content of dinitolmide (DOT) in feeding stuffs, concentrates and premixtures. Nitrofuran derivatives may interfere. The lower limit of the determination is 40 mg/kg. 2. Principle The sample is extracted with acetonitrile. The extract is purified on aluminium oxide and filtered. An aliquot of the filtrate is evaporated to dryness. The residue is dissolved in dimethylformamide and treated with ethylenediamine forming a purple complex. Dinitolmide is determined by spectrophotometry at 560 nm. 3. Reagents 3.1 Acetonitrile 85% (v/v): mix 850 ml of pure acetonitrile and 150 ml of water. Before use, distil the mixture and collect the fraction which boils between 75—77°C. 3.2 Aluminium oxide for column chromatography: heat at 750°C for at least 2 hours, cool in desiccator and keep in a brown glass bottle with a ground glass stopper. Before use humidify as follows: place in a brown glass bottle 10 g of aluminium oxide and 0.7 ml of water, stopper, heat for 5 minutes in a bath of boiling water, shaking vigorously, let it cool, still shaking. Check strength by subjecting to analysis, starting from point 5.1, a determined quantity of standard solution (3.6). The recovery rate of the dinitolmide must be 100%±2%. 3.3 N, N-dimethylformamide 95% (v/v): mix 95.0 ml of N, N-dimethylformamide and 5.0 ml of water. 3.4 Diaminoethane, maximum water content: 2.0%. 3.5 Standard substance: pure 3, 5-dinitro-o-toluamide complying with the characteristics below: melting point: 177°C; molecular extinction coefficient at 248 nm in acetonitrile: 13.1 x 103 molecular extinction coefficient at 266 nm in N, N-dimethylformamide: 10.1 x 103. 3.6 Standard solution: weigh, to the nearest 0.1 mg, 40 mg of standard substance (3.5), dissolve with acetonitrile (3.1) in a 200 ml volumetric flask, make up to volume with the same solvent and mix. Dilute 20.0 ml to 100 ml with acetonitrile (3.1) in a volumetric flask and mix. 1 ml of this solution contains 40 µg of dinitolmide. 4. Apparatus 4.1 Conical flask with ground glass stopper. 4.2 Reflux condenser with ground glass joint. 4.3 Filtering crucible porosity G3, diameter 60 mm. 4.4 Vacuum filter (such as Witt apparatus). 4.5 Water bath, set at 50°C. 4.6 Spectrophotometer with suitable cells (10 mm). 5. Procedure 5.1 Extraction and purification Weigh, to the nearest mg, approximately 10 g of the finely divided and mixed sample. For concentrates and premixtures, weigh approximately 1 g, to the nearest mg. Place the test portion in a conical flask (4.1) and add 65 ml of acetonitrile (3.1). Mix, fit reflux condenser (4.2) to the flask and heat in the water bath (4.5) for 30 minutes, shaking continuously. Cool under stream of cold water. Add 20 g of aluminium oxide (3.2), shake for 3 minutes, leave to settle. Place a 100 ml volumetric flask in the vacuum filter (4.4), fit filtering crucible (4.3) and filter the solution, using suction. Then transfer the remaining solids into the crucible with the aid of a few ml of acetonitrile (3.1) and suck the residue dry. Release the vacuum, suspend the residue again in a few ml of acetonitrile (3.1) and again apply vacuum. Repeat these last operations until the volume of the filtrate reaches about 95 ml. Make up to 100 ml with acetonitrile (3.1) and mix. If necessary, dilute an aliquot with acetonitrile (3.1) to obtain a solution containing 5—15 µg of dinitolmide per ml. 5.2 Development of color and measurement of the optical density Pipette into three beakers A, B and C respectively, 4.0 ml of the solution obtained in 5.1. Also add to beaker C only 1.0 ml of standard solution (3.6). Place the three beakers on the water bath (4.5), placed under a well ventilated hood, and evaporate until dry in a current of dry air. Cool the three beakers to room temperature. Add 10.0 ml of N, N-dimethylformamide (3.3) in beaker A and 2.0 ml in beakers B and C respectively, leave in contact for a few minutes, stirring a little, until the residue completely dissolves. Then add 8.0 ml of diaminoethane (3.4) in beakers B and C and mix. Exactly 5 minutes after adding the diaminoethane measure the optical density of the three solutions in the spectrophotometer (4.6) at 560 nm using the N, N-dimethylformamide (3.3) as a blank. 6. Calculation of results The dinitolmide content in mg per kg of sample is given by the formula: in which EA = optical density of solution A (blank); EB = optical density of solution B (sample); EC = optical density of solution C (internal standard); W = weight of test portion in grams; F = coefficient of dilution (see 5.1). Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed: 10 mg/kg in absolute value, for contents of dinitolmide below 100 mg/kg; 10%, relative to the higher result, for contents between 100—5,000 mg/kg; 500 mg/kg in absolute value, for contents between 5,000—10,000 mg/kg; 5%, relative to the higher result, for contents above 10,000 mg/kg. 11. DETERMINATION OF NICARBAZIN. (equimolecular mixture of 4, 4-dinitrocarbanilide and 2-hydroxy-4, 6-dimethylpyrimidine). 