As a result of the full integration of Spain into the European Community, and of the consequent need to harmonise Spanish legislation with Community legislation, the Order of 23 May 1989 was published, approves the official methods of analysis of feed or food for animals and their first subjects.

The subsequent enactment of Commission Directive 92/89/EEC of 3 November amending Annex I to the fourth Directive 73 /46/EEC determining the methods of Community analysis for the control of the (a) official food for animals; Commission Directives 92/95/EEC of 9 November and 94 /14/EC of 29 March amending the Annex to the Seventh Directive 76 /372/EEC establishing Community methods of analysis for the official control of feedingstuffs; and of Commission Directives 93 /70/EEC of 28 July 1993, 93 /117/EEC, of the Commission Decision of 17 December laying down Community methods of analysis for the official control of feedingstuffs and 93 /28/EEC of the Commission of 4 June amending Annex I to the third Directive 72 /199/EEC determining Community methods of analysis for the official control of food for animals, make it necessary to adopt within the internal scope of the relevant transposition standard in order to incorporate the methods of analysis In the case of the Community, the Commission has been in a position to (a) the order of 23 May 1989, cited above, or adding others which are not covered by it.

This Royal Decree is dictated by the provisions of article 40.2 of Law 14/1986 of 25 April, General of Health, and of Article 149.1.16.

In the process of this Royal Decree, the entities and organizations of the sector affected by it have been consulted.

In its virtue, on the proposal of the Ministers of Agriculture, Fisheries and Food, of Health and Consumer Affairs, of Economy and Finance and of Industry and Energy, prior to the report of the Inter-Ministerial Committee for Food and Agriculture agreement with the Council of State and after deliberation by the Council of Ministers at its meeting on 25 November 1994,

DISPONGO:

Article 1. Object.

The animal feed or food analysis methods and their first subjects as detailed in the Annex are approved as official.

Article 2. Supposed of non-existence of official methods.

Where there are no official methods for certain animal feed or food analyses and their first subjects, and until approved, those laid down in existing national rules or those laid down may be used. international methods of recognised solvency.

Single additional disposition. Basic character.

The provisions of this Royal Decree have the character of basic State regulations in the field of bases and coordination of the general planning of economic activity and health, contained in Article 149.1.16. Constitution.

Single repeal provision. Regulatory repeal.

The provisions of the same or lower rank which are opposed to this Royal Decree and, in particular, the Order of 23 May 1989, for which the official methods of analysis of feedingstuffs or foodstuffs are approved, are hereby repealed. animals and their first subjects.

Final disposition first. Powers of development.

the Ministers for Agriculture, Fisheries and Food, Health and Consumer Affairs, Economic and Finance and Industry and Energy Ministers are empowered to make the necessary arrangements for the compliance and application of the provisions of this Royal Decree.

Final disposition second. Entry into force.

This Royal Decree shall enter into force on the day following that of its publication in the "Official Gazette of the State".

Given in Madrid on November 25, 1994.

JOHN CARLOS R.

The Minister of the Presidency,

ALFREDO PEREZ RUBALCABA

ANNEX

Official feed analysis methods

and its first subjects

1. Preparing the sample for analysis

and general provisions

1.1 Principle. The preparation of the samples for the analysis should ensure that they are as homogeneous and representative as possible.

In general, the methods of analysis are applicable to all types of feed and their raw materials. Certain feedingstuffs require particular analytical methods, which are provided for in the description of the methods concerned.

Where two or more methods are indicated for the determination of the same component of a feed, the choice of the applicable method, unless otherwise specified, shall be the responsibility of the control laboratory; however, the analysis bulletin.

1.2 Material and apparatus. In the description of the methods of analysis only special instruments or instruments are indicated or which require special rules. It is considered superfluous to mention all the appliances or utensils that are part of the current instrumentation of the control laboratories.

For the rest, when reference is made to water for dilutions or washing, it is always understood that it is distilled water. Similarly, when reference is made to a solution, without further indication, it will be understood that this is a solution in distilled water.

1.3 Reassets. All reagents must be of a contrasting quality for the method of analysis (p.a.). For the analysis of trace elements the purity of the reagents must be controlled by a blank test. Depending on the result, further purification may be required.

1.4 Preparation of the sample-procedure. Chemical analysis should necessarily be done on a homogeneous sample. On the contrary, certain macroscopic determinations, as well as the determination of humidity, should be made on the sample in the state in which it is found when arriving in the laboratory. To take account of this double requirement, the sample will be divided into two parts. One of them will be removed in the state in which it is located; the other will be prepared for chemical analysis as follows:

Split the sample with the help of a mechanical device or manually, after having carefully mixed the whole on a clean, dry surface. In the latter case, it is appropriate to apply the method of the rooms, which consists in successively taking samples in two opposing sectors. Finally, take for the analysis a portion of approximately 100 g and to crush, if necessary, for the totality to pass through a 1 mm mesh light sieve.

Immediately enter this sample into a dry container provided with hermetic closure and cover.

If the sample is wet, a pre-desiccation must be carried out, so that the degree of humidity is in a value between 8 and 12 per 100. To this end, desiccate the sample at a suitable temperature in the precise time.

1.5 Results. The result to be mentioned in the analysis bulletin shall be the average value obtained from two determinations at least. Unless otherwise provided, it shall be expressed as a percentage of the original sample, as arrived at the laboratory. The result should not include more significant numbers than the accuracy of the analysis method.

1.6 References. First Community Directive of 15 June 1971. 17 /250/EEC. 'Official Journal of the European Communities' number L 155 of 12 July 1971.

2. Alkaloids in lupins

2.1 Principle. The alkaloids are placed in solution in a mixture of diethyl ether and chloroform, extracting by hydrochloric acid. The alkaloids are precipitated by the silico-tungstic acid, incinerated and heavy.

2.2 Material and apparatus.

2.2.1 Mechanical Agitator.

2.2.2 Platinum, quartz or porcelain incineration capsule.

2.2.3 Electrical muffle.

2.3 Reassets.

2.3.1 Diethyl Ether.

2.3.2 Chloroform.

2.3.3 Sodium hydroxide solution 4N.

2.3.4 Hydrochloric acid 0.3N.

2.3.5 Sodium chloride.

2.3.6 Solution to 10 per 100 (w/v) silico-tungstic acid (SiO12WO. 26 HO).

2.4 Procedure. Weigh, with an approximation of 5 mg, 15 g of the sample and introduce it into a container of about 200 ml with a frosted stopper. Add 100 ml of diethyl ether and 50 ml of chloroform exactly as measured and immediately and with the help of a graduated burette, 10 ml of the sodium hydroxide solution. Shake vigorously at the beginning to avoid the formation of clumps, continuing the agitation at intervals; leave to rest until the next day. If the supernatant liquid is not totally clean add a few drops of water; filter the layer of ether-chloroform. Collect 50 ml of the filtrate in a 50 ml volumetric flask and transfer it quantitatively with the aid of 50 ml of diethyl ether to a 150 ml decantation ampoule. Extract three times successive with 20 ml of hydrochloric acid, allow to decant and collect the acid extract after each extraction. Gather the acid extracts in a 250 ml flask and remove the last traces of ether and the heated chloroform slightly. Add around 1 g of sodium chloride, allow to cool and precipitate the alkaloids by solution of the silico-tungstic acid (2.6.1). Shake mechanically for 30 minutes, leave to rest for one night, filter on known ash filter and wash the precipitate successively twice with 10 ml and twice with 5 ml of hydrochloric acid.

Position the filter containing the precipitate into an incineration capsule and incinerate at 900 ° C. Leave to cool and weigh.

2.5 Calculations. Percentage of alkaloids = 0.2P.

Being:

P = Weight of the ashes.

Express the results as a percentage of the sample.

2.6 Remarks. Add silicone-tungstic solution until it is seen that no more white milky alkaloid precipitate is formed.

2.7 References. First Community Directive of 15 June 1971 (71/250/EEC). Official Journal of the European Communities No L 155 of 12 July 1971.

3. Crude protein (total protein)

3.1 Principle. The sample is mineralized with sulphuric acid in the presence of a catalyst. The acid solution is alkalized by a sodium hydroxide solution. The ammonia is distilled and collected in a measured amount of sulphuric acid, the excess of which is called by a standard solution of sodium hydroxide.

This method makes it possible to determine the raw protein content of feed from the nitrogen content, determined according to the Kjeldahl method.

3.2 Material and apparatus. Apparatus suitable for mineralizing, distilling and holder according to the Kjeldahl procedure.

3.3 Reassets.

3.3.1 potassium sulphate.

3.3.2 Catalyst: Cupric Oxide, CuO, or Crystallized Cupric Sulfate Pentahydrate, CuSO.5HO.

3.3.3 Zinc granules.

3.3.4 sulphuric acid q = 1.84 g/l.

3.3.5 Sulphuric Acid C (½ HSO) = 0.5 mol/l.

3.3.6 Sulphuric Acid C (½ HSO) = 0.1 mol/l.

3.3.7 methyl red indicator: Dissolve 300 mg of methyl red in 100 ml of ethanol, R = 95-96 per 100 (v/v).

3.3.8 Sodium hydroxide solution (technical grade may be used) or = 40 g/100 ml (m/v: 40 per 100).

3.3.9 Sodium hydroxide solution, c = 0.25 mol/l.

3.3.10 Sodium hydroxide solution, c = 0.1 mol/l.

3.3.11 Stone pumice in granules, washed with hydrochloric acid and calcined.

3.3.12 Acetanilide (melting point = 114 ° C, nitrogen = 10,36 per 100)

3.3.13 Sucrose (free of nitrogenous compounds).

3.4 Procedure.

3.4.1 Mineralization. Weigh 1 g of sample with an accuracy of 0,001 g and transfer that quantity to the flask of the mineralising apparatus. Add 15 g potassium sulphate (3.3.1), an appropriate amount of catalyst (3.3.2) (0.3 to 0.4 g of copper oxide or 0.9 to 1.2 g of copper sulphate pentahydrate), 25 ml of sulphuric acid (3.3.4) and some granules of pumice stone (3.3.11). Homogenize. Heat the flask first with moderation and stirring occasionally, if necessary, until the carbonization of the mass and the disappearance of the foam; then, more intensely until the regular boiling of the liquid. The heating is suitable when the boiling acid condenses into the walls of the flask. Avoid overheating of walls and adhesion on them of organic particles. When the solution becomes clear, light green, keep boiling for another two hours. Then leave to cool.

3.4.2 Distillation. Add sufficient water to completely dissolve the sulfates. Allow to cool and then add some zinc granules (3.3.3).

Introduce 25 ml, measured with accuracy, of sulphuric acid (3.3.5 or 3.3.6) into the collector flask of the distiller apparatus, depending on the content of the nitrogen content and some drops of the red methyl indicator (3.3.7).

Connect the mineralization flask to the coolant of the distiller apparatus and submerge the coolant limb in the liquid from the collector flask to a minimum depth of 1 cm (see observation 3.7.3). Slowly enter into the digestion flask 100 ml of sodium hydroxide solution (3.3.8) without loss of ammonia (3.7.1).

Heat the flask until complete distillation of the ammonia.

3.4.3 Titulation. Holder the excess sulphuric acid of the collector bottle by solution of sodium hydroxide (3.3.9 or 3.3.10), depending on the concentration of the sulphuric acid used, until the end point is reached.

3.4.4 Blank test. To confirm that the reagents are free of nitrogen, perform a blank test (mineralisation, distillation and titration) using 1 g of sucrose (3.3.13) instead of the sample.

3.5 Calculation of results. The raw protein content is calculated according to the following formula:

(V-V) x c x 0.014 x 100 x 6.25

m

Being:

V = Volume (ml) of NaOH (3.3.9 or 3.3.10) used in the blank test.

V = Volume (ml) of NaOH (3.3.9 or 3.3.10) used in the sample titration.

c = Concentration (mol/l) of sodium hydroxide (3.3.9 or 3.3.10).

m = Mass (g) of the sample.

3.6 Method checking.

3.6.1 Repetibility. The difference between the results of two parallel determinations made with the same sample must not exceed:

-0,2 per 100 in absolute value, for crude protein content of less than 20 per 100.

-1.0 per 100 in relative value over the highest value, for protein content between 20 and 40 per 100.

-0.4 per 100 in absolute value, for contents greater than 40 per 100.

3.6.2 Exattitude. Carry out the analysis (mineralisation, distillation and titration) in 1,5 to 2,0 g of acetanilide (3.3.12) in the presence of 1 g of sucrose (3.3.13); 1 g of acetanilide consumes 14.80 ml of sulphuric acid (3.3.5). The recovery must be 99 per 100, at least.

3.7 Remarks.

3.7.1 The device can be manual, semi-automatic or automatic. If the apparatus requires a transfer between mineralisation and distillation, such transfer must be carried out without loss. If the flask of the distillation apparatus is not fitted with a key funnel, add the hydroxide solution immediately before connecting the flask to the refrigerant, allowing the liquid to slowly slip through the walls.

3.7.2 If the product of the mineralization is solidified, start the determination again using an amount of sulphuric acid (3.3.4) greater than that specified above.

3.7.3 For products with a low nitrogen content, the volume of sulphuric acid (3.3.6) to be introduced into the collector bottle may be reduced, if necessary, to 10 or 15 ml and to be completed with water up to 25 ml.

3.8 References. Directive 93 /28/EEC. 'Official Journal of the European Communities' L 179 of 4 June 1993.

4 (a) Gross grase (no prior hydrolysis)

4 (a) .1 Principle. The sample is extracted with petroleum ether. The solvent is distilled and the residue is dried and weighed.

The method makes it possible to determine the raw fat content in feed. It is not applicable to the analysis of oilseeds and oleaginous fruits as defined in Regulation No 136 /66/EEC of the Council of 22 September 1966.

Applicable to raw materials of plant origin, except for those in which their fats are not completely removable with petroleum ether without prior hydrolysis. These exceptions include, among other things, glutene, yeasts, soya proteins and potatoes. This procedure also applies to compound feed, with the exception of those containing milk in plovo or whose fats are not wholly removable with petroleum ether without prior hydrolysis.

4 (a) .2 Material and apparatus.

4 (a) .2.1 Extractor. If the apparatus carries a siphon (Soxhlet type) to regulate the reflux volume so that at least 10 cycles per hour are obtained. If the apparatus does not have siphon, the volume of liquid under reflux shall be around 10 ml per minute.

4 (a) .2.2 Extraction cartridges, free of oil-soluble materials, and the porosity of which is compatible with the requirements of point 4 (a) .2.1.

4 (a) .2.3 Desiccation study, either empty at 75 ° C ± 3 ° C or at atmospheric pressure at 100 ° C ± 3 ° C.

4 (a) .3 Reassets.

4 (a) .3.1 Ether oil, boiling range: 40 to 60 ° C. The bromine index must be less than 1 and the evaporation residue less than 2 mg/100 ml.

4 (a) .3.2 Sodium sulphate, anhydrous.

4 (a) .3.3 pumice stone or glass beads.

4 (a) .4 Procedure.

4 (a) .4.1 Pesar, with precision of 1 mg, 5 g of the sample and introduce it with a defatted cotton stopper.