1. Purpose and scope To determine the content of nicarbazin in feeding stuffs, concentrates and premixtures containing not more than 5% grassmeal. Nitrofuran derivatives, acetylenheptine and carbadox may interfere. The lower limit of the determination is 20 mg/kg. 2. Principle The sample is extracted with N, N-dimethylformamide. The extract is purified by chromatography on a column of aluminium oxide; the nicabazine is eluted with ethanol. The eluate is treated with ethanolic sodium hydroxide, forming a yellow colour. Nicarbazin is determined by spectrophotometry at 430 nm. 3. Reagents 3.1 N, N-dimethylformamide. 3.2 Aluminium oxide for column chromatography: heat at 750°C for at least 2 hours, cool in desiccator and keep in a brown glass bottle with a ground glass stopper. Before use, check strength by subjecting to analysis, starting from point 5.2, a determined quantity of standard solution (3.8.3). The recovery rate of the nicarbazin must be 100% ± 2%. 3.3 Ethanol solution, 95% (v/v). 3.4 Ethanol solution, 80% (v/v). 3.5 Sodium hydroxide solution, 50% (w/v). 3.6 Ethanolic sodium hydroxide solution, 1% (w/v): put 1 ml of sodium hydroxide solution (3.5) in a 50 ml volumetric flask; make up the volume with 80% ethanol (3.4). Prepare at the time of use. 3.7 Standard substance: pure nicarbazine, moleucular extinction coefficient at 362 nm in N, N-dimethylformamide: 37.8x103. 3.8 Standard solutions: 3.8.1 Solution of 1.25 mg of nicarbazin per ml: weigh out to within 0.1 mg, 125 mg of standard substance (3.7). Dissolve in 75 ml of N, N-dimethylformamide (3.1) in a 100 ml volumetric flask, heating slightly. Allow to cool, make up the volume with the same solvent and mix. Keep away from light. 3.8.2 Solution of 0.125 mg of nicarbazin per ml: dilute 10.0 ml of the solution (3.8.1), to 100 ml with N, N-dimethylformamide (3.1) in a volumetric flask and mix. 3.8.3 Solution of 0.025 mg of nicarbazin per ml: dilute 20.0 ml of the solution (3.8.2), to 100 ml with N, N-dimethylformamide (3.1) in a volumetric flask and mix. 4. Apparatus 4.1 Conical flask with ground glass stopper. 4.2 Reflux condenser with ground glass joint. 4.3 Boiling water bath. 4.4 Centrifuge. 4.5 Glass tube for chromatography (interior diameter: approximately 25 mm, length: approximately 300 mm). 4.6 Spectrophotometer with suitable cells (10 mm). 4.7 Burette marked in 1/10th ml. 5. Procedure 5.1 Extraction Weigh, to the nearest mg, approximately 10 g of the finely divided and mixed sample. For concentrates and premixtures, weigh approximately 1 g, to the nearest mg. Place the test portion in a conical flask (4.1) and add exactly 100 ml of N, N-dimethylformamide (3.1). Mix, fit reflux condenser (4.2) on the flask and heat on the water bath (4.3) for 15 minutes, shaking from time to time. Cool under a stream of cold water. Then pour the supernatant layer into a centrifuge tube (4.4) and centrifuge for about 3 minutes. If necessary, dilute 25.0 ml of the supernatant layer with N, N-dimethylformamide (3.1), to obtain a solution containing 2.0—10 µg of nicarbazin per ml. 5.2 Chromatography Run into a tube for chromatography (4.5) a slurry of 30 g of aluminium oxide (3.2) in N, N-dimethylformamide (3.1). Let the liquid level fall to 1 cm above the column of aluminium oxide and then put into the column 25.0 ml of the extract obtained in 5.1. Allow the liquid to flow through, not letting the column get dry, and wash the column with three 10 ml portions of N, N-dimethylformamide (3.1). Then elute with 70 ml of ethanol (3.3). Eliminate the first 10 ml of the eluate and collect the rest, dividing it up as follows: one 5 ml portion (a); one 50 ml portion (
2. b)in a volumetric flask; one 5 ml portion (c). Check that portions (
3. a)and (
4. c)do not turn yellow when ethanolic sodium hydroxide (3.6) is added. Continue the operations on portion (
5. b)as shown in 5.3. 5.3 Development of colour and measurement of the optical density Pipette 20.0 ml of portion (
6. b)of the eluate in two separate 25 ml volumetric flasks A and B. Add to flask A 5.0 ml of ethanolic sodium hydroxide (3.6) and to flask B 5.0 ml of ethanol (3.3). Mix well. Within the next 5 minutes measure the optical density of both solutions at 430 nm, using a mixture of 20.0 ml of ethanol (3.3) and 5.0 ml of ethanol solution of sodium hydroxide (3.6) as a blank. Subtract the value of the optical density of solution B from that of solution A. From this value determine the quantity of nicarbazin referring to the calibration curve (5.4). 5.4 Calibration curve Subject 25.0 ml of the standard solution (3.8.3) to chromatography as shown in 5.2. Transfer 2.0, 4.0, 6.0, 8.0 and 10.0 ml (corresponding to 0.025, 0.050, 0.075, 0.100 and 0.125 mg of nicarbazin respectively) of portion (
7. b)into 25 ml graduated flasks from a burette (4.7). To each flask add 5.0 ml of ethanolic sodium hydroxide (3.6), make the volume up with ethanol (3.3) and mix well. Within the next 5 minutes measure the optical density of the solutions at 430 nm, using a mixture of 20.0 ml of ethanol (3.3) and 5.0 ml of ethanolic sodium hydroxide (3.6) as a control. Trace the calibration curve, using the optical density values as the ordinates and the corresponding quantities of nicarbazin in mg as the abscissae. 6. Calculation of results The nicarbazin content in mg per kg of sample is given by the formula in which: A = quantity of nicarbazin in mg as determined by photometric measurement; W = weight of test portion in grams; F = coefficient of dilution (see 5.1). Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed: 10 mg/kg in absolute value, for contents of nicarbazin below 100 mg/kg; 10%, relative to the higher result, for contents between 100 — 5 000 mg/kg; 500 mg/kg in absolute value, for contents between 5 000 — 10 000 mg/kg; 5%, relative to the higher result, for contents above 10 000 mg/kg. 12 DETERMINATION OF VITAMIN A (RETINOL). 1. Purpose and scope To determine the content of Vitamin A in feeding stuffs, concentrates and premixtures. The lower limit of the determination is 10 000 IU/kg for highly pigmented feeds and 4 000 IU/kg for others1. Products are classified in two groups, according to their presumed Vitamin A contents: 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 in hot ethanolic potassium hydroxide solution 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 Group B products, chromatography is only required in certain cases). For Group A products the Vitamin A 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.

(1)1 IU = 0.3 g of Vitamin A. 3. Reagents ( a ) used for analysing products of Groups A and B. 3.1 Ethanol, 96% (v/v). 3.2 Sodium ascorbate solution, 10% (w/

1. v)or 3.3 Purified nitrogen. 3.4 Potassium hydroxide solution, 50% (w/v). 3.5 Potassium hydroxide solution, 1 N. 3.6 Potassium hydroxide solution, 0.5 N. 3.7 1, 2-dichlorethane. 3.8 Light petroleum, boiling range: 40—60°C: 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/
2. 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, standardised according to Brockmann: ash for 8 hours at 750°C, 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 0.9 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 Vitamin A (3.17) (approximately 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 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) containing 4, 8, 12, 16 and 20% (v/
3. v)diethyl ether (3.11). 3.13 Sodium sulphide solution 0.5 molar in 70% (v/
4. v)glycerine, prepared from sodium sulphide. ( b ) used exclusively for analysing Group A products. 3.14 Crystallizable benzene. 3.15 Chloroform: 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 (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. Keep in a refrigerator in a brown glass bottle with ground glass stopper. The solution keeps for several weeks. 3.17 Vitamin A — 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: approximately 300 mm; internal diameter: approximately 13 mm). 4.4 Spectrophotometer. Measurements in the UV require silica cells. 4.5 UV lamps suitable for 364 nm. 5. Procedure NB 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 Vitamin A content, thus: 0.1— 1.0 g for concentrates (contents greater than 20 000 IU/g); 3.0 — 5.0 g for premixtures (contents of between 400 — 20 000 IU/g); 10 — 20 g for mineral mixtures; 30 g for Group A products. Immediately place the test sample in a flask with a ground glass stopper. 5.2 Hydrolysis and extraction1 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°C 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°C. 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 alumium 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 2-3 drops per second. The carotene is eluted first3. The Vitamin A 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 Vitamin A is clearly separated from the yellow xanthophyll zones following it. Collect the eluate fraction containing the Vitamin A in an Erlenmeyer flask. 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. 3 Carotene content may be determined by optical density measurement at 450 nm: 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 Vitamin A, 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 Vitamin A 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 Vitamin A content by reference to a standard curve obtained from benzene solutions of increasing Vitamin A-standard concentrations treated with Carr-Price reagent (2-16 IU Vitamin A-standard (3.17) per 0.3 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 Vitamin A. 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 Vitamin A content of the solution is calculated as follows: E325 . 18.30 = IU of Vitamin A/ml However, the ratio of the optical densities E310 : E325 and E334: E325 must be 6 : 7 = 0.857. If one of these ratios differs appreciably from this value ( 0.880), 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 above mentioned ratios still differ appreciably from the value of 0.857 ( 0.880), the determination must be carried out in accordance with the method given for Group A products. 6. Calculation of results Calculate the Vitamin A 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 Vitamin A per kg of feeding stuff, concentrate or premixture. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed: 20%, relative to the higher result, for Vitamin A contents lower than 75,000 IU/kg; 15 000 IU for contents between 75 000 — 150 000 IU/kg; 10%, relative to the higher result, for contents between 150 000 — 250 000 IU/kg; 25 000 IU for contents between 250 000 — 500 000 IU/kg; 5%, relative to the higher result, for contents greater than 500 000 IU/kg. 13. DETERMINATION OF THIAMINE (VITAMIN B1, ANEURINE) 1. Purpose and scope To determine the content of thiamine (aneurine, Vitamin B1 ) in feeding stuffs, concentrates and premixtures. The lower limit of the determination is 5 mg/kg. 