Put the cartridge into the extractor (4 (a) .2.1) by extracting for six hours with petroleum ether (4 (a) .3.1). Collect the extract in a previously tarted dry flask, in which there are some fragments of pumice stone.

Evaporate the solvent by distillation. Dry the residue by introducing the flask for an hour and a half into a drying stove (4 (a) .2.3). Allow to cool in desiccator and weigh. Dry again for 30 minutes to ensure that the fat content remains constant (the difference by weight between two successive heavy ones will be less than 1 mg).

4 (a) .4.2 For products of high fat content, difficult to crush or not suitable for the taking of a homogeneous reduced sample, proceed as follows:

weigh, with precision of 1 mg, 20 g of the sample and mix with 10 g or more of anhydrous sodium sulfate.

Extract with petroleum ether (4 (a) .3.1) as indicated in 4 (a) .4.1.

Complete the extract obtained up to 500 ml with petroleum ether (4 (a) .3.1) and homogenize. Insert 50 ml of the solution into a small dry and tarred flask containing some fragments of pumice stone (4 (a) .3.3). Remove the solvent by distillation, dry and continue with the operative method indicated in the last paragraph of point 4 (a) .4.1. Value (a).

Remove the solvent from the extraction residue that is in the cartridge. Crush the residue to a particle size of 1 mm. Place it again in the extraction cartridge (do not add sodium sulphate) and continue with the operative method, as indicated in the second and third paragraphs of the point (4 (a) .4.1.). Value (b).

4 (a) .5 Calculations. According to 4 (a) .4.1, express the result as a percentage of the sample.

According to 4 (a) .4.2, the raw fat content as a percentage of the sample is given by the formula:

(10 a + b) x 5

Being:

a = Mass, in grams, of the residue of the first extraction (aliquot part of the extract).

b = Mass, in grams, of the residue of the second extraction.

The difference between the results of two parallel determinations performed on the same sample by the same analyst should not exceed:

-0,2 per 100 in absolute value, for gross fat content less than 5 per 100.

-4.0 per 100 of the highest result for contents 5 to 10 per 100.

-0.4 per 100 in absolute value, for contents greater than 10 per 100.

4 (a) .6 References. Commission Directive of 20 December 1983 amending Directive 71 /393/EEC. 'Official Journal of the European Communities' number L 15/28 of 18 January 1984. Method number 4.

4 (b) Gross grase (with prior hydrolysis)

4 (b) .1 Principle. The sample is treated as hot with hydrochloric acid. Allow to cool the mixture and filter. After washing and drying, it is extracted with petroleum ether. The solvent is distilled and the residue is dried and weighed.

The method makes it possible to determine the raw fat content of feed. It is not applicable to the analysis of oilseeds and oleaginous fruits as defined in Regulation No 136 /66/EEC of the Council of 22 September 1966.

Applicable to simple feed of animal origin, as well as those referred to in Method 4 (a) .1, as exceptions to that procedure.

4 (b) .2 Material and apparatus. The same as in 4 (a) .2.

4 (b) .3 Reassets.

4 (b) .3.1 Ether oil, boiling range: 40 to 60 ° C. The bromine index must be less than 1 and the evaporation residue less than 2 mg/100 ml.

4 (b) .3.2 Hydrochloric acid 3N.

4 (b) .3.3 Filtering aid, for example diatom land.

4 (b) .3.4 pumice stone or glass beads.

4 (b) .4 Procedure. Weigh, with an approximation of 1 mg, 2.5 g of sample (for poor fat samples can be increased to 5 g), introduce them into a 400 ml beaker, or in a 300 ml erlenmeyer, add 100 ml of 3N hydrochloric acid (4 (b) .3.2) and some fragments of pumice stone. Cover the glass with a watch glass or connect to the erlenmeyer a reflux coolant. Bring the mixture to a smooth boil, using a small flame or a heating plate; keep the boil boiling for an hour. Prevent the product from adhering to the container walls.

Cool and add sufficient filtration aid (4 (b) .3.3) to prevent the loss of fat during filtration. Filter with double wetted filter paper, free of fat. Wash the residue with cold water until the filter neutrality. Check that the fat does not contain fat. Their presence would indicate the need for an extraction of the sample with petroleum ether, prior to hydrolysis, according to method 4 (a).

Put the double filter paper containing the residue on a watch glass and dry it for an hour and a half in the stove at 100 ° C ± 3 ° C.

Introduce the double filter containing the dry residue in the extraction cartridge (4 (a) .2.2) and cover it with a defatted cotton stopper. Put the cartridge into the extractor (4 (a) .2.1) and follow the operative mode indicated in the second and third paragraphs of point 4 (a) .4.1.

4 (b) .5 Calculations. As in 4 (a) .5.1.

4 (b) .6 References. Same as in method 4 (a).

5. Chlorides

5.1 Principle. The chlorides are solubilized in water, the solution being defecated if it contains organic matter, subsequent acidification of the same with nitric acid and precipitation of the chlorides with silver nitrate. The excess nitrate is valued with an ammonium sulfocyanide solution. Applicable to all feed.

5.2 Material and apparatus.

5.2.1 Agitator from 35 to 40 r.p.m.

5.3 Reassets.

5.3.1 0.1N ammonium sulfocyanide solution.

5.3.2 Silver nitrate solution 0.1N.

5.3.3 A saturated solution of ammonium-ferric sulfate.

5.3.4 Nitric acid, d = 1.38.

5.3.5 Ethyl Ether.

5.3.6 Acetone.

5.3.7 Carrez I solution: Dissolve in water 24 g of zinc acetate dihydrate (Zn (CHCOO) .2HO) and 3 g of glacial acetic acid. Complete up to 100 ml with water.

5.3.8 Carrez II solution: Dissolve in water 10.6 g of potassium ferrocyanide trihydrate (K (FeCN)) .3HO. Complete 100 ml with water.

5.3.9 Active carbon, free of chlorides.

5.4 Procedure.

5.4.1 Preparing the solution.

5.4.1.1 Samples without organic matter. Weigh, with precision of 1 mg, 0 to 10 g of the sample so that it does not contain more than 3 g of chlorine in the form of chloride and introduce it into a 500 ml graduated flask with 400 ml of water at approximately 20 ° C. Shake for thirty minutes, make up, homogenise and filter.

5.4.1.2 Samples with organic matter (minus those cited in 5.4.1.3). Weigh to the nearest 1 mg, approximately 5 g of sample and enter it with 1 g of activated carbon in a 500 ml graduated flask. Add 400 ml of water at approximately 20 ° C and 5 ml of Carrez solution I, shake and then add 5 ml of the Carrez II solution. Shake for thirty minutes, make up, homogenise and filter.

5.4.1.3 Torta and flax flour, products rich in flax flour and other products rich in mucilages or in colloidal substances (e.g. hydrolysed starch).

Prepare the solution as indicated in 5.4.1.2, but unfiltered. Decant (if necessary centrifuge), separate 100 ml of the supernatant liquid and introduce it into a 200 ml flask. Mix with acetone and make up with this solvent, homogenize and filter.

5.4.2 Valuation. Take 25 to 100 ml of filtration (with chlorine content less than 150 mg) obtained in 5.4.1.1, 5.4.1.2 or 5.4.1.3, and introduce it into an erlenmeyer, dilute if necessary, to 50 ml with water. Add 5 ml of nitric acid, 20 ml of saturated solution of ferric ammonium sulphate and two drops of the ammonium sulfocyanide solution, added by a full burette to the zero stroke. Add silver nitrate solution up to a 5 ml excess using a burette. Add 5 ml of ethyl ether and shake strongly to collect the precipitate. Assess the excess of silver nitrate by solution of ammonium sulfocyanide until the dark red turn persists for one minute.

5.5 Calculations. The amount of chlorine (p) expressed in sodium chloride present in the volume of separate filtering for the assessment is given by the formula:

P = 5,845 (V1-V) mg

Being:

V = Volume, in ml, of added silver nitrate solution.

V = Volume, in ml, of 0.1N ammonium sulfocyanide solution used in the assessment.

Perform a blank test without the sample to be analyzed and if it consumes silver nitrate solution 0.1N subtract this value to the volume (V-V).

Express the result as a percentage of the sample.

5.6 Remarks. For products rich in fat, degreasing previously by ethyl ether according to 4 (a).

5.7 References. First Commission Directive of 15 June 1971. (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

6. Moisture

6.1 Principle. The sample is desegated under defined conditions, varying according to the nature of the product. The loss of mass is determined by heavy. It is necessary to proceed to a pre-desiccation when it comes to solid substances, with high moisture content. It is not applicable to milk products considered as simple feed, mineral substances, compound feed consisting essentially of mineral substances and seeds and oleaginous fruits. For cereals and their products, except for hydrolysed cereal products and barley raicilla (6.3.2.2), the official method for cereals and derivatives shall apply.

6.2 Material and apparatus.

6.2.1 Grinding mill, easy to clean, allowing quick and uniform grinding, without causing sensitive heating or condensations, avoiding maximum contact with air.

6.2.2 Precision analytical balance 0.5 mg.

6.2.3 Stainless or glass-metal dry-ups, provided with a lid that ensures a tight seal; useful surface to obtain a sample distribution of the order of 0.3 g per cm.

6.2.4 Electrical heating isothermal (± 1 ° C), which ensures fast temperature regulation and suitably ventilated.

6.2.5 Empty, adjustable electric heating, provided with an oil pump or a device for the introduction of dehydrated hot air or a dehydrator.

6.2.6 Decker with metal or porcelain plate, containing an effective dehydrator.

6.3 Procedure.

6.3.1 Preparation of the sample.

6.3.1.1 All samples, except those mentioned in 6.3.1.2. Pre-separate at least 50 g of the sample, by crushing or treating it in advance in an appropriate manner, if necessary, to avoid any variation of the moisture content.

6.3.1.2 Liquid or pasty food, consisting essentially of fat. Separate previously and weigh, with approximation of 10 mg around 25 g of sample. Add an appropriate amount of anhydrous, heavy sand with 10 mg approximation and mix until a homogeneous product is obtained.

6.3.2 Desecation.

6.3.2.1 All foods, except those mentioned in 6.3.2.2. Tarar, with a 0.5 mg approach, a container provided with a lid. Weigh, with an approximation of 1 mg, in the tarado container about 5 g of sample and spread it evenly. Place the container, without a lid, in a stove previously heated to 103 ° C. To prevent the temperature of the stove from falling too low, enter the container quickly. Allow to dry for four hours from the moment the stove reaches again the temperature of 103 ° C. Place the lid on the container, remove it from the stove, allow to cool thirty to forty-five minutes in a desiccator and weigh with 1 mg.

In the case of samples consisting essentially of fat, a complementary drying of thirty minutes in the stove at 103 ° C. The difference between the two heavy ones must not exceed 0,1 per 100 humidity.

6.3.2.2 Compounds containing more than 4 per 100 sucrose or lactose, hydrolysed cereal products, barley raicilla, garrofa, beetroot head, sugar and fish soluble and compound feed contain more than 25 per 100 of mineral salts with crystallization water. Tarar, with an approximation of 0.5 mg, a container with a lid. Weigh, with an approximation of 1 mg in the tarado container approximately 5 g of sample and spread it evenly. Place the container in the previously heated vacuum stove at a temperature of 80 to 85 ° C, without a lid. To prevent the temperature of the stove from falling too low, enter the container quickly. Bring the pressure to 100 torts, and let this pressure dry for four hours, under a hot and dry air stream or with the help of a dehydrator (300 g for about 20 samples). In the latter case, cut the connection with the vacuum pump when the prescribed pressure is reached. Count the drying time from the moment when the stove reaches again the temperature from 80 to 85 ° C. Take the stove with caution to the atmospheric pressure.

Open the stove, immediately plug the canister and remove it. Allow to cool for thirty to forty-five minutes in the desiccator and weigh with an approximation of 1 mg. Proceed to a complementary drying of thirty minutes in the vacuum stove at the temperature of 80 to 85 ° C and weigh again. The difference between the two heavy ones must not exceed 0,1 per 100 humidity.

6.3.3. Pre-desiccation. Solid foods, whose moisture content is high and make the grinding difficult, should be pre-dried as follows:

Weigh with an approximation of 10 mg about 50 g of unground sample (if necessary, a prior split may be made in the case of granules or agglomerates) in a suitable container.

Let dry on a stove, at a temperature of 60 to 70 ° C, until the moisture content is reduced to a value ranging from 8 to 12 per 100.

Remove from the stove, allow air to cool in the laboratory for one hour and weigh with a 10 mg approach. Crush immediately after as indicated in 6.3.1, and perform the desiccation as in 6.3.2.1.

6.4 Calculations. The moisture content, as percent of the sample, is obtained by the following formulas:

6.4.1 Desecation without predesiccation:

100/M (M-m)

Being:

M = Initial Mass, in g, of the sample.

m = Mass, in g, of the dry sample.

6.4.2 Desecation with pre-desiccation:

(M'-m) M/M' + E-M) 100/E = 100 (1-M m/E M ')

Being:

E = Initial Mass, in g, of the sample.

M = Masa, in g, of the pre-dried sample.

M ' = Mass, in g, of the sample after grinding.

m = Mass, in g, of the dry sample.

The difference between the results of two simultaneous determinations carried out on the same sample must not exceed 0,2 per 100 humidity.

6.5 References. Second Commission Directive of 18 November 1971. (71/393/EEC) 'Official Journal of the European Communities', number L 279 of 20 December 1971.

7. Determination of gross fibre (raw cellulose)

7.1 Principle. Determination in feed of organic substances free of fat and insoluble in acid and alkaline medium, conventionally called brute fibre.

The sample, in its case degreased, is successively treated with boiling solutions of sulphuric acid and potassium hydroxide, of determined concentrations. The residue is separated by filtration by porous glass filter, is washed, dried, weighed and calcated at a temperature between 475 and 500 ° C. The weight loss due to the calcination corresponds to the gross fibre of the test sample.

7.2 Material and appliances.

7.2.1 Heating apparatus for digestion with sulphuric acid or potassium hydroxide solution, equipped with a support for the filter crucible (7.2.2) and with an outlet tube provided with a tap to make the vacuum and Evacuate the liquid and, where appropriate, compressed air to apply back pressure. Each day, before use, heat it up previously with boiling water for five minutes.

7.2.2 Glass filter crucible, 50 ml, with a glass filter plate, of porosity between 40 and 90 xm. Before use for the first time, heat to 500 ° C for a few minutes and cool (7.7.6).

7.2.3 Boiling-resistant column, at least 270, with reflux condenser.

7.2.4 Thermostat drying.

7.2.5 Mufla, with thermostat.

7.2.6 Cold extraction apparatus, composed of a support for the filter crucible (7.2.2) and a discharge tube provided with a faucet for making the vacuum and evacuating the liquid.

7.2.7 Connection boards for joining the heating apparatus (7.2.1), the crucible (7.2.2), and the column (7.2.3) and connecting the cold extractor (7.2.6) and the crucible.

7.3 Reassets.

7.3.1 sulphuric acid, c = 0.13 mol/l.