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 µg/ml standard thiamine solution: dissolve 127.1 mg pure thiaminium dichloride, previously dried under vacuum to constant weight, or 113.4 mg pure thiaminium chloride, treated in the same fashion, in 1000 ml of sulphuric acid 0.2 N (3.2). If stored in a cool, dark place, this solution keeps for 1 month. 3.2 Sulphuric acid 0.2 N. 3.3 Sodium bisulphite. 3.4 Potassium ferricyanide solution, 20% (w/v). 3.5 Potassium hydroxide solution, 25% (w/v). 3.6 Oxidizing mixtures: 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. 3.8 Sodium acetate solution, 2.5 N. 3.9 Multienzymatic preparation containing protease, phosphatase and amylase (e.g. Clarase). 3.10 Ethanol, 96% (v/v). 4. Apparatus 4.1 Water bath. 4.2 Centrifuge (3 500 rpm). 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 µg thiamine and 125 ml sulphuric acid (3.2). Also add, to flask A only, 1.0 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°C. Add to each flask 20 ml sodium acetate solution (3.8) and 0.5 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, mix 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 1 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 mix (= 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 formula: 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 grams; d = amount of thiamine in µg 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%, relative to the higher result, for contents lower than 500 mg/kg, and 5%, relative to the higher result, for contents equal to or greater than 500 mg/kg. 14. DETERMINATION OF ASCORBIC ACID AND DEHYDROASCORBIC ACID (VITAMIN C) 1. Purpose and scope To determine the total content of ascorbic and dehydroascorbic acids (Vitamin C) in feeding stuffs, concentrates and premixtures. The lower limit of the determination is 5 mg/kg. 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 L-ascorbic acid solution, 0.05%: dissolve 50 mg L-ascorbic acid in approximately 20 ml metaphosphoric acid solution (3.2) and make up to 100 ml with water. Prepare immediately before use. 3.2 Metaphosphoric acid solution 10% (w/v): after grinding it in a mortar, dissolve in water 200 g metaphosphoric acid and make up to 2000 ml with water. Keep at 4°C. This is stable for 1 week. 3.3 Chloroform. 3.4 2, 6-dichlorophenol-indophenol solution, 0.5% (w/v). Prepare immediately before use. 3.5 Filtration aid (S. and S. No. 121 or equivalent). 3.6 2, 4-dinitrophenylhydrazine solution, 2% (w/v): dissolve 2 g 2, 4-dinitrophenylhydrazine in 100 ml dilute sulphuric acid (25 ml sulphuric acid, d: 1.84, diluted by making up to 100 ml with water). Stored at a cool temperature this solution keeps for 1 week. 3.7 Nitrogen, or 3.8 Carbon dioxide. 3.9 Mixture of ethyl acetate/glacial acetic acid/acetone: 96/2/2 in volume. 3.10 Mixture of dichloromethane/glacial acetic acid: 97/3 in volume. 3.11 Silica gel, particle size: 0.05—0.2 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, d: 1.84. 3.14 Eluting solvent for thin layer chromatography: mix 75 ml diethyl ether, 25 ml ethyl acetate and 4.0 ml 96% (w/
5. v)acetic acid. Renew after 2—3 chromatographs. 4. Apparatus 4.1 Water bath fitted with a thermostat set at 20°C. 4.2 Centrifuge (3 500 rpm). 4.3 Rotary vacuum evaporator. 4.4 Glass chromatography tubes (length: approximately 100 mm, internal diameter: approximately 20 mm), with a sintered disc (e.g. Allihn tubes). 4.5 Spectrophotometer or colorimeter with filters. 4.6 Apparatus for thin layer chromatography, with silica gel plates (3.12) coated to a depth of 0.5—0.6 mm. (Ready-made plates are appropriate). Dry the plates for 2½—3 hours in the drying oven at 120—130°C. Leave to cool and then keep in a desiccator for at least 24 hours before use. 4.7 Drying oven set at 120—130°C. 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 µg vitamin C. Add to flask A only 0.4 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°C. Shake briefly and then leave to stand for 10—15 minutes. Add 25 ml water, stopper the flasks, shake vigorously for 10 seconds and leave to stand for 10—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 0.5—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 reheating at 50°C, 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—4 lots of approximately 5
6. 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) 0.5 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 pink-coloured 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 1.5 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 4.0 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—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 formula: 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 grams, of the test sample. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed: 10%, relative to the higher result, for vitamin C contents lower than 10 g/kg, and 5%, relative to the higher result, for contents equal to or greater than 10 g/kg. 15. DETERMINATION OF MENADIONE (VITAMIN K3) 1. Purpose and scope To determine the quantity of menadione (vitamin K3) in feeding stuffs, concentrates and premixtures. The lower limit of the determination is 1mg/kg. 2. Principle The sample is extracted with diluted ethanol. The mixture is clarified with tannin solution and centrifuged. The extract is treated with a solution of sodium carbonate; the liberated menadione is extracted with 1, 2-dichloroethane. The dichloroethane extract is treated, according to its menadione content, either directly or after evaporation, with 2, 4-dinitrophenylhydrazine in solution in ethanol acidified with hydrochloric acid. The obtained hydrazone treated with ammonia in excess gives rise to a blue-green coloured complex the optical density of which is measured at 635 nm. 3. Reagents 3.1 Ethanol, 96% (v/v). 3.2 Ethanol (3.1) diluted to 40% with water. 3.3 Tannin solution, 10% (w/v), prepared from purified powdered tannin. 3.4 1, 2-dichloroethane. 3.5 Anhydrous sodium carbonate solution, 10% (w/v). 3.6 Hydrochloric acid solution, 37% (w/v), d: 1.19. 3.7 Absolute ethanol. 3.8 2, 4-dinitrophenylhydrazine reagent: dissolve 40 mg 2, 4-dinitrophenylhydrazine in about 40 ml boiling absolute ethanol (3.7), allow to cool and transfer into a 50 ml volumetric flask. Add 1 ml hydrochloric acid (3.6) and make up to volume with absolute ethanol (3.7). Prepare immediately before use. 3.9 Ammonia solution, 25% (w/v), d: 0.91. 3.10 Ammoniacal ethanol: mix one volume of ethanol (3.7) with one volume of ammonia (3.9). 3.11 Standard solutions of menadione: dissolve 20 mg menadione (vitamin K3) in 1, 2-dichloroethane (3.4) and make up to 200 ml. Dilute aliquots of this stock solution with 1, 2-dichloroethane (3.4) to obtain a series of solutions with menadione concentrations between 2—10 µg per ml. These solutions must be freshly prepared. 4. Apparatus 4.1 Mechanical shaker. 4.2 Centrifuge (3,000—5,000 rpm). 4.3 Separators with ground glass stoppers. 4.4 Rotary vacuum evaporator. 4.5 Water bath. 4.6 Spectrophotometer. 5. Procedure NB · All operations must be carried out away from direct light, using apparatus of amber glass where necessary. 5.1 Test Sample From the finely divided sample, take a test sample according to the presumed menadione content, e.g.: 0.1—5.0 g for concentrates and premixtures; 20—30 g for feeding stuffs. Transfer the test sample to a flask with ground glass stopper without delay. 5.2 Extraction Add to the test sample exactly 96 ml dilute ethanol (3.2) and shake mechanically for 15 minutes at room temperature. Then add 4.0 ml tannin solution (3.3), mix, transfer the extract into a centrifuge tube, centrifuge at 3,000—5,000 rpm and decant. Place 20—40 ml, accurately measured, of the extract in a seperator, add by pipette 50 ml 2, 3-dichloroethane (3.4), mix and add by pipette 20 ml sodium carbonate solution (3.5). Shake vigorously for 30 seconds and then collect the dichloroethane phase in a separator (4.3). Add 20 ml water, shake again for 15 seconds, collect the dichloroethane phase and remove traces of water with strips of filter paper. For concentrates and premixtures, take an aliquot part of the extract and dilute with 1, 2-dichloroethane (3.4) to obtain a menadione concentration of 2—10 µg per ml. For feeding stuffs, evaporate to dryness an aliquot part of the extract under reduced pressure in an atmosphere of nitrogen on a water bath at 40°C. Rapidly treat the residue with 1, 2-dichloroethane (3.4) to obtain a solution containing 2—10 µg menadione per ml. 5.3 Hydrazone formation Transfer 2.0 ml of the dichloroethane extract obtained in 5.2 to a 10 ml volumetric flask and add 3.0 ml 2, 4-dinitrophenylhydrazine reagent (3.8), securely stopper the flask with a cork or teflon or other suitable stopper so as to prevent evaporation, and heat for 2 hours at 70°C on a water bath (4.5). Allow to cool, add 3.0 ml ammoniacal ethanol (3.10), mix, make up to volume with absolute ethanol (3.7) and mix again. 5.4 Measurement of the optical density Measure the optical density of the blue-green coloured complex by the spectrophotometer (4.6) at 635 nm by comparison with a reagent blank obtained by treating 2.0 ml 1, 2-dichloroethane (3.4) as indicated in 5.3. Determine the quantity of menadione by reference to a calibration curve established for each series of analyses. 5.5 Calibration curve Treat 2.0 ml of the menadione standard solutions (3.11) as described in 5.3. Measure the optical density as indicated in 5.4. Plot the calibration curve with the optical density values as ordinates and the corresponding quantities of menadione in µg