7.3.2 Anti-foaming agent (e.g. n-octanol).

7.3.3 Celite filtration adjuvant 545 or equivalent, heated to 500 ° C for four hours (see 7.7.6).

7.3.4 Acetone.

7.3.5 Ether oil-boiling range 40-60 ° C.

7.3.6 Hydrochloric acid, c = 0.5 mol/l.

7.3.7 Potassium hydroxide solution, c = 0.23 mol/l.

7.4 Procedure. Weigh 1 g of sample with an approximation of 1 mg, if any, after preparation (see 7.7.1, 7.7.2 and 7.7.3), and put it in a crucible (7.2.2) and add a gram of filtration aid (7.3.3).

Coupling the heating apparatus (7.2.1) and the filter crucible (7.2.2), and then join the column (7.2.3) and the crucible. Put in the glass 150 ml of sulphuric acid (7.3.1) previously heated to the boiling point and add some antifoaming drops (7.3.2) if necessary. Bring the liquid to a boil in 5 ± 2 minutes and boil vigorously for 30 minutes.

Open the tap of the discharge tube (7.2.1) and filter out the sulphuric acid through the filter crucible. Wash the residue of the filter crucible three times using 30 ml of boiling water each time. After each wash, dry the residue of the suction filter.

Close the outlet tap and add to the column 150 ml of boiling potassium hydroxide solution (7.3.7). Add anti-foaming drops (7.3.2). Bring the liquid to a boil in 5 ± 2 minutes and boil vigorously for 30 minutes. Filter the residue and wash as indicated for treatment with sulphuric acid.

After the last wash, dry the residue by aspiration, disconnect the crucible and its contents and connect it to the cold extractor (7.2.6). Apply vacuum and wash the residue three times, in the crucible, using 25 ml of acetone each time, drying it by aspiration after each washing.

Dry the filter crucible at 130 ° C in the stove until reaching a constant weight. After each drying, cool in the desiccator and weigh quickly. Then place the crucible in the muffle oven and burn the contents at a temperature between 475 and 500 ° C for at least 30 minutes.

After each calcination, cool, first in the oven and then in the desiccator, before weighing.

Perform a blank test without the sample. Weight loss due to calcination should not exceed 4 mg.

7.5 Calculations.

The raw fiber content in percent of the sample is expressed by the formula:

(b-c) x 100/a

Being:

a = Mase of the sample in g.

b = Loss of mass by calcination of the residue of the sample after drying at 130 ° C.

c = Mass loss by calcination of the blank test residue after drying at 130 ° C.

7.6 Repetibility. The difference between two parallel determinations carried out within the same sample must not exceed:

-0.3 in absolute value for gross fibre content less than 10 per 100.

-3 per 100 relative to the highest result, for gross fibre content equal to or greater than 10 per 100.

7.7 Remarks.

7.7.1 Feed with a gross fat content greater than 10 per 100 must be degreased with petroleum ether (7.3.5) before performing its analysis. Connect the filter crucible (7.2.2), with the previously heavy sample, to the cold extractor (7.2.6) and wash three times to the vacuum using 30 ml of petroleum ether (7.3.5) each time. Dry the sample by aspiration, connect the crucible with its contents to the heating apparatus (7.2.1) and continue according to paragraph 7.4.

7.7.2 Feed containing fats which cannot be extracted directly with petroleum ether (7.3.5) must be degreased according to paragraph 7.7.1 and undergo a new degreasing after having been treated with acid in boiling.

After the boiling and washing acid treatment, join the crucible and its contents to the cold extractor (7.2.6), wash three times using 30 ml of acetone each time and then another three times using 30 ml of ether. of oil every time. Dry the filter by aspiration and continue the analysis according to point 7.4, starting with the potassium hydroxide treatment.

7.7.3 If feed contains more than 5 per 100 carbonates, expressed as calcium carbonate, connect the crucible (7.2.2), with the heavy sample, to the heating apparatus (7.2.1). Wash the sample three times with 30 ml of hydrochloric acid (7.3.6). After each addition, leave the sample to rest for about one minute before filtering. Wash once with 30 ml of water and then follow it according to paragraph 7.4.

7.7.4 If a battery of appliances (several crucibles attached to the same heating device) is used, two determinations of the same sample must not be performed in the same series.

7.7.5 If, after boiling, it is difficult to filter the acidic and alkaline solutions, introduce compressed air into the discharge tube of the heating apparatus and continue to filter below.

7.7.6 In order to lengthen the duration of glass filter crucibles, the calcination temperature should not exceed 500 ° C. Also, abrupt thermal changes in heating and cooling cycles should be avoided.

7.8 References. Directive 92 /89/EEC. 'Official Journal of the European Communities', L 344 of 26 November 1992.

8. Sugars

8.1 Principle. The method makes it possible to determine the reducing sugars and the total sugars prior to inversion, expressed in glucose or, if appropriate, in sucrose, by conversion with the aid of factor 0.95. This method is applicable to compound feed. Special modalities for other foods are provided. Where appropriate, the lactose should be determined separately and taken into account when calculating the results.

Defecation from the solutions of Carrez I and II, after dissolution of the sugars in diluted ethanol. Elimination of ethanol and valuation before and after investment according to the Luff-Schoorl method.

8.2 Material and appliances. Mechanical agitator.

8.3 Reassets:

8.3.1 Ethanol at 40 per 100 (v/v) (d = 0.948 at 20 ° C).

8.3.2 Carrez I. Dissolve in water 24 g of zinc acetate dihydrate and 3 ml glacial acetic acid and add distilled water up to 100 ml.

8.3.3 Carrez Solution II. Dissolve in water 10,6 g hexacane (II) potassium (K (FeCN)-3HO) and add distilled water up to 100 ml.

8.3.4 methyl orange solution at 0.1 per 100 (w/v).

8.3.5 Hydrochloric acid 4N.

8.3.6 Hydrochloric acid 0.1N.

8.3.7 Sodium hydroxide solution 0.1N.

8.3.8 Copper sulphate solution II. Dissolve 25 g of copper sulphate (CuSO-5HO) p.a., free of iron in 100 ml of water.

8.3.9 Citric acid solution. Dissolve 50 g of citric acid (CHO-HO) p.a. in 50 ml of water.

8.3.10 Sodium carbonate solution. Dissolve 143.8 g of anhydrous sodium carbonate (NaCO) p.a. in 300 ml of hot water, allow to cool.

8.3.11 Sodium thiosulfate solution 0.1N.

8.3.12 Starch solution. Add a mixture of 5 g of soluble starch in 30 ml of water to a litre of boiling water. Let boil for three minutes. Leave to cool. Add 10 mg of iodide mercury (II) as a conservative agent.

8.3.13 6N sulphuric acid.

8.3.14 Potassium iodide solution (KI) to 30 per 100 (w/v).

8.3.15 pumice stone boiled with hydrochloric acid and rinsed with water.

8.3.16 Isopentanol.

8.3.17 Reactive of Luff-Schoorl. Carefully agitate the citric acid solution (8.3.9) on the sodium carbonate solution (8.3.10). Immediately add the copper sulphate solution (8.3.8) and complete one litre with water. Leave to rest one night and filter. Control the normal of the reagent obtained (Cu 0.1N; NaCO2N). The pH of the solution should be approximately 9.4.

8.4 Procedure.

8.4.1 Sample Preparation. Weigh with 1 mg 2.5 g of the sample and introduce it into a 250 ml graduated flask. Add 200 ml of ethanol to 40 per 100 (v/v) and mix for an hour in the agitator. Add 5 ml of the Carrez I solution and shake for one minute, add and shake during the same time with 5 ml of the Carrez II solution.

Scratch 250 ml with the ethanol solution 8.3.1, homogenize and filter. Take 200 ml of the filtrate and evaporate approximately up to half of the volume to remove most of the ethanol. Transfer the evaporation residue in full with the aid of hot water to a 200 ml graduated flask and cool, then make up to the water and filter if necessary. This solution will be used for the determination of reducing sugars and after the inversion for the determination of total sugars.

8.4.2 Determination of reducing sugars. Take at most 25 ml of the solution prepared according to 8.4.1 and contain less than 60 mg of reducing sugars, expressed as glucose. If necessary, complete the volume up to 25 ml with distilled water and determine the amount of reducing sugars according to Luff-Schoorl. The result will be expressed as a percentage of glucose.

8.4.3 Determination of total sugars prior to investment. Take 50 ml of solution 8.4.1 and take a 100 ml graduated flask. Add a few drops of the orange methyl solution and add slowly by stirring 4N hydrochloric acid solution to red. Add 15 ml of 0.1N hydrochloric acid and dip it into a boiling hot water bath for thirty minutes. Refrigerate up to 20 ° C and then add 15 ml of the sodium hydroxide solution 0.1N (8.3.7). Make up to 100 ml with water and homogenise.

Take an amount that does not exceed 25 ml and contains less than 60 mg of reducing sugars expressed in glucose. If it is necessary to complete the volume up to 25 ml with distilled water and determine the amount of reducing sugars according to Luff-Schoorl. The result will be expressed as a percentage of glucose. To express it in sucrose, it must be multiplied by the factor 0.95.

8.4.4 Luff-Schoorl Valuation. Take 25 ml of the Luff-Schoorl reagent (8.3.17) and take it to a 300 ml erlenmeyer add 25 ml exactly measured from the defecated solution of sugars, add some pumice stone and heat agitating on the mechero flame. Immediately place the erlenmeyer on a metal fabric, perforated by a 6 cm diameter opening and regulating the flame so that only the bottom of the erlenmeyer is heated. Immediately adapt a reflux coolant over the erlenmeyer, from this moment on to boil the solution and keep on boiling for ten minutes exactly. Immediately refrigerate the cold water jet for five minutes and proceed to its assessment as follows:

Add 10 ml of the potassium iodide solution (8.3.14) immediately after and with care 25 ml of 6N sulphuric acid (8.3.13). Rate below by solution of sodium thiosulfate 0.1N (8.3.9) until the appearance of yellow color, add at this time the starch solution and finish valuing.

Make the same assessment of a mixture containing 25 ml exactly as measured from the Luff-Schoorl reagent, 25 ml of water, 10 ml of the potassium iodide solution (8.3.14) and 25 ml of the 6N sulphuric acid solution (8.3.13). without bringing to the boil.

8.5 Calculations. Establish by means of Table I the amount of glucose in mg corresponding to the difference between the two valuations, according to the ml of sodium thiosulfate 0.1N spent in each of the valuations.

Express results as percent of the sample.

8.6 Remarks.

8.6.1 In case of very rich feed in molasses or unhomogeneous feed, weigh 20 g and introduce them into a one litre graduated flask with 500 ml of water. Mix for an hour in the agitator. Defecate by the Carrez I and II reagents (8.3.2 and 8.3.3) as described in 8.4.1, using all the four-fold higher dose reagents. Lead to 1000 ml with 80 per 100 ethanol (v/v) (8.3.1). Homogenize and filter, then eliminate ethanol according to 8.4.1.

In the absence of starch free from hydrolysate products, make up to 100 ml with distilled water.

8.6.2 In the case of simple molasses and feed, rich in sugars and practically free of starch, weigh 5 g and introduce them into a 250 ml graduated flask, add 200 ml of distilled water and mix for an hour or more in the agitator. Defecate immediately by means of the Carrez I and II reagents (8.3.2 and 8.3.3), according to 8.4.1.

Take 250 ml with water, homogenise and filter, to determine total sugars, continue as 8.4.3.

8.6.3 It is recommended to add approximately 1 ml of isopentanol (regardless of volume) before boiling, with the Luff-Schoorl reagent to prevent foaming.

8.6.4 The difference between the amount of total sugars after the investment, expressed in glucose and the amount of reducing sugars expressed equally in glucose, multiplied by 0.95 gives the amount as much as sucrose.

8.6.5 To calculate the amount of reducing sugars, excluding lactose, it can be determined in the following ways:

8.6.5.1 For an approximate calculation, multiply by 0.675 the amount of lactose obtained, by separate determination, and subtract the result obtained from the amount in reducing sugars.

8.6.5.2 For the precise calculation of reducing sugars, excluding lactose, it is necessary to start from the same sample 8.4.1 for the final two determinations. One of the analyses is carried out from the solution obtained in 8.4.1 and the other one on a part of the solution obtained for the assessment of lactose according to the method for the determination of lactose.

In cases 8.6.5.1 and 8.6.5.2 the amount of sugars present is determined by the Luff-Schoorl method, expressed in mg of glucose.

The difference between the two values is expressed as a percent of the sample.

8.7 References. First Commission Directive of 15 June 1971. (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

TABLE I

For 25 ml Luff-Schoorl reagent

Na2S2O30.1N: Ml/Glucose fructose sugars inverted C6H12O6: mg/Difference/Lactose C12H22O11: mg/Difference/Maltose C12H22O11: mg/Difference

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23/2.4

4.8

7.2

9.7

12.2

14.7

17.2

19.8

22.4

25.0

27.6

30.3

33.0

35.7

38.5

41.3

44.2

47.1

50.0

53.0

56.0

59.1

62.2/2.4

2.4

2.5

2.5

2.5

2.5

2.6

2.6

2.6

2.6

2.7

2.7

2.7

2.8

2.8

2.9

2.9

2.9

3.0

3.0

3.1

3.1/3.6

7.3

11.0

14.7

18.4

22.1

25.0

29.5

33.2

37,0

40.8

44.6

48.4

52.2

56.0

59.9

63.8

67.7

71.7

75.7

79.8

83.9

88.0/3.7

3.7

3.7

3.7

3.7

3.7

3.7

3.7

3.8

3.8

3.8

3.8

3.8

3.8

3.9

3.9

3.9

4.0

4.0

4.1

4.1

4.1/3.9

7.8

11.7

15.6

19.6

23.5

27.5

31.5

35.5

39.5

43.5

47.5

51.6

55.7

59.8

63.9

68.0

72.2

76.5

80.9

85.4

90.0

94.6/3.9

3.9

3.9

4.0

3.9

4.0

4.0

4.0

4.0

4.0

4.0

4.1

4.1

4.1

4.1

4.1

4.2

4.3

4.4

4.5

4.6

4.6

9. Acidity of the fat

This determination shall apply to those fats which are used as raw materials.

The procedure will be the one in method number 10 of the Official Methods of Analysis of Aceites and Grases approved by Order of January 31, 1977

(Official State Gazette of 14 July 1977).

In the event that it is necessary to prepare the sample, the procedure set out in the method number 1 of the above order will be followed.

10. Calcium

10.1 Principle. The method makes it possible to determine the total calcium content of the feed.

The sample is incinerated, the ashes are treated with hydrochloric acid and the calcium is precipitated in the form of calcium oxalate. After dissolving the precipitate in sulphuric acid, the formed oxalic acid is valued by a potassium permanganate solution.

10.2 Material and apparatus.

10.2.1 Capacity 250 ml capacity.

10.2.2 Erlenmeyer 250 ml capacity.

10.2.3 Platinum, quartz or porcelain crisol.

10.2.4 Glass filter crucibles, G4 porosity.

10.2.5 Electrical air with air circulation and automatic regulator, to regulate at 550 ° C.

10.2.6 Bath of water.

10.3 Reassets.

10.3.1 Hydrochloric acid d = 1.14, p.a.

10.3.2 Nitric acid d = 1.40, p.a.

10.3.3 sulphuric acid d = 1.13, p.a.