7. as)abscissae. 6. Calculation of results Calculate the menadione content of the sample by taking account of the weight of the test sample and of the dilutions carried out in the course of analysis. Express the result in mg menadione per kg. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed: 20%, relative to the higher result, for menadione contents less than 10 mg/kg; 2 mg/kg, in absolute value, for contents between 10—14 mg/kg; 15%, relative to the higher result, for contents between 14—100 mg/kg; 15 mg/kg, in absolute value, for contents between 100—150 mg/kg; 10%, relative to the higher result, for contents greater than 150 mg/kg. 16. DETERMINATION OF HYDROCYANIC ACID 1. Purpose and scope To determine the content of hydrocyanic acid, free and combined in the form of glucosides, in feeding stuffs and in particular in products derived from linseed, manioc flour and certain species of beans. 2. Principle The sample is suspended in water. The hydrocyanic acid is released by the action of enzymes, entrained by steam distillation and collected in a specific volume of acidified silver nitrate solution. The silver cyanide is separated by filtration and the excess silver nitrate is titrated with a solution of ammonium thiocyanate. 3. Reagents 3.1 A suspension of sweet almonds: crush twenty blanched sweet almonds in 100 ml of water at 37-40°C. Check that there is no hydrocyanic acid in 10 ml of the suspension using sodium picrate paper or by carrying out a blank test as described in the last paragraph of 5. 3.2 Sodium acetate solution, 10% (w/
8. v)neutral to phenolphthalein. 3.3 Anti-foaming emulsion (e.g. silicone). 3.4 Nitric acid, d: 1.40. 3.5 Silver nitrate solution, 0.02 N. 3.6 Ammonium thiocyanate solution, 0.02 N. 3.7 Ammonium ferric sulphate saturated solution. 3.8 Ammonia, d: 0.958. 4. Apparatus 4.1 Oven with thermostat set at 38°C. 4.2 Apparatus for distillation by entrainment in steam fitted with a condenser with a curved extension piece. 4.3 1000 ml flat-bottomed flasks with ground glass stoppers. 4.4 Oil bath. 4.5 Burette graduated in 1/20 ml. 5. Procedure Weigh, to the nearest 5 mg, approximately 20 g of the sample. Place in 1 litre flat-bottomed flask and add 250 ml of water and 10 ml of sweet almond suspension (3.1). Stopper the flask and transfer to the oven for 16 hours at 38°C. Next, cool to room temperature and add 80 ml of water, 10 ml of sodium acetate solution (3.2) and a drop of anti-foaming emulsion (3.3). Connect the flask to the steam distillation apparatus (4.2) and place in an oil bath (4.4) which has first been brought to a temperature slightly above 100°C. Distil 200—300 ml of liquid by passing a powerful current of steam through the flask and gently heating the oil bath (4.4). Collect the distillate in an Erlenmeyer flask protected from the light and containing exactly 50 ml of silver nitrate solution 0.02 N (3.5) and 1 ml of nitric acid (3.4). Make sure that the condenser's delivery adaptor is immersed in the silver nitrate solution. Transfer the contents of the Erlenmeyer flask to a 500 ml volumetric flask, make up to volume with water, stir and filter. Remove 250 ml of the filtrate, add approximately 1 ml solution of ammonium ferric sulphate (3.7) and back-titrate the excess silver nitrate with the solution of ammonium thiocyanate 0.02 N (3.6) taken from the burette graduated in 1/20 ml (4.5). A blank test may, if required, be carried out by applying the same procedure to 10 ml of sweet almond suspension (3.1), omitting the sample to be analysed. 6. Calculation of results If the blank test indicates that silver nitrate solution 0.02 N has been consumed, subtract the value of this from the volume consumed by the distillate of the sample. 1 ml of AgNO3 0.02 N corresponds to 0.54 mg of HCN. Express the result as a percentage of the sample. 7. Observation If the sample contains a large quantity of sulphides (eg beans), a black precipitate of silver sulphide is formed which is filtered together with the silver cyanide deposit. The formation of this precipitate causes a loss of silver nitrate solution 0.02 N, the volume of which must be subtracted from the volume used to calculate the HCN content. To do this, proceed as follows: Treat the deposit left on the filter with 50 ml of ammonia (3.8) in order to dissolve the silver cyanide. Wash the residue in dilute ammonia and then determine its silver content. Convert the value obtained into ml of silver nitrate solution 0.02 N. The sample's HCN content may also be determined by titrating the acidified ammoniacal filtrate with nitric acid. 17. DETERMINATION OF FREE AND TOTAL GOSSYPOL 1. Purpose and scope To determine the levels of free gossypol, total gossypol and chemically related substances in cottonseed, cottonseed meal and cottonseed cake and in compound feedingstuffs containing these substances where more than 20 mg/kg are present. 2. Principle The gossypol is extracted in the presence of 3-aminopropan-1-ol, either with a mixture of propan-2-ol and hexane, for the determination of free gossypol, or with dimethylformamide, for the determination of total gossypol. The gossypol is converted by aniline into gossypol-dianiline, the optical density of which is measured at 440 nm. 3. Reagents 3.1 Propan-2-ol-hexane mixture: mix 60 parts by volume of propan-2-ol with 40 parts by volume of n-hexane. 3.2 Solvent A: place in a 1 litre graduated flask approximately 500 ml of propan-2-ol-hexane mixture (3.1), 2 ml of 3-aminopropan-1-ol, 8 ml of glacial acetic acid and 50 ml of water. Make up to volume with the propan-2-ol-hexane mixture (3.1). This reagent is stable for one week. 3.3 Solvent B: pipette 2 ml of 3-aminopropan-1-ol and 10 ml of glacial acetic acid into a 100 ml graduated flask. Cool to room temperature and make up to volume with N, N-dimethylformamide. This reagent is stable for one week. 3.4 Aniline: if the optical density in the blank test exceeds 0.022, distil the aniline over zinc dust, discarding the first and last 10% fractions of the distillate. Refrigerated and stored in a brown, stoppered glass flask, this reagent will keep for several months. 3.5 Standard gossypol solution A: place 27.9 mg of gossypol acetate in a 250 ml graduated flask. Dissolve and make up to volume with solvent A (3.2). Pipette 50 ml of this solution into a 250 ml graduated flask and make up to volume with solvent A. The gossypol concentration of this solution is 0.02 mg per ml. Leave to stand for one hour at room temperature before use. 3.6 Standard gossypol solution B: place 27.9 mg of gossypol acetate in a 50 ml graduated flask. Dissolve and make up to volume with solvent B (3.3). The gossypol concentration of this solution is 0.5 mg per ml. Standard gossypol solutions A and B will remain stable for 24 hours if protected from the light. 4. Apparatus 4.1 Shaker: approximately 35 rpm. 4.2 Spectrophotometer. 5. Procedure 5.1 Test sample The amount of test sample used depends on the presumed gossypol content of the sample. It is preferable to work with a small test sample and a relatively large aliquot part of the filtrate, so as to obtain sufficient gossypol for precise photometric measurement to be possible. For the determination of free gossypol in cottonseed, cottonseed meal and cottonseed cake, the test sample should not exceed 1 g; for compound feedingstuffs, it may be as much as 5 g. A 10 ml aliquot part of filtrate is suitable in most cases; it should contain 50—100 ug of gossypol. For the determination of total gossypol, the test sample should be between 0.5—5 g, so that a 2 ml aliquot part of filtrate will contain 40—200 µg of gossypol. The analysis should be carried out at a room temperature of about 20°C. 5.2 Determination of free gossypol Place the test sample in a ground-necked 250 ml flask, the bottom of the flask having been covered with crushed glass. Using a pipette, add 50 ml of solvent A (3.2), stopper the flask and mix for one hour in the shaker. Filter through a dry filter and collect the filtrate in a small ground-necked flask. During filtration, cover the funnel with a watch glass. Pipette identical aliquot parts of filtrate containing 50—100 µg of gossypol into each of two 25 ml graduated flasks (A and B). If necessary, make up the volume to 10 ml with solvent A (3.2). Then make the contents of flask (A) up to volume with the propan-2-ol-hexane mixture (3.1). This solution will be used as a reference solution against which to measure the sample solution. Pipette 10 ml of solvent A (3.2) into each of two other 25 ml graduated flasks (C and D). Make the contents of flask (C) up to volume with the propan-2-ol-hexane mixture (3.1). This solution will be used as a reference solution against which to measure the blank test solution. Add 2 ml of aniline (3.4) to each of flasks (D) and (B). Heat for 30 minutes over a boiling water bath to develop the colour. Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture (3.1), mix and leave to stand for one hour. Determine the optical density of the blank test solution (D) by comparison with the reference solution (C), and the optical density of the sample solution (B) by comparison with the reference solution (A), in the spectrophotometer at 440 nm using 1 cm glass cells. Substract the optical density of the blank test solution from that of the sample solution (= corrected optical density). From this value calculate the free gossypol content as indicated in 6. 5.3 Determination of total gossypol Place a test sample containing 1—5 mg of gossypol in a 50 ml graduated flask and add 10 ml of solvent B (3.3). At the same time, prepare a blank test, placing 10 ml of solvent B (3.3) in another 50 ml graduated flask. Heat the two flasks for 30 minutes over a boiling water bath. Cool to room temperature and make the contents of each flask up to volume with the propan-2-ol-hexane mixture (3.1). Mix and leave to settle for 10—15 minutes, then filter and collect the filtrates in ground-necked flasks. Pipette 2 ml of the sample filtrate into each of two 25 ml graduated flasks, and 2 ml of the blank test filtrate into each of two other 25 ml flasks. Make the contents of one flask from each series up to 25 ml with the propan-2-ol-hexane mixture (3.1). These solutions will be used as reference solutions. Add 2 ml of aniline (3.4) to each of the other two flasks. Heat for 30 minutes over a boiling water bath to develop the colour. Cool to room temperature, make up to 25 ml with the propan-2-ol-hexane mixture (3.1), mix and leave to stand for one hour. Determine the optical density as indicated in 5.2 for free gossypol. From this value calculate the total gossypol content as indicated in 6. 6. Calculation of results Results may be calculated either from the specific optical density (6.1), or by reference to a calibration curve (6.2). 