10.3.4 Ammonia d = 0.98, p.a.

10.3.5. Saturated solution of cold ammonium oxalate, p.a.

10.3.6 Solution to 30 per 100 (w/v) citric acid, p.a.

10.3.7 Solution to 5 per 100 (w/v) ammonium chloride, p.a.

10.3.8 Solution to 0.04 per 100 (w/v) bromoresol green.

10.3.9 Potassium permanganate solution; 0.1N, p.a.

10.4 Procedure. Weigh with 1 mg, 5 g of the sample to be analysed (or more if necessary), calcined at 550 °C and transfer the ashes to a 250 ml erlenmeyer. Add 40 ml of hydrochloric acid (10.3.1), 60 ml of water and some drops of nitric acid (10.3.2). Bring to the boil and keep it that way for thirty minutes. Cool, transfer the solution to a 250 ml graduated flask, rinse the erlenmeyer and complete the volume with water, homogenise and filter.

Take with a pipette based on the presumed amount of calcium, an aliquot that contains 10 to 40 mg of calcium and introduce it into a 250 ml erlenmeyer. Add 1 ml of the citric acid solution (10.3.6) and 5 ml of ammonium chloride solution (10.3.7). Complete the volume to approximately 100 ml with water. Bring to the boil, adding 8 to 10 drops of bromoresol green solution (10.3.8) and 30 ml of hot ammonium oxalate solution (10.3.5). If a precipitate appears, dissolve it by adding a few drops of hydrochloric acid (10.3.1).

Neutralize after very slowly with ammonia (10.3.4), stirring constantly, until obtaining a pH 4.4-4.6 (turn of the indicator). Place the erlenmeyer in a boiling water bath for thirty minutes, leaving the formed precipitate to rest. Remove it from the bathroom, allow it to rest for an hour and filter in a filter crucible G4. Wash the erlenmeyer and the crucible with water until the total elimination of the excess ammonium oxalate (the absence of chlorides in the washing water indicates that the washing is sufficient).

Dissolve the precipitate on the filter with 50 ml of hot sulphuric acid (10.3.3), rinse the crucible with hot water until the filter is approximately 100 ml. Heat to 70-80 ° C and value by solution of potassium permanganate (10.3.9) until the obtention of a persistent pink colouring for one minute.

10.5 Calculation. 1 ml of potassium permanganate 0.1N corresponds to 2,004 mg of calcium.

Express the result obtained as a percentage of the sample.

10.6 Remarks.

10.6.1 For small amounts of calcium, proceed as in 10.5. Filter the calcium oxalate precipitate on a filter paper without ashes and burn it in a crucible at 550 ° C. Recover the residue with some drops of sulphuric acid (10.3.3), evaporate to dry, calcined again at 550 ° C and weigh.

If P represents the weight of calcium sulfate obtained, the amount in calcium of the aliquot taken will be equal to P x 0.2944.

10.6.2 If the sample consists exclusively of mineral materials, proceed to dissolution by hydrochloric acid without prior incineration. For products such as aluminic-calcium phosphates difficult to dissolve in acids, proceed to an alkaline fusion before dissolution. Mix the part taken with five times the weight of a mixture composed of equal parts of potassium carbonate and sodium carbonate in a crucible. Heat with caution until the complete fusion of the mixture. Refrigerate and dissolve with hydrochloric acid.

10.6.3 If the amount in magnesium of the sample is high, proceed to a second precipitation of calcium oxalate.

10.7 References. First Commission Directive of 15 June 1971. (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

11. Fat

11.1 Principle. Determination of the fat by the Gerber method.

11.2 Material and apparatus.

11.2.1 Contrast Butirometers, with graduations of 0 to 6 per 100 of fat and with divisions of 0.1 per 100. Butirometers with graduations of 0 to 5 per 100 and divisions of 0,1 per 100 may be used when the sample has a fat content of less than 5 per 100.

11.2.2 Appropriate rubber or other rubber-conical plugs.

11.2.3 Contrast pipettes of 1 and 10 ml.

11.2.4 Watch glass.

11.2.5 Bath of adjustable water at 65 ± 1 ° C.

11.2.6 Centrileak capable of reaching 1,200 r.p.m.

11.2.7 Embudo devoid of neck and stem, for the introduction of the sample in the butirometer.

11.3 Reassets.

11.3.1 Density sulphuric acid d = 1.82.

11.3.2 Isoamyl alcohol of density d = 0.815 and distillation range of 128 ° C to 132 ° C.

11.4 Procedure.

11.4.1 Pesar, with precision of 0.1 mg, 1,100 g of product and introduce it into the butirometer through the funnel. Add, through the funnel, 10 ml of warm water (40 ° C), dragging what has been able to adhere to it. Cap and shake tightly to dissolve the milk.

Then add 10 ml of sulphuric acid, making it slip gently through the walls of the butirometer. Add 1 ml of isoamyl alcohol. Close the butirometer with the rubber stopper, gently shake and centrifuge at 1,200 r.p.m. for three to five minutes. Remove the butirometer from the centrifuge and enter the water bath at 65 ° C. Allow a few minutes to pass and read.

11.5 Expression of the results. Read on the butirometer, on the split scale, the height that has reached the fat column, having adjusted to zero the liquid that does not contain fat. The degrees of the scale indicate the tens, and the tenths of the units per cent. A 0.5 per 100 can be perfectly appreciated.

11.6 Remarks.

11.6.1 In the case of serums devoid of casein, they can produce carbonizations that make it difficult to read (because the sulphuric acid of density d = 1.82 is too concentrated). In this case it can be diluted to 10 per 100 (v/v) with distilled water.

11.6.2 In products with very micronized fat, it is necessary to repeat for three times the centrifuges, the heating at 65 ° C and the readings, until obtaining constant results.

11.7 References. Institute for Rationalization and Standardisation of Work. A Spanish Standard, 64,029.

12. Gross ash

12.1 Principle. Incineration of the sample at 550 ° C and heavy of the residue up to constant weight.

12.2 Material and apparatus.

12.2.1 Calefactor plate.

12.2.2 Electrical temperature with temperature regulation.

12.2.3 Platinum or quartz Crisols, rectangular (60 x 40 x 25 mm) or round (diameter: 60 to 75 mm, height: 20 to 25 mm).

12.2.4 Ammonium Nitrate: Solution to 20 per 100 (w/v).

12.3 Procedure. Weigh about 5 g of sample, with an approximation of 1 mg (for products that have a tendency to sponges weigh 2.5 g in a crucible previously calcined and tarado). Place the crucible on the heating plate until the sample is carbonized. Introduce the crucible in the regulated oven to 550 ± 5 ° C. Keep this temperature until white, light gray or reddish ashes are obtained, apparently devoid of carbonaceous particles. Place the crucible in a desiccator, allow to cool and weigh immediately.

12.4 Calculations. The percentage of ashes on natural matter is obtained by the following formula:

Ashes% (natural matter) = (P1-P2) 100/P-P2 100

Being:

P = Weight, in g, of the capsule with the sample.

P = Weight, in g, of the capsule with the ashes.

P = Weight, in g, of the empty capsule.

12.5 Remarks.

12.5.1 The hard to incinerate materials must undergo a first incineration of three hours, cool and add a few drops of a solution to 20 per 100 of ammonium nitrate. Continuing the incineration after drying on the stove.

Eventually repeat the operation until full incineration.

12.5.2 For materials resistant to previous treatment, operate as follows: After a three-hour incineration, drag the ashes with hot water and filter on a small known ash filter. Incinerate the filter and its contents in the initial crucible.

Bring filtering to cold crucible, evaporate dryness, incinerate and weigh.

12.5.3 In the case of oils and fats, weigh 25 g in an appropriate capacity melting pot. Carbonize by inflaming the sample by means of a filter paper wick without ashes. After the combustion moistens with the minimum amount of water possible. Desiccate and continue as indicated in 12.5.

12.6 References. First Commission Directive of 15 June 1971. (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

13 (a) Volatile Nitrogen Bases (nitrogen

ammoniacal) (by microdiffusion)

13 (a) .1 Principle. The method makes it possible to determine the content of volatile nitrogenous bases, expressed in ammonia, from animal feed.

The sample is extracted with water, the solution is defecated and filtered. The volatile nitrogenous bases are displaced with a solution of potassium carbonate by microdiffusion, are collected in a solution of boric acid and are valued with sulfuric acid.

13 (a) .2 Material and apparatus.

13 (a) .2.1 Mixer: from about 35 to 40 oscillations per minute.

13 (a) .2.2 Conway cells (v. scheme), glass or plastic material.

13 (a) .2.3 Microstools, graduated to 1/100 ml.

(FIGURE OMITTED)

13 (a) .3 Reassets.

13 (a) .3.1 Solution to 20 per 100 (w/v) trichloroacetic acid.

13 (a) .3.2 Indicator: dissolve 33 mg of bromoresol green and 65 mg of methyl red in 100 ml of ethanol at 95-96 per 100.

13 (a) .3.3 Boric acid solution. In a 1 litre graduated flask, dissolve 10 g of boric acid p.a. in 200 ml of ethanol at 95-96 per 100 and 700 ml of water. Add 10 ml of indicator (13 (a) .3.2.). Mix and adjust if necessary, the colouring of the clear red solution by addition of a sodium hydroxide solution. 1 ml of this solution allows you to fix at most 300 g NH3.

13 (a) .3.4 Potassium carbonate saturated solution: dissolve 100 g of potassium carbonate p.a. in 100 ml of boiling water. Leave to cool and filter.

13 (a) .3.5. Sulphuric acid 0,02N.

13 (a) .4 Procedure. Weigh 10 g of the sample and introduce it into a 200 ml volumetric flask with 100 ml of water, with a precision of 1 mg. Mix for 30 minutes in the mixer. Add 50 ml of trichloroacetic acid solution (13 (a) .3.2), complete the volume with water, shake vigorously and filter on a fold filter.

Introduce with the pipette into the central part of the Conway cell 1 ml of boric acid solution (13 (a) .3.3) and in the cell crown 1 ml of the sample filtrate. Cover partially with aid from the greased cap. Quickly drop in the crown 1 ml of saturated potassium carbonate solution (13 (a) .3.4) and close the lid tightly. Remove the cell with caution by providing a rotation motion to the horizontal plane in order to ensure the mixing of the two reagents. Leave to incubate either for four hours at least at room temperature or for one hour at 40 ° C.

Assess the volatile bases in the solution of boric acid with sulphuric acid 0.02N (13 (a) .3.5) using a microstool (13 (a) .2.3).

Perform a blank test by applying the same operating method.

13 (a) .5 Calculations of results. 1 ml of sulphuric acid 0,02N corresponds to 0,34 mg of ammonia.

Express the result as a percentage of the sample.

The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 per 100 in relation to the ammonia content of less than 1,0 per 100; 0,1 in absolute value for ammonia content equal to or greater than 1,0 per 100.

13 (a) .6 Remarks. If the ammonia content of the sample is greater than 0,6 per 100 dilute the initial filtering.

13 (a) .7 References. Second Commission Directive of 18 November 1971. (71/393/EEC). 'Official Journal of the European Communities' number L 279 of 20 December 1971.

13 (b) Volatile nitrogen Bases

13 (b) .1 Principle. The method makes it possible to determine the content of volatile nitrogenous bases, expressed in ammonia, of fish flours that do not contain practically urea. It is only applicable for ammonia content of less than 0,25 per 100.

The sample is extracted with water, the solution is defecated and filtered. The volatile nitrogenous bases are displaced by boiling by addition of magnesium oxide and are collected in a known amount of sulphuric acid whose excess is valued with a sodium hydroxide solution.

13 (b) .2 Material and apparatus.

13 (b) .2.1 Mixer from 35 to 40 oscillations per minute approximately.

13 (b) .2.2 Kjeldahl type distillation apparatus.

13 (b) .3. Reagents.

13 (b) .3.1 Solution to 20 per 100 (w/v) trichloroacetic acid.

13 (b) .3.2 Magnesium oxide, p.a.

13 (b) .3.3 Antifoam emulsion (e.g., silicone).

13 (b) .3.4 sulphuric acid 0.1N.

13 (b) .3.5 Sodium hydroxide solution 0.1 N.

13 (b) .3.6 Solution to 0.3 per 100 (w/v) methyl red in ethanol from 95-96 per 100.

13 (b) .4 Procedure. Weigh to the nearest 1 mg, 10 g of the sample and introduce them with 100 ml of water in a 200 ml graduated flask. Mix for 30 minutes in the mixer. Add 50 ml of trichloroacetic acid solution (13 (b) .3.1), complete the volume with water, shake with force and filter on a fold filter.

Taking a number of clear filtering based on the supposed content in volatile nitrogenous bases (100 ml is sufficient, in general). Dilute in 200 ml and add 2 g magnesium oxide (13 (b) .3.2) and some anti-foam emulsion drops (13 (b) .3.3). The solution should be alkaline in the role of tornasol; if not, add magnesium oxide (13 (b) .3.2). Distil approximately 150 ml of the solution into an apparatus of the type of the type of the lldahl and collect the distillate into an erlenmeyer containing an exact volume (25 to 50 ml) of 0,1N sulphuric acid (13 (b) .3.4). During distillation, avoid overheating of the walls. Bring the sulphuric solution to the boil for two minutes, cool and rate the excess sulphuric acid with the sodium hydroxide solution 0.1N (13 (b) .3.5) in the presence of the red methyl indicator (13 (b) .3.6).

Perform a blank test using the same operative method.

13 (b) .5 Calcles. 1 ml of sulphuric acid 0.1N corresponds to 1.7 mg of ammonia.

Express the result in percent sample.

The difference between the results of two parallel determinations carried out on the same sample must not exceed, in relative value, 10 per 100 of ammonia.

13 (b) .6 References. Second Commission Directive of 18 November 1971. (71/393/EEC). 'Official Journal of the European Communities' number L 279 of 20 December 1971.

14. Carbonates

14.1 Principle. Decomposition of the carbonates by the action of the hydrochloric acid and comparison of the volume of carbon dioxide released against a known amount of calcium carbonate measured under the same conditions.

14.2 Material and appliances.

14.2.1 Scheibler-Dietrich's Apparatus, as shown in 14.1.

14.3 Reassets.

14.3.1 Hydrochloric acid, d = 1.10.

14.3.2 Calcium Carbonate.

14.3.3 Sulfuric acid solution 0.1N or approximate, colored with methyl red.

14.4 Procedure. According to the concentration in carbonates of the sample weigh: 0,5 g when the richness is between 50 and 100 per 100 expressed in calcium carbonate.

1 g when the richness is between 10 and 50 per 100 expressed in calcium carbonate.

2 g to 3 g when the richness is less than 10 per 100 expressed in calcium carbonate.

Once the ideal quantity of sample is weighed to be analyzed, it will be inserted into the bottle (4) of the Scheibler-Dietrich device, which will be provided with a small tube of unbreakable material containing 10 ml of the solution 14.3.1. Then connect the bottle with the device. Turn the three-way key (5) so that the tube (1) communicates with the outside.