6.1 From the specific optical density The specific optical densities, under the conditions described, will be the following: free gossypol: total gossypol: The free or total gossypol content of the sample is calculated by using the following formula: % gossypol where: E = corrected optical density, determined as indicated in 5.2; p = test sample in grams. a = aliquot part of the filtrate in ml. 6.2 From a calibration curve 6.2.1 Free gossypol Prepare 2 series of five 25 ml graduated flasks. Pipette aliquots of 2.0, 4.0, 6.0, 8.0 and 10.0 ml of standard gossypol solution A (3.5) into each series of flasks. Make up the volumes to 10 ml with solvent A (3.2). Complete each series with a 25 ml graduated flask containing only 10 ml of solvent A (3.2) (blank test). Make the volume of the flasks in the first series (including the flask for the blank test) up to 25 ml with the propan-2-ol-hexane mixture (3.1) (reference series). Add 2 ml of aniline (3.4) to each flask in the second series (including the flask for the blank test). Heat for 30 minutes over a boiling water bath to develop the colour. Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture (3.1), mix and leave to stand for one hour (standard series). Determine as indicated in 5.2 the optical density of the solutions in the standard series by comparison with the corresponding solutions in the reference series. Trace the calibration curve by plotting the optical densities against the quantities of gossypol (in µg). 6.2.2 Total gossypol Prepare six 50 ml graduated flasks. In the first flask place 10 ml of solvent B (3.3), and in the others 2.0, 4.0, 6.0, 8.0 and 10.0 ml of standard gossypol solution B (3.6) respectively. Make the contents of each flask up to 10 ml with solvent B (3.3). Heat for 30 minutes over a boiling water bath. Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture (3.1) and mix. Place 2.0 ml of these solutions in each of two series of six 25 ml graduated flasks. Make the contents of the flasks in the first series up to 25 ml with the propan-2-ol-hexane mixture (3.1) (reference series). Add 2 ml of aniline (3.4) to each flask in the second series. Heat for 30 minutes over a boiling water bath. Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture (3.1), mix and leave to stand for one hour (standard series). Determine as indicated in 5.2 the optical density of the solutions in the standard series by comparison with the corresponding solutions in the reference series. Trace the calibration curve by plotting the optical densities against the quantities of gossypol (in µg). 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 for gossypol contents of less than 500 mg/kg; —75 ppm, in absolute value, for contents of not less than 500 ppm and not more than 750 mg/kg; —10% relative to the higher result, for contents of more than 750 mg/kg. 18. DETERMINATION OF THEOBROMINE 1. Purpose and scope To determine the content of theobromine in the by-products of the processing of cocoa beans. 2. Principle The theobromine is extracted with chloroform. The extract is evaporated until dry, dissolved in water and treated with a specific quantity of silver nitrate solution. The nitric acid liberated is titrated with a solution of sodium hydroxide. 3. Reagents 3.1 Chloroform. 3.2 Ammonia, d: 0.958. 3.3 Sodium sulphate, anhydrous. 3.4 Sodium hydroxide solution, 0.1 N. 3.5 Silver nitrate solution, 0.1 N. 3.6 Ethanolic solution of phenol red, 1% (w/v). 3.7 Light petroleum, boiling range 40—60°C. 4. Apparatus Flat-bottomed 500 ml flasks with ground glass stoppers. 5. Procedure Weigh, to the nearest mg, a sample of not more than 10 g containing not more than 80 mg of theobromine, place in a 500 ml flat-bottomed flask with a ground glass stopper and add 270 ml of chloroform (3.1) and 10 ml of ammonia (3.2). Stopper the flask and shake vigorously for 5 minutes. Add 12 g of anhydrous sodium sulphate (3.3), shake again and leave to settle until the following day. Filter into a 500 ml Erlenmeyer and wash the residue with 100 ml of chloroform (3.1). Distil the solvent and eliminate the last traces over a boiling water bath. Redissolve the extract in 50 ml of water and bring to the boil. Cool, neutralise exactly with the sodium hydroxide solution (3.4) using 0.5 ml of phenol red solution (3.6). Add 20 ml of silver nitrate solution (3.5). Titrate the nitric acid liberated with a solution of sodium hydroxide (3.4) until the indicator changes colour (pH 7.4). 6. Calculation of results 1 ml of 0.1 N NaOH = 18 mg of theobromine. Express the results as a percentage of the sample. 7. Observation Products containing more than 8% of crude fatty matter must first be defatted by a 6-hour extraction with light petroleum (3.7). 19. DETERMINATION OF VOLATILE MUSTARD OIL 1. Purpose and scope To determine the content of mustard oil, separated under steam and expressed as allyl isothiocyanate, in cakes made from the Brassica and Sinapis species, and in compound feeding stuffs which contain cakes made from those species. 2. Principle The sample is suspended in water. The mustard oil is released by the action of enzymes, separated by distillation with ethanol and collected in dilute ammonia. The solution is treated while warm with a given volume of silver nitrate s