By means of the mobile tube (2), which is filled with colored sulphuric acid (14.3.3) and attached to the graduated tube (1), bring the level of the liquid to the graduation of zero. Turn the key (5) so that you can communicate the tubes (1) and (2) and check the level to zero.

Prior inclination of the bottle (4), let all hydrochloric acid (14.3.3) slowly pass over the sample. Match the pressure by lowering the tube (2). Shake the bottle (4) until the release of the carbonic gas completely ceases.

Reset the pressure by reducing the liquid to the same level in the tubes (1) and (2). Make the reading after a few minutes, until the gaseous volume remains constant.

Perform a comparative test on the same conditions with 0.5 g of calcium carbonate (14.3.2).

14.5 Calculations.

% CO3Ca = V. 100: T. 2P Being:

V = Volume, in ml, of CO released by the sample.

T = Volume, in ml, of CO detached by 0.5 grams of COCa (14.3.2).

P = Weight, in grams, of the sample.

14.6 Remarks.

14.6.1 When the sample taken is greater than 2 grams, pre-insert 15 ml of distilled water into the bottle (4) and mix before starting the analysis. Use the same volume of water for comparative analysis.

14.6.2 If an apparatus of a volume other than Scheibler-Dietrich is used, it is necessary to adapt the quantity of sample taken from the boil and the substance to be compared, as well as the calculation of results.

14.7 References. First Commission Directive of 15 June 1971. 71 /250/EEC. 'Official Journal of the European Communities' number L 155 of 12 July 1971.

(FIGURE OMITTED)

FIGURE 14.1. (Scale 1/8 in mm)

Appliance according to Scheibler-Dietrich to determine CO

15 (a) Aflatoxin B (chromatography

monodimensional thin layer)

15 (a) .1 Principle. The method makes it possible to determine the aflatoxin content of the raw materials and feed. This method cannot be used in the presence of citrus pulps. The lower limit of determination is 0 ' 01 mg/kg (10 ppb).

In the presence of interfering substances that hinder determinations, the analysis shall be reinitiated according to method 15 (b) (by high resolution liquid chromatography).

15 (a) .2 Material and apparatus.

15 (a) .2.1 Triturator-mixer.

15 (a) .2.2 Apparate for shaking or magnetic agitator.

15 (a) .2.3 Folded filters, Schleicher and Schull number 588 or equivalent, diameter: 24 cm.

15 (a) .2.4 Columns for chromatography (inside diameter: 22 mm, length 300 mm), with teflon tap and capacity of 250 ml.

15 (a) .2.5 Daily vacuum evaporation apparatus, with 500 ml flask.

15 (a) .2.6 Conic bottles of 500 ml, with a grinding stopper.

15 (a) .2.7 Equipment for thin layer chromatography.

15 (a) .2.8 Glass plates for thin-layer chromatography, 200 x 200 mm, prepared as follows (with the indicated amounts can be coated five plates): enter 30 g of silica gel G-HR (15 (a) .3.1.15) in a Conical bottle, add 60 ml of water, cover and shake for one minute. Extend the suspension on the plates in order to obtain a uniform layer of 0,25 mm thick. Allow to dry in the air and then store in a desiccator provided with silica gel. At the time of use, activate the plates by holding them for an hour in the stove at 110 ° C. The plates already ready for use are practical to the extent that they yield results similar to those of the prepared plates as indicated.

15 (a) .2.9 Long-wave ultraviolet Lamp (360 nm). The irradiation intensity should allow to distinguish, even with clarity, a spot of 1.0 ng from aflatoxins B in a plate for thin-layer chromatography, at a distance of 10 cm from the lamp.

15 (a) .2.10 10 ml Tubes, graduated, with polyethylene stoppers.

15 (a) .2.11 Espectrophotometer ultraviolet.

15 (a) .2.12 Fluordensitometer (eventually).

15 (a) .3 Reassets.

15 (a) .3.1 Acetone.

15 (a) .3.2 Chloroform stabilized by 0.5 to 1 per 100 ethanol from 96 per 100 (v/v).

15 (a) .3.3 n-Hexane.

15 (a) .3.4 Methanol.

15 (a) .3.5 Ether diethyl anhydrous, free of peroxides.

15 (a) .3.6 Mix of benzene and acetonitrile 98/2 (v/v).

15 (a) .3.7 Chloroform mixture (15 (a) .3.2.) and methanol 97/3 (v/v).

15 (a) .3.8 Gel of silica, for granulometric column chromatography 0.05 to 0.20 nm.

15 (a) .3.9 Cotton previously degreased, by chloroform, or glass wool.

15 (a) .3.10 Sodium sulphate anhydrous.

15 (a) .3.11 inert gas, e.g. nitrogen.

15 (a) .3.12 1N hydrochloric acid solution.

15 (a) .3.13 sulphuric acid solution at 50 per 100 (v/v).

15 (a) .3.14 Acid-washed diatoms.

15 (a) .3.15 silica gel for thin layer chromatography.

15 (a) .3.16 Standard solution of 0.1 micrograms, about aflatoxin B per milliliter in chloroform (15 (a) .3.2) or in the mixture of benzene/acetonitrile (15 (a) .3.6), prepared and controlled as indicated in 15 (a) .6.

15 (a) .3.17 A qualitative standard solution of 0.1 micrograms of aflatoxins B and B per ml by chloroform (15 (a) .3.2) or in the benzene/acetonitrile mixture (15 (a) .3.6). These concentrations are given as an indication and must be adjusted in order to obtain the same fluorescence intensity for the two aflatoxins.

15 (a) .3.18 Development solvents.

15 (a) .3.18.1 Chloroform (15 (a) .3.2)/acetone (15 (a) .3.1): 9/1 (v/v), not saturated.

15 (a) .3.18.2 Ether diethyl (15 (a) .3.5)/methanol (15 (a) .3.4)/water: 96/3/1 (v/v/v), not saturated.

15 (a) .3.18.3 Ether diethyl (15 (a) .3.5)/methanol (15 (a) .3.4)/water: 94/4,5/1, 5 (v/v/v), saturated bucket.

15 (a) .3.18.4 Chloroform (15 (a) .3.2)/methanol (15 (a) .3.4): 94/6 (v/v), saturated bucket.

15 (a) .3.18.5 Chloroform (15 (a) .3.2)/methanol ((15 (a) .3.4): 97/3 (v/v), saturated bucket.

15 (a) .4 Procedure.

15 (a) .4.1 Sample preparation. Proceed as indicated in the official method number 1.

Samples containing more than 5 per 100 fat must be degreased with petroleum ether (boiling point 40-60 ° C) after the preparation indicated in 15 (a) .5.1. In such cases, the results of the analysis shall be expressed by weight of the non-degreased sample.

15 (a) .4.2 Extraction. Introduce 50.0 g of the milled and homogenised sample into a 500 ml conical bottle (15 (a) .2.6). Add 25 g of diatoms (15 (a) .3.14), 25 ml of water and 250 ml of chloroform (15 (a) .3.2.). Cap the bottle, shake or shake for 30 minutes with the aid of the apparatus (15 (a) .2.2) and filter by folding filter (15 (a) .2.3). Remove the first ten ml of the result of the leak and then collect 50 ml.

15 (a) .4.3 Purification in column. Provide the lower extremity of the column for chromatography (15 (a) .2.4) of a cotton or glass wool stopper (15 (a) .3.9), fill the thirds of the chloroform tube (15 (a) .3.2) and add 5 g of sodium sulphate (15 (a) .3.10).

Check that the top surface of the sodium sulfate layer is flat; add then, in small portions, 10 g of silica gel (15 (a) .3.8).

Remove with caution after each addition in order to remove air bubbles. Let it pose for fifteen minutes and then add, with caution, 15 g of sodium sulfate (15 (a) .3.10). Leave the liquid down to the immediate proximity of the upper surface of the sodium sulfate layer.

Mix the 50 ml of extract collected in 15 (a) .4.2 with 100 ml of n-hexane (15 (a) .3.3) and quantitatively transfer the mixture into the column. Let the liquid down to the top surface of the sodium sulfate layer. Remove the filtered liquid. Then add 100 ml of diethyl ether (15 (a) .3.5) and let the liquid down again to the top surface of the sodium sulphate layer. During these operations, ensure that the flow is 8 to 12 ml per minute and that the column is not emptied. Remove the filtered liquids. Elude afterwards by 150 ml of the chloroform-methanol mixture (15 (a) .3.7) and collect the whole eluid.

Evaporate is almost dry under an inert gas stream (15 (a) .3.11) and a temperature that does not exceed 50 ° C, by means of the rotating vacuum evaporation apparatus (15 (a) .2.5). Quantitatively enter the residue by means of chloroform (15 (a) .3.2) or the benzene/acetonitrile mixture (15 (a) .3.6) in a 10 ml tube (15 (a) .2.10). Concentrate the solution under an inert gas stream (15 (a) .3.11) and then take the volume to 2.0 ml by chloroform (15 (a) .3.2) or the benzene/acetonitrile mixture (15 (a) .3.6).

15 (a) .4.4 Fine-layer chromatography. Deposit on a plate for thin-layer chromatography (15 (a) .2.8), 2 cm from the bottom edge and at intervals of 2 cm, the volumes of the standard solution and the extract indicated below:

-10, 15, 20, 30 and 40 microlitres of the pattern solution of aflatoxin B (15 (a) .3.16).

-10 microlitres of the extract obtained at (15 (a) .4.3) and, in overlap at the same point, 20 microlitres of the standard solution (15 (a) .3).

-10 and 20 microlitres of the extract obtained at (15 (a) .4.3).

Develop the chromatogram outside the range of light, with the help of one of the development solvents (15 (a) .3.16). The choice of solvent should be determined in advance by depositing 25 microlitres of the qualitative standard solution (15 (a) .3.17) and ensuring that, during development, the aflatoxins B and B are completely separated:

-25 per 100 of the highest result for aflatoxin B contents of 10 to 20 micrograms/kilogram.

-5 micrograms, in absolute value, for contents of 20 to 50 micrograms/kilogram.

-10 per 100 of the highest result for contents above 50 micrograms/kilogram.

15 (a) .5 Preparation and control of the standard solution (15 (A) .3.16).

15 (a) .5.1 Determination of concentration in aflatoxin B.

Prepare a pattern solution of aflatoxin B in chloroform (15 (a) .3.2) or in the benzene/acetonitrile mixture (15 (a) .3.6) whose concentration is 8 to 10 micrograms per millilitre. Determine the absorption spectrum between 330 and 370 nm with the aid of a spectrophotometer (15 (a) .2.11).

Measure the absorbance (A) at 363 nm in the case of the chloroformic solution and at 348 nm in the case of the solution in the benzene/acetonitrile mixture.

Calculate the concentration in micrograms of aflatoxin B per milliliter of solution from the following formulas:

312-A-1000 for chloroformic solution

312-A-1000 for chloroformic solution

206000 for the chloroformic solution

312-A-1000

312-A-1000 for solution in the mix

19800 benzene/acetonitrile

Outside the range of light, perform the appropriate dilutions to obtain a working standard solution whose concentration in aflatoxin B is approximately 0.1 micrograms per millilitre.

In the refrigerator, the solution remains stable for two weeks.

15 (a) .5.2 .Control of chromatographic purity.

Deposit on a plate (15 (a) .2.8), 5 microlitres of the 8-10 microgram standard solution of aflatoxin B per millilitre (15 (a) .6.1).

Develop the chromatogram as indicated in (15 (a) .4.4). With ultraviolet light, fluorescence should only result in the perception of a single stain and no fluorescence should be perceived in the area of the origin deposit.

(FIGURE OMITTED)

15 (a) .5.3 Reproducibility of the results of method A. The reproducibility of the results, that is, the variation between the results obtained by two or more laboratories with the same sample, has been calculated in:

± 50 per 100 of the mean value of the results for the average aflatoxin B values of 10 to 20 xg/kg

± 10 xg/kg of mean value for mean values between 20 to 50 xg/kg.

± 20 per 100 of the mean value for mean values greater than 50 xg/kg.

15 (a) .5.4 References.

Directive 92/95 EEC. 'Official Journal of the European Communities' L 327 of 9 November.

Directive 94 /14/EEC. 'Official Journal of the European Communities' L 94 of 13 April.

15 (b) Determination of aflatoxin B, by

High-resolution liquid chromatography

15 (b) .1 Principle. The method is based on separation by high resolution liquid chromatography with fluorescence detection. The extraction of the sample is done with chloroform. The extract is filtered and an aliquot portion of the extract is purified in a florisil cartridge and then in a cartridge C. The final separation and determination are performed by high resolution liquid chromatography (CLAR) using a Reverse phase C column followed by a post-column reaction with aqueous iodine solution and fluorescence detection.

The method allows for the determination of aflatoxin B in feed, including those containing citrus pulp. The lower limit of determination is 0,001 mg/kg.

Note: Mycotoxins are extremely toxic. The manipulations must be carried out in the exhaust hood. Special precautions should be taken when the toxins are in solid form, as due to their electrostatic nature they tend to disperse in the work areas.

15 (b) .2 Material and apparatus. Attention: The use of glass material that has not been washed with acid, for the aqueous solutions of aflatoxins, may cause losses of aflatoxins. Special precautions must be taken with the new or disposable glass material. For example, the bottles for automatic sampler and the Pasteur pipettes. Therefore, the glass material to be in contact with the aqueous solutions of aflatoxins should be immersed for several hours in a diluted acid (e.g. sulphuric acid, c = 2 mol/l) and then wash thoroughly with water. distilled to remove all other acid (for example, three rinses, followed by a pH paper check). In particular, this treatment should be applied to round flasks (15 (b) .2.4), to the flasks, specimens, to the flasks or tubes used for calibration solutions and final extracts (in particular, the flasks for sampler). automatic) and Pasteur pipettes if used to transfer solutions for calibration or extracts.

15 (b) .2.1 Triturator/Mixer.

15 (b) .2.2 1,0 mm mesh size (ISO R 565).

15 (b) .2.3 Mechanical Agitator.

15 (b) .2.4 Empty rotary evaporator provided with a round flask of 150 ml at 250 ml.

15 (b) .2.5 High-resolution liquid chromatograph, with injection loop to allow 250 xl to be injected.

View manufacturer's instructions for partial or total looping.

15 (b) .2.6 Analytical column for CLAR: 3 or 5 xm C fill.

15 (b) .2.7 Pulse-free pump providing the iodinated reagent for post-column reaction, e.g., pump for CLAR or conceived for post-column reaction.

15 (b) .2.8 Null dead volume, stainless steel (1/16 " x 0.75 mm) connection in T.

15 (b) .2.9 Serpentin reaction, teflon or stainless steel. Dimensions between 3,000 x 0,5 mm and 5,000 x 0,5 mm are appropriate in combination with 5 or 3 xm CLAR columns. 15 (b) .2.10 Bane of thermostatic water at 60 ° C, with a temperature variation of less than 0,1 ° C.

15 (b) .2.11 fluorescence detector that provides approximately 365 nm excitation wavelengths and approximately 435 nm emission. (For filter devices: emission wavelength 400 nm). It shall be possible to detect 0,05 ng of B at least. It is recommended to apply some back pressure (e.g., a constrictor or a teflon or stainless steel coil connected to the detector outlet orifice) in order to suppress air bubbles in the flow cell.

15 (b) .2.12 Paper band recorder.

15 (b) .2.13 Electronic integrator (optional).

15 (b) .2.14 folds filter 24 cm. Machey-Nagel 617 1/4 or equivalent.

15 (b) .2.15 membrane filter with a pore size of 0.45 xm, Millipore HAWP 04700 or equivalent.

15 (b) .2.16 Erlenmeyer 500 ml with glass stopper.

15 (b) .2.17 Glass column (with an inside diameter of approximately 1 cm and a length of approximately 3 cm) provided with a Luer tip.

15 (b) .2.18. Chloroform-resistant nylon key Luer (e.g. Bio-rad 7328017, Analytichem A1 6078, J.T. Baker 4514 or equivalent).

15 (b) .2.19 10 ml chemical resistant Jeringa, with Luer key.

15 (b) .2.20 250 xl syringe suitable for injection into CLAR (See 15 (b) .2.5).

15 (b) .2.21. 100 xl microsyringe for the preparation of calibration solutions (check, by heavy, that its accuracy is 2 per 100).

15 (b) .2.22 calibrated 10.0 ml tubes with glass stopper.

15 (b) .2.23 Espectrophotometer suitable for performing measurements in the U.V. spectrum region.

15 (b) .2.24. Equipment for carrying out the confirmatory test (15 (b) .5).

15 (b) .2.24.1 100 ml decantation (with teflon key), acid wash.

15 (b) .2.24.2 Heat source at 40-50 ° C.

15 (b) .3 Reassets.

15 (b) .3.1 Chloroform stabilized with 0.5 to 1.0 per 100 ethanol, en masse.

View 15 (b) .9.2.

15 (b) .3.2 Methanol, CLAR grade for preparation of 15 (b) .3.6.

15 (b) .3.3 Acetone.

15 (b) .3.4 Acetonitrile, CLAR grade.

15 (b) .3.5 Elution solvents; prepare a day before use or by ultrasonic removal of the air containing them.

15 (b) .3.5.1 Acetone mixture (15 (b) .3.3) and water, 98 + 2 (v/v).

15 (b) .3.5.2 Water mix and methanol (15 (b) .3.2), 80 + 20 (v/v).

15 (b) .3.5.3 Water mix and acetone (15 (b) .3.3), 85 + 15 (v/v).

15 (b) .3.6 Mobile phase for CLAR. Mixture of water, methanol (15 (b) .3.2) and acetonitrile (15 (b) .3.4), 130 + 78 + 40 (v/v/v).

Note: It may be necessary to adjust the composition of the solvents of the mobile phase, according to the characteristics of the column CLAR used.

15 (b) .3.7 Iodine Saturated Aqueous Solution. Add 2 g of iodine to 400 ml of water. Mix for at least 90 minutes and filter through a membrane filter (15 (b) .2.15). Protect from light the saturated solution in order to avoid photodegradation.

15 (b) .3.8 Celite 545 washed with acid, or equivalent.

15 (b) .3.9 Florisil cartridge (Waters SEP-PAK) or equivalent.

15 (b) .3.10 Cartridge C18 (Waters SEP-PAK) or equivalent.

15 (b) .3.11 inert gas, e.g. nitrogen.

15 (b) .3.12 Standard solution of aflatoxin B in chloroform with a concentration of 10 xg/ml. Check the concentration of the solution as follows: Determine the absorption spectrum of the solution cited between 330 and 370 nm by means of the spectrophotometer (15 (b) .2.23). Measure the absorbance (a) at the maximum close to 363 nm. Calculate the concentration of Aflatoxin B in micrograms per milliliter of solution, using the following formula:

Concentration (xg/ml) = 312-A-1000 = 13.991-A

Concentration (xg/ml) = 312-A-1000 = 13.991-A

Concentration (xg/ml) = 22300 = 13.991-A

15 (b) .3.12.1 Mother solution of aflatoxin B in chloroform. Transfer quantitatively 2.5 ml of aflatoxin-standard solution B (15 (b) .3.12) to a 50 ml graduated flask and make up with chloroform. Store this solution in a cool place (4 ° C) in the light coat, properly covered and wrapped in an aluminium foil (15 (b) .3.13). Aflatoxin B solutions for CLAR calibration.

Note: For the preparation of these solutions acid-washed glass material must be used (see point 15 (b) .2. Apparatus).

15 (b) .3.13.1 Solution for calibration of 4 ng/ml. Leave to rest (some hours) the stock solution (15 (b) .3.12.1), contained in the graduated flask wrapped in the aluminium foil, until it reaches the ambient temperature. Transfer 400 xl of the stock solution (200 ng aflatoxin B) to a 50 ml graduated flask and evaporate the solution until dry in the inert gas stream (15 (b) .3.11). Dissolve the residue obtained in approximately 20 ml of water mixture and acetone (15 (b) .3.5.3), make up to the same mixture and homogenise.

15 (b) .3.13.2 Solution for calibration of 3 ng/ml. Transfer quantitatively 7.5 ml of solution for calibration (15 (b) .3.13.1) to a 10 ml graduated flask with the mixture of water and acetone (15 (b) .3.5.3) and homogenise.

15 (b) .3.13.3 Solution for calibration of 2 ng/ml. Transfer quantitatively 25 ml of the solution for calibration (15 (b) .3.13.1) to a 50 ml graduated flask, make up to the mixture of water and acetone (15 (b) .3.5.3) and homogenise. This solution is also called 'Reference Pattern' and is used, in particular, for repeated injections during the CLAR procedure.

15 (b) .3.13.4 Solution for calibration of 1 ng/ml. Transfer quantitatively 2.5 ml of solution for calibration (15 (b) .3.13.1) to a 10 ml graduated flask, make up to the mixture of water and acetone (15 (b) .3.5.3) and homogenise.

15 (b) .3.14. A mixture of aflatoxin B, B, G and G in approximate concentrations of 1; 0.5; 1 and 0.5 xg/ml, respectively, in 1ml of chloroform.

15 (b) .3.14.1 Chromatographic Check Solution. Transfer the mixture (15 (b) .3.14) to a test tube with a glass stopper or to a bottle with a screw cap. Transfer 40 æl of this solution to a test tube with glass stopper-acid wash-(15 (b) .2.22). Evaporate the chloroform into an inert gas stream (15 (b) .3.11) and dissolve again in 10 ml of the water and acetone mixture (15 (b) .3.5.3).

15 (b) .3.15 Reassets for the confirmatory test (15 (b) .5). 15 (b) .3.15.1 Sodium chloride saturated aqueous solution.

15 (b) .3.15.2 anhydrous sodium sulphate, granular.

15 (b) .4 Procedure.

15 (b) .4.1 Sample preparation. Crush the sample so that it can pass through the sieve (15 (b) .2.2).

15 (b) .4.2 Test Portion. Weigh on the erlenmeyer (15 (b) .2.16) 50.0 g of the sample problem prepared.

15 (b) .4.3 Extraction. Add to the test portion 25 g of Celite (15 (b) .3.8) 250 ml of chloroform (15 (b) .3.1) and 25 ml of water. Cap the flask and shake for thirty minutes in the mechanical shaker (15 (b) .2.3). Filter through pleat filter (15 (b) .2.14). Collect 50 ml of filtering. If necessary, take an aliquot of the filtrate and dilute to 50 ml with chloroform so that the concentration of aflatoxin B is not greater than 4 ng/ml.

15 (b) .4.4. Purification (the procedure should be followed without significant interruptions). The following precautions should be taken:

-conveniently protect the analysis laboratory from natural light.

To do this, they can be used:

1) Hojas that absorb UV rays to cover windows and a screen light (avoid direct sunlight).

2) Cortinas or blinds in combination with artificial light (fluorescent tubes may be used).

-Protect as much as possible from light solutions that contain aflatoxin (keep in the dark and use aluminium foil).

15 (b) .4.4.1 Purification with florisil SEP-PAK.

15 (b) .4.4.1.1. Preparing the column-cartridge assembly. Attach a key (15 (b) .2.18) to the shorter branch of a florisil cartridge (15 (b) .3.9) (see figure 15 (b) .1).

Wash the cartridge and remove the air by using a syringe (15 (b) .2.19) 10 ml of chloroform (15 (b) .3.1) and quickly pass 8 ml of chloroform through the cartridge. Attach the longest branch of the cartridge to the glass column (15 (b) .2.17) and insert the remaining 2 ml of chloroform into the column through the cartridge. Close the key and draw the syringe. 15 (b) .4.4.1.2. Purification. Insert in the column-cartridge the filter collected according to the point (15 (b) .4.3) and leave to drain by gravity. Wash with 5 ml of colroform (15 (b) .3.1) and then with 20 ml of methanol (15 (b) .3.2). Discard the eluates. During these operations, prevent the column-cartridge assembly from becoming dry. Elude aflatoxin B with 40 ml of the acetone/water mixture (15 (b) .3.5.1) and collect the entire eluate in the round flask (150 ml) of the rotary evaporator (4.4). Concentrate eluate in the rotary evaporator at a temperature of 40-50 ° C until the distillation of acetone ceases.

Note: At that time, approximately 0.5 ml of liquid is left in the flask. It has been shown experimentally that continuing evaporation has no harmful consequences and that when 0.5 ml of liquid remains the amount of acetone present is not significant. The presence of acetone residues could lead to losses of aflatoxin B in the cartridge C. Add 1 ml of methanol (15 (b) .2.2), shake the flask to dissolve the aflatoxin B attached to its walls, add 4 ml of water and mix. Disconnect and discard the cartridge. Wash the glass column with water and keep it for purification C.

15 (b) .4.4.2 Purification with SEP-PAK C.

15 (b) .4.4.2.1 Preparation of the column-cartridge assembly. Attach a key (15 (b) .2.18) to the shortest branch of a C cartridge (15 (b) .3.10). (See Figure 1 (b) .1.)

Purge the cartridge and remove the air by passing quickly with a syringe (15 (b) .2.19) 10 ml of methanol (15 (b) .3.2) by the key through the cartridge. (The air bubbles of the cartridge are visible in the form of light spots on a grayish background.) Take 10 ml of water and make 8 ml pass through the cartridge (avoid introducing air when passing from methanol to water). Attach the longest branch of the cartridge to a glass column and insert the remaining 2 ml of water into the column through the cartridge. Close the key and draw the syringe.

15 (b) .4.4.2.2 Purification. Transfer quantitatively to the column (15 (b) .2.17) the extract collected at the point (15 (b) .4.4.1.2), twice washing the flask with 5 ml of the water and methanol mixture (15 (b) .3.5.2) and leave to drain by gravity. During these operations, prevent the column-cartridge assembly from becoming dry. (If air bubbles form in the narrowing close to the cartridge, stop the flow and hit the top of the glass column to remove the bubbles. Resume operations immediately.) Elude with 25 ml of the mixture of water and methanol (15 (b) .3.5.2). Discard the eluates. Elude aflatoxin B with 50 ml of the mixture of water and acetone (15 (b) .3.5.3) and collect the whole eluate in a 50 ml graduated flask. Make up to 50 ml with water and mix, the test solution obtained is used for chromatography (15 (b) .4.5).

Attention: You do not normally need to filter the final extract before you make CLAR. When filtering is necessary, cellulose filters should be avoided, since they may result in loss of aflatoxin B. Teflon filters may be used.

15 (b) .4.5 High-resolution liquid chromatography. (See Figure 2 for the assembly of the equipment.)

Allow sufficient time for the instruments to be heated and stabilized before use.

Note 1: The flow rates quoted for the mobile phase of CLAR and the post-column reagent are merely indicative. An adjustment may be required based on the characteristics of the CLAR column.

Note 2: The detector response for aflatoxin B depends on the temperature, so the drift compensation (see figure 3) should be performed. The injection of a fixed amount of the reference pattern of aflatoxin B (15 (b) .3.13.3) at regular intervals (for example, every three injections) makes it possible to correct, using the mean response, the values of the peaks of aflatoxin B between these reference patterns, as long as the difference between consecutive reference pattern responses is very small (R 10 per 100). Therefore, injections should be carried out without interruption. If an interruption is necessary, the last injection before the interruption and the first injection after the interruption must be injections of the reference pattern (15 (b) .3.13.3). Since the calibration curve is linear and passes through the origin, the amounts of aflatoxin B present in the sample extracts are determined directly by reference to the adjacent patterns.

15 (b) .4.5.1 CLAR pump adjustment. Adjust the pump CLAR (15 (b) .2.5) so that a flow rate of 0.5 or 0.3 ml/min is obtained for a CLAR column of 5 xm or 3 xm (15 (b) .2.6), respectively, using the mobile phase (15 (b) .3.5).

15 (b) .4.5.2 Pump adjustment for post-column reaction. Adjust the pump (15 (b) .2.7) so as to obtain a flow rate of 0.2-0.4 ml/min of iodine saturated aqueous solution (15 (b) .3.7). Indicative information: flow rates of approximately 0.4 or 0.2 ml/min are recommended in association with flow rates of 0.5-0.3 ml/min of the mobile phase, respectively.

15 (b) .4.5.3 fluorescence detector. Set the detector (15 (b) .2.11) to an excitation wavelength of 365 nm and emission of 435 nm (filter apparatus: T 400 nm). Adjust the detector attenuator so that the 80 per 100 approximately of the maximum path of the registrant rainfall corresponds to 1 ng of aflatoxin B.

15 (b) .4.5.4 Inject. For all solutions, inject amounts of 250 xl following the instructions of the manufacturer of the device.

15 (b) .4.5.5 Check for chromatographic separation. Inject the chromatographic solution (15 (b) .3.14.1).

The valleys shall be less than 5 per 100 of the sum of the heights of the adjacent peaks.

15 (b) .4.5.6 System stability check. Before carrying out each of the test series, repeat injections of the reference pattern (15 (b) .3.13.3) until the areas of the peaks are stabilized.

Note: The peaks produced by aflatoxin B between consecutive injections should present differences of less than 6 per 100. Proceed immediately to carry out the linearity check (15 (b) .4.5.7).

15 (b) .4.5.7 Check linearity. Inject the aflatoxin B solutions for calibration (15 (b) .3.13.1) and (15 (b) .3.14.4). Use the reference pattern (15 (b) .3.13.3) at intervals of three injections, in order to correct the drift in the responses.

Note: The responses of the peaks of the reference pattern must present differences of less than 10% in 90 minutes: Correct the drift by applying the formula given in paragraph 15 (b) .6. The calibration graph must be linear and pass through the origin, within the limits of 2 times the value of the standard deviation of the Y estimate. The values found must differ by less than 3% of the nominal values. If these conditions are met, continue operations immediately. Otherwise, identify and correct the causes of the problem before continuing.

15 (b) .4.5.8 Injection of sample extracts. Inject the purified sample extracts (15 (b) .4.4.2.2). Repeat the injection of the reference pattern (15 (b) .3.13.3) after two injections of sample extract according to the following sequence: reference pattern, extract, extract, reference pattern, extract, extract, reference pattern, etc. ..

15 (b) .5 Confirmation test.

15 (b) .5.1 Further treatment of extract (15 (b) .4.4.2.2). Add 5 ml of the sodium chloride solution (15 (b) .3.15.1) to the final extract obtained as described in point (15 (b) .4.4.2.2). Extract three times with 2 ml of chloroform for 1 minute, using the decantation funnel (15 (b) .2.24.1). Pour the combined chloroform extracts into a 10 ml test tube through 1 g of approximately sodium sulphate (15 (b) .3.15.2). (A small funnel (4 cm in diameter) can be used by placing in the narrowing a cotton coated with 1 g of sodium sulphate.)

Wash the sodium sulfate layer with a few ml of chloroform and collect the washes in the same test tube. Evaporate the chloroform extract in the said tube until dryness using the heat source (15 (b) .2.24.2) and dissolve it again in 1 ml of chloroform.

15 (b) .5.2 Preparation of derivatives and fine-layer chromatography. See Council Directive 76 /372/EEC, Annex, Method A, point 5.6.2 (Method 15 (a), point 15 (a) .4.4).

15 (b) .6 Calculations. Calculate the content of aflatoxin B: (xg/kg) of the sample using the following formula:

Aflatoxin B Content in lg/Kg = m-V

Aflatoxin B Content in lg/Kg = m-V

Aflatoxin B Content in lg/Kg = m-VV

Aflatoxin B Content in lg/Kg = V-M-V

Aflatoxin B Content in lg/Kg = m-VV

Being:

m = amount, in ng, of aflatoxin B represented by peak B of the sample, calculated as follows:

m = P (sample)-2 r (st)

P (st) + P (st)

P (sample) = area of the peak of the aflatoxin B of the sample.

P (st1) = peak area of the aflatoxin B of the previous reference pattern (15 (b) .3.13.3).

P (st2) = peak area of aflatoxin B of the following reference pattern (15 (b) .3.13.3).

r (st) = injected quantity of the reference pattern (15 (b) .3.13.3), expressed in ng.

V = volume of the injected sample extract, in ml.

V = final volume of the sample extract, in ml (15 (b) .3.13.3).

M = mass of the sample in g.

V = volume of the filtrate transferred to the florisil cartridge (15 (b) .4.4.1.2), in ml.

V = volume of chloroform used for the extraction of the sample, in ml.

If the exposed procedure is applied, the formula is reduced to:

Alatoxin B content, in lg/kg = 20 x m.

15 (b) .6.1 The calculation of the results can also be done by measuring the height of the peaks.

15 (b) .7 Repetibility. See point 15 (b) .9.1 (comments).

15 (b) .8 Reproducibility. See point 15 (b) .9.1.

15 (b) .9 Remarks.

15 (b) .9.1 Precision.

Table 1 shows the results of the repeatability and reproducibility obtained in a collaborative study (1) on compound feed carried out at international level. The term repeatability (r) used here is defined as the largest non-significant difference, with a probability of 95 per 100, between two readings of the same sample carried out in the same laboratory and under similar conditions. The term reproducibility (R) is defined in a similar way and refers to the comparison between the results obtained in two different laboratories. In accordance with ISO 3534-1977, 2.35 (2) and Commission Decision 89 /610/EEC (3), both R and R are also given in Table 1 in the form of coefficients of variation.

TABLE 1

Repeatability (r) and reproducibility (R) expressed

in differences and in variation coefficients

15. Laboratories

Level (lg/kg)/r/R/CVt (*) Percentage/CVr Percentage

8-14/1.4/1.7/11/18

(*) CV = Variation coefficient.

(1) Egmond, H.P. van. Heisterkamp, S.H. and Paulsch, W.E. (1991), Food Additives and Contamination 8, 17-29.

(2) ISO 3534-1977.

(3) OJ No L 351, 2.12.1989, p. 39.

15 (b) .9.2 chloroform stability (15 (b) .3.13.3). The absorption characteristics of the florisil cartridge may be modified if a different stabilizer of ethanol is used. This should be verified in accordance with point 15 (b) .9.3 when the described chloroform is not available.

15 (b) .9.3 Exattitude. The correct application of the method should be checked by making repeated measurements with certified reference materials. If these materials are not available, the validity of the method must be checked by means of recovery experiments on blank samples. The difference between the mean and the actual value must not exceed the limits of -20 to 10 per 100 of the actual value.

15 (b) .10 References. Directive 92 /95/EEC. 'Official Journal of the European Communities' number L 327 of 9 November 1992.

(OMITIDES FIGURES)

16. Peroxides index

This determination shall apply to those fats which are used as raw materials.

The procedure will be the one in method number 21 of the official methods of analysis of Oils and Grases approved by Order of 31 January 1977 ("Official State Gazette" of July 14).

If the sample is to be prepared, the procedure set out in method number 1 of the above order will be followed.

17. Total phosphorus

(Spectrophotometric method)

17.1 Principle. Determination of the phosphorus of a mineralized sample, by the transformation of its phosphorated compounds into orthophosphorates.

The mineralization is done by dry (calcination) and subsequent acid dissolution, or by acid digestion.

Measure of the absorbance at 430 nm of the complex formed with the nitro-molar-vanadate reagent.

17.2 Material and apparatus.

17.2.1 Spectrophotometer, capable of performing readings of 430 nm, with 10 mm light-passing buckets.

17.2.2 Quartz or porcelain incineration Crisols.

17.2.3 Test tubes 25 to 30 ml with frosted glass mouth.

17.2.4 Double-root pipettes of 5, 10, 15, 20, 25 and 50 ml.

17.2.5 Kjeldahl of 250 to 500 ml capacity.

17.2.6 Graduated matrices of 100, 250, 500 and 1000 ml.

17.3 Reassets.

17.3.1 Calcium Carbonate.

17.3.2 Hydrochloric acid d = 1,19 g/ml.

17.3.3 Nitric acid d = 1.38-1.42 g/ml.

17.3.4 Nitric acid d = 1,045 g/ml (10 per 100 m/v).

17.3.5 sulphuric acid of d = 1.84 g/ml.

17.3.6 Ammonia concentrate of d = 0.910 g/ml.

17.3.7 Solving heptamolibdata solution. Dissolve in hot water 100 g of heptamolibdate ammonium, tetrahydrate (NH) MoO-4HO. Add 10 ml of ammonia (17.3.6), transfer to a 1000 ml volumetric flask, and once cold is complete with water to the root.

17.3.8 ammonium metavanadate solution. Dissolve 2,35 g of ammonium metavanadate (NHVO) in a 500 ml erlenmeyer with 400 ml of hot distilled water. Add slowly and agitate 20 ml of a solution containing 7 ml of nitric acid (17.3.3) and 13 ml of distilled water. Take a 1000 ml volumetric flask and, once cold, make up to scratch with distilled water.

17.3.9 Nitro-molybdo-vanadate solution. In graduated litre flask, mix 200 ml of ammonium heptamolibdate solution (17.3.7) with 200 ml of ammonium metavanadate solution (17.3.8) and 134 ml of nitric acid (17.3.3). Complete with distilled water to the root.

17.3.10 phosphorus standard solution containing 1 mg phosphorus per milliliter. Dissolve in volumetric flask 4,387 g of monopotassium phosphate (KHPO) (previously dried in a 100 ° C stove until constant weight) in distilled water and lead to the enrasar.

17.4 Procedure.

17.4.1 Calibration curve.

In 100 ml graduated flask and from the phosphorus standard solution (17.3.10) prepare solutions containing 0, 5, 10, 20, 30 and 40 micrograms of phosphorus per millilitre.

In six erlenmeyer or test tubes (17.2.3) take with double-root pipettes 10 ml of each phosphorus standard solution. Add to each of them, also with double-rooted pipette, 10 ml of the nitro-molybovanadato reagent (17.3.9), shake to homogenise and leave at rest ten minutes at 20º C.

Perform the photometric readings at 430 nm, using 10 mm light-passing buckets, using the white phosphorus solution as the reference solution.

Graphically represent the absorbances obtained against the microgram/millilitres or the milligrams of phosphorus existing in each reading.

17.4.2 Preparation of the sample.

17.4.2.1 Mineralization by calcination (for samples containing phosphate-free organic substances that give insoluble products when incinerating).

weigh 2.5 g of the sample, with precision of 1 mg, in quartz or porcelain capsules.

Mix with 1 g of calcium carbonate (17.3.1.). Put in mufla at 550 ° C-5 ° C until white or grey ashes are obtained (a small part of coal does not interfere). Transfer the ashes to a 150 ml glass. Add 10 ml of water by washing the crucible with hydrochloric acid (17.3.2) until the effervescence ceases. Add another 10 ml of hydrochloric acid (17.3.2). Evaporate the hydrochloric acid in a boiling sand bath until dryness. Cool and dissolve the residue with 10 ml of nitric acid (17.3.4), boil in sand bath for five minutes, without getting dry. Filter through paper on 500 ml volumetric flask, wash with distilled water the glass and make up once cold.

17.4.2.2 Mineralization by acid digestion (for mineral compounds and liquid feed). Weigh 1 g of sample, with precision of 1 mg, and lead to flask Kjeldahl (17.2.5), add 20 ml of sulphuric acid (17.3.5), shake the flask circularly to prevent the sample from adhering to the walls and boil for ten minutes. Allow to cool a little and add 2 ml of nitric acid (17.3.3), heat and bring again to the boiling point. Repeat this procedure until discoloration of the solution. Cool, add some water and transfer the liquid, filtering if necessary, to 500 ml graduated flask, wash the Kjeldahl flask with hot water, and attach the washing liquids if necessary through the filter initially used. Once cold, add water to the root.

17.4.3 Development of the color and measure of the absorbance. Dilute an aliquot of the problem filtering to achieve a phosphorus concentration of not more than 40 micrograms/ml.

Transfer 10 ml of this solution to erlenmeyer or test tube (17.2.3) and add 10 ml of the nitro-molybovanadate reagent (17.3.9). The two solutions taken with two-frame pipette. Mix well and leave ten minutes at rest. Transfer an aliquot to the cell and measure the absorbance in the spectrophotometer at 430 nm, using as a reference a 10 ml solution of white solution with 10 ml of the nitro-molar-vanadate reagent.

Always carry out a blank test with the reagents used, following the same exprelimental procedure. Use this as a reference in each spectrophotometric reading.

17.5 Calculations. Determine the phosphorus concentration in the diluted aliquot portion of the problem solution or the microgram/millilitres of that solution, by reference to the calibration curve, calculating the percentage by weight of phosphorus in the sample.

% P = S-C-C-... C

G-10,000-A-A-... A

Being:

G = Sample weight in grams.

C C ... C = Dilutions to which the aliquots were taken in millilitres.

A A ... A = Alicuotas taken for successive dilutions in millilitres.

S = Micrograms/millilitres of phosphorus measured from the solution problem in relation to the calibration curve.

17.6 Remarks. The difference between the result of two successive determinations must not exceed:

3 per 100 (relative value), with phosphorus content less than 5 per 100 (m/m).

0,1 per 100 (absolute value) for phosphorus content equal to or greater than 5 per 100 (m/m).

17.7 References. Second Commission Directive of 18 November 1971 (71/383/EEC). 'Official Journal of the European Communities' number L 279 of 20 December.

18. Crude protein soluble in hydrochloric acid

and pepsin

18.1 Principle. The method makes it possible to determine the fraction of the raw proteins solubilized by the pepsin and the hydrochloric acid in the given conditions. It applies to flours of animal origin.

The sample is subjected to digestion for forty-eight hours at 40 ° C for a chlorhydric solution of pepsin. The suspension is filtered or centrifuged and the nitrogen content of the filtrate or supernatant is determined, according to the official method number 3.

18.2 Material and apparatus.

18.2.1 Bath water or regulated incubation stove at 40 ° C ± 1 ° C.

18.2.2 Mineralizer and distiller Kjeldahl.

18.3 Reassets.

18.3.1 Hydrochloric acid (d = 1,125).

18.3.2 Hydrochloric acid solution 0.075N.

18.3.3 Pepsin 2.0 U/mg. The activity should be controlled according to the official method number 19.

18.3.4 Recently prepared solution of pepsin 0.02 per 100 (w/v) in hydrochloric acid solution (18.3.2). Activity 400 U/l.

18.3.5 Antifoaming.

18.3.6 Those listed in 3.3.

18.4 Procedure. Weigh, with precision of 1 mg, 2 g of sample. Introduce the sample into 500 ml volumetric flask and add 450 ml of the chlorhydric solution of pepsin (18.3.4), previously taken at 40 ° C. Shake in such a way as to prevent the formation of agglomerates. Check that the pH of the suspension is less than 1.7. Take the flask to the water bath or the hatchery (18.2.1) and keep it for forty-eight hours at 40 ° C ± 1 ° C, shake at eight, twenty-four and thirty-two hours. After 48 hours add 15 ml of hydrochloric acid (18.3.1), refrigerate up to 20 ° C, make up the flask with water and filter.

Take 250 ml of the filtrate and introduce it into the Kjeldahl digestion flask and proceed as in the official method number 3. Perform a blank test.

18.5 Calculations. The content of the protein soluble in hydrochloric and pepsin for 100 g of sample is given by the following formula:

Soluble protein/100 g sample = 2-1,4-6.25 (VN V' N ')

P

Being:

P = Weight, in g, of the sample.

V = Volume, in ml, of sulphuric acid.

N = Sulphuric acid normality.

V ' = Volume, in ml, of sodium hydroxide consumed in the assessment.

N' = Normal of the sodium hydroxide solution.

18.6 Remarks. Products whose fat content exceeds 10 per 100 must be previously degreased by extraction with petroleum ether.

18.7 References. Third Commission Directive of 27 April 1972 (72/199/EEC). 'Official Journal of the European Communities' number L, 123 of 29 May.

19. Pepsin activity

19.1 Principle. The method is used to test the activity of the pepsin used in the determination of the protein soluble in pepsin and hydrochloric acid.

The haemoglobin is treated in the conditions defined with pepsin and hydrochloric acid. The non-hydrolysed fraction of the proteins is precipitated by trichloroacetic acid. The addition of the sodium hydroxide solution and the Folin-Ciocalteu reagent is added to the filtrate. The absorbance of this solution is measured at 750 nm and the corresponding tyrosine amount is read on a pattern curve.

The pepsin unit is defined as the amount of said enzyme that releases per minute, in the conditions of the method, a quantity of hydroxyaryl compounds whose colouring by the Folin-Ciocalteu reagent has an absorbance. corresponding to that of a tyrosine mole, under the same conditions.

19.2 Material and apparatus.

19.2.1 Bath water, regulated at 25 ° C ± 0,1 ° C by ultrathermostat.

19.2.2 Spectrophotometer.

19.2.3 Cronometer; precision: 1 second.

19.2.4 pH-meter.

19.3 Reassets.

19.3.1 Hydrochloric acid 0.2N.

19.3.2 Hydrochloric acid 0.26N.

19.3.3 Hydrochloric acid 0.025N.

19.3.4 Solution to 5 per 100 (w/v) trichloroacetic acid.

19.3.5 Sodium hydroxide solution 0.5N.

19.3.6 Reactive of Folin-Ciocalteu. Introduce 100 g of sodium wolframate dihydrate (NaWO-2HO), 25 g of sodium molybdate dihydrate (NaMO-2HO) and 700 ml of water in a round-bottom flask of 2 litres of grinding closure. Add 50 ml of phosphoric acid (d = 1,71) and 100 ml of concentrated hydrochloric acid (d = 1,19), adjust the flask to a reflux coolant, heat to the boil and keep the solution in a smooth boiling for 10 h. Leave to refrigerate, separate the reflux coolant, add 175 g of lithium sulfate dihydrate (LiSO-2HO), 50 ml of water and 1 ml of bromine. Boil for 15 minutes to remove excess bromine.

Let refrigerate, transfer the solution into a 1 litre volumetric flask, complete the volume with water, homogenise and filter. The reactant obtained must not be coloured. Before your use, dilute 1 volume of the reagent with two volumes of water.

19.3.7 Hemoglobin solution: Pesar a quantity of hemoglobin, protein substrate according to Anson (2 g approximately) corresponding to 354 mg of nitrogen and enter into a 200 ml flask of grinding closure. Determine the nitrogen content by means of a semi-microkjeldahl (theoretical content: 17,7 per 100 nitrogen). Add some ml of hydrochloric acid (19.3.2) connect the flask to the vacuum pump and shake up to complete haemoglobin dissolution. Stop making the vacuum and add, stirring constantly, hydrochloric acid (19.3.2) to complete 100 ml. Prepare immediately before your employment.

19.3.8 Tyrosine standard solution: Dissolve 181.2 mg tyrosine in hydrochloric acid (19.3.2) and complete 1 litre with the same hydrochloric acid.

19.3.9 Work pattern solution: Take 20.0 ml of solution (19.3.8) and dilute to 100 ml with hydrochloric acid (19.3.1). One ml of said solution contains 0.2 micromoles of tyrosine.

19.4 Procedure.

19.4.1 Preparation of solution (see 19.6.1). Dissolve 150 mg of pepsin in 100 ml of hydrochloric acid (19.3.2). Take 2 ml of the solution using pipette, insert them into a 50 ml volumetric flask and complete by volume using hydrochloric acid (19.3.3). The pH, controlled by the pH-meter, should be 1.6 ± 0.1. Keep the flask in a water bath at 25 ° C (19.2.1).

19.4.2 Hydrolysis. Insert by pipette into test tube 5,0 ml of haemoglobin solution (19.3.7) lead to 25 ° C in the water bath (19.2.1) add 1,0 ml of the pepsin solution obtained in (19.4.1) and mix, with the aid of a glass rod widened in one end, by approximately 10 vaiven movements. Keep the test tube in the water bath at 25 ° C for ten minutes exactly, counted from the time of the addition of the pepsin solution (duration and temperature must be perfectly respected). Add 10,0 ml of trichloroacetic acid solution (19.3.4) previously taken at 25 ° C. Homogenize and filter on a dry filter.

19.4.3 Development of the coloration and measurement of the absorbance. Take 5.0 ml of the filtrate by pipette, introduce them into a 50 ml erlenmeyer, add 10.0 ml of sodium hydroxide solution (19.3.5) and continuously agitate, 3.0 ml of the diluted reagent of Folin-Ciocalteu (19.3.6). After five to ten minutes, determine the absorbance of the solution at 750 nm in buckets of 1 cm thick, using as white water.

19.4.4 Blank test. For each determination, proceed to a blank test as follows. Pipette into a test tube 5,0 ml of haemoglobin solution (19.3.7), lead to 25 ° C in the water bath (19.2.1.), add 10.0 ml of trichloroacetic acid solution (19.3.4) previously taken at 25 ° C, homogenise and add to continuation 1.0 ml of the pepsin solution obtained at 19.4.1. Mix with the aid of a glass rod and keep the test tube ten minutes exactly in the water bath (19.2.1.) at 25 ° C. Homogenize and filter on a dry filter.

Continue the operating method as indicated in 19.4.3.

19.4.5 Calibration curve. Introduce in erlenmeyers 50 ml volumes of 1.0; 2.0; 3.0; 4.0; 5.0 ml tyrosine pattern solution (19.3.9) corresponding respectively to a tyrosine amount of 0.2; 0.4; 0.6; 0.8; and 1.0 micromoles. Complete the series by means of a tyrosine-free witness. Bring the volumes to 5.0 ml with the help of hydrochloric acid (19.3.1). Add 10.0 ml of sodium hydroxide solution (19.3.5) and constantly agitating, 3.0 ml of the diluted Folin-Ciocalteu reagent (19.3.6). Measure the absorbance as indicated in the last sentence of the paragraph (19.4.3). Draw the calibration curve by placing the tyrosine concentrations against the obtained absorbances.

19.5 Calculations. Read on the calibration curve the amount of tyrosine, in micromoles corresponding to the absorbance of the coloured solution, corrected from the blank test.

The activity of pepsin, in tyrosine micromoles, per mg and per minute at 25 ° C, is given by the following formula:

Units per mg (U/mg) = 0.32-a

P

Being:

a = Amount of tyrosine in micromoles read on the pattern curve.

P = Weight in mg of the amount of pepsin added in 19.4.2.

19.6 Remarks.

19.6.1 The amount of pepsin to be placed in the solution should be such that it can obtain a final absorbance of 0.35 ± 0.035.

19.6.2 Two units per mg obtained by this method correspond to 3.64 units millionths Anson/mg (tyrosine micromoles/mg per minute at 35.5 ° C), or 36,400 commercial units/g, (myrosin/g micromol in 10 minutes) 35.5 ° C).

19.7 References. Third Commission Directive of 27 April 1972 (72/199/EEC). 'Official Journal of the European Communities' number L, 123 of 29 May.

20. Total casein

20.1 Principle. It is based on precipitating (after reconstituted the milk in warm water), the casein to its isoelectric point. By washing, pure casein is collected by eliminating the serum and impurities. The protein level N x 6,38 is determined via Kjeldahl and the corresponding target is expressed as casein.

This method is applicable to dairy raw materials (uncreamed, whole or semi-creamed milk powder).

20.2 Material and apparatus.

20.2.1 Material required for the determination of crude protein.

20.2.2 Water bath with thermostat for 40 ° C.

20.2.3 Centrileak capable of reaching 4,000 r.p.m.

20.3 Reassets.

20.3.1 Reassets required for gross protein determination.

20.3.2 acetic acid. Dilution to 10 per 100 (w/v).

20.3.3 Dilution of sodium acetate 1N.

20.3.4 Albet filter paper number 240 or similar.

20.4 Procedure. Weigh 5 g of sample, with precision of 1 mg. Dissolve in 100 ml of heated deionised water at 40-45 ° C. Centrifuge at 4,000 r.p.m. for 15 minutes. Take 20 ml of the solution and heat in a water bath at 40 °C for five or ten minutes. Add 5 ml of acetic acid (20.3.2) and let it precipitate for 10 minutes.

Add 5 ml of sodium acetate solution (20.3.3) and wait 10 minutes for the casein precipitate to evolve and agglomerate. Filter on filter paper (20.3.4).

Wash with acidic water with a few drops of acetic acid to remove the remaining milk soluble proteins or not, until you get a curd of pure casein.

Then determine the nitrogen content of the precipitate, according to the Kjeldahl method (official method number 3).

20.5 Calculation. The casein content shall be given in weight procentage according to:

Casein% = N x 6.38

Being:

N = Total Nitrogen of the precipitate expressed as a percentage of sample.

20.6 Remarks. The washing of the curd should be done with acidic water and with great care, on the one hand so that it does not subtract in it serum proteins, from another so that it does not lose casein for tampering.

20.7 References. Official Methods of Analysis of Leches. Method number 3.

21. Urea

21.1 Principle. Defecation of the sample and measure of the absorbance at 420 nm of the compound formed by adding p-dimethylaminobenzaldehyde.

21.2 Material and apparatus.

21.2.1 Mixer or agitator capable of 35 to 40 turns per minute.

21.2.2 Test tubes of approximately 160 x 16 mm with screw plugs.

21.2.3 Spectrophotometer.

21.3 Reassets.

21.3.1 Solution of p-dimethylaminobenzaldehyde (D.M.A.B.).

Dissolve 1.6 g of (D.M.A.B.) in 100 ml of 96 per 100 ethanol and add 10 ml of hydrochloric acid (d = 1,19 g/ml). This reagent is retained for up to two weeks.

21.3.2 Carrez Solution I.

Dissolve 24 g of zinc acetate trihydrate (Zn (CH-COO)-3HO) and 3 ml of glacial acetic acid in distilled water and complete up to 100 ml.

21.3.3 Carrez Solution II.

Dissolve 10,6 g of potassium trihydrate (KFe (CN)-3HO) in distilled water and complete up to 100 ml.

21.3.4 Active carbon not absorbing urea.

21.3.5 urea standard solution at 0.1 per 100 (w/v).

21.4 Procedure.

21.4.1 Calibration curve. Bring volumes of 1, 2, 4, 5, and 10 ml of the urea standard solution (21.3.5) to 100 ml graduated flasks and make up to be drawn with distilled water. Take 5 ml of each solution, take the tubes (21.2.2) and add 5 ml of the DMAB solution respectively (21.3.1). Mix and put the tubes in water bath at 20 ° C for 15 minutes. Measure the absorbance of each solution at 420 nm against a target obtained by taking 5 ml of distilled water and following the same procedure. Get the calibration curve.

21.4.2 Sample Preparation. Weigh with 1 mg, 2 g of sample and introduce them into a 500 ml graduated flask and add 1 g of activated carbon (21.3.4). Add 400 ml of distilled water, 5 ml of Carrez solution I and 5 ml of Carrez II solution. Put the flask in the agitator (21.2.1) for 30 minutes. Make up with distilled water, stir and filter.

21.4.3 Determination. Take 5 ml of the filtrate and take to the tubes (21.2.2). Add 5 ml of the DMAB solution (21.3.1) and continue as in (21.4.1).

21.5 Calculations. Determine the urea content by comparing the absorbances obtained from the sample against those of the calibration curve. Express the result in percent sample.

21.6 References. First Commission Directive of 15 June 1971 (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1973.

22. Cyanhydride acid

22.1 Principle. The sample is suspended in water. The hydrocyanic acid is released under the ferment action, is dragged by water vapor distillation and is collected in a determined volume of acidified silver nitrate solution. The silver cyanide is separated by filtration and the excess silver nitrate is valued by an ammonium thiocyanate solution.

The method makes it possible to determine the free and combined cyanhydride acid content in the form of glycosides existing in certain feed and, in particular, the products of flax seed, cassava flour and certain species of leguminous plants.

22.2 Material and apparatus.

22.2.1 Estufa provided with a regulated thermostat at 38 ° C.

22.2.2 Water vapor stream distillation apparatus provided with a curved elongated refrigerant.

22.2.3 100 ml flat bottom matricas of a grinding plug.

22.2.4 Bane oil.

22.2.5 Bureta graduated to 1/20 ml.

22.3 Reassets.

22.3.1 Sweet Almond Suspension:

Crush 20 sweet almonds peeled in 100 ml of water at a temperature of 37 to 40 ° C. Check the absence of hydrocyanic acid on 10 ml of the suspension, with the help of a picro-sodium paper or by using a blank test as indicated in the last paragraph of 22.4.

22.3.2 Solution to 10 per 100 (w/v) sodium acetate, neutral to phenolphthalein.

22.3.3 Antifoam Emulsion (silicone, for example).

22.3.4 Nitric acid, d = 1.40.

22.3.5 Silver nitrate solution 0.02N.

22.3.6 0.02N ammonium thiocyanate solution.

22.3.7 Ammonium Ferric Sulfate Saturated Solution.

22.3.8 Ammonia d = 0.958.

22.4 Procedure. Weigh, with precision of 5 mg, 20 g of the sample, enter them in a flat-bottom 1 litre flask and add 50 ml of water and 10 ml of suspension of sweet almonds (22.3.1). Cap the flask and keep it for sixteen hours in the stove at 38 ° C. Cool below to room temperature and add 8 ml of water and 10 ml of sodium acetate solution (22.3.2) and one drop of anti-foam emulsion (22.3.3).

Connect the flask to the steam distillation apparatus and place it in an oil bath previously carried at a temperature of slightly more than 100 ° C. Distill from 200 to 300 ml of liquid by passing in the flask a strong steam current and gently heating the oil bath. Collect the distillate in an erlenmeyer located at the back of the light and contain 50 ml exactly of silver nitrate solution 0,02N (22.3.5) and 1 ml of nitric acid (22.3.4). Ensure that the coolant elongation is submerged in the silver nitrate solution.

Transfer the contents of the erlenmeyer to a 500 ml volumetric flask, complete the volume with water, shake and filter. Take 250 ml of the filtrate, add 1 ml approximately of ammonium ferric sulfate solution (22.3.7) and value in recoil the excess of silver nitrate by the ammonium thiocyanate solution 0.02N (22.3.6.) supplied by the graduated burette to 1/20 ml

Make in the case where a blank test is required applying the same operative method to 10 ml of sweet almond suspension (22.3.1), in the absence of the sample to be analysed.

22.5 Calculations. If the blank test indicates a consumption of the silver nitrate solution 0,02N, subtract this value from the volume consumed by the distillate from the sample.

1 ml of silver nitrate 0,02N corresponds to 0,54 of hydrocyanic acid. Express the result as a percentage of the sample.

22.6 Remarks. If the sample contains a significant amount of sulphides (e.g. beans), a black silver sulfide precipitate is formed that is filtered with the silver cyanide sediment. The formation of this precipitate involves a loss of silver nitrate solution 0,02N, the volume of which must be subtracted from the volume taken into account for the calculation of the content of hydrocyanic acid. To this end, proceed as follows:

Treat the sediment over the filter with 50 ml of ammonia (22.3.8) to dissolve the silver cyanide. Wash the residue by diluted ammonia and proceed to the determination of its silver content. Convert the value obtained in ml of silver nitrate solution 0.02N.

The cyanhydride acid content of the sample may also be determined by titration of the ammoniacal filtrate acidified by nitric acid.

22.7 References. First Commission Directive of 15 June 1971 (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July.

23. Determination of the insoluble ashes

in hydrochloric acid

23.1 Principle. The method makes it possible to determine the content of mineral substances insoluble in hydrochloric acid from feed and its raw materials. Two procedures are foreseen according to the nature of the sample.

23.1.1 Procedure A. Applicable to organic raw materials and to most compound feed.

23.1.2 Procedure B. Applicable to mineral mixtures, as well as to compound feed whose content in hydrochloric insoluble, determined according to Procedure A, is greater than 1 per 100.

23.1.3 Procedure A. The sample is incinerated, the ashes are treated by boiling in hydrochloric acid and the insoluble residue is filtered and weighed.

23.1.4 Procedure B. The mixture is treated by hydrochloric acid. The solution is filtered, the residue is incinerated and the ashes obtained are treated as Procedure A.

23.2 Material and appliances.

23.2.1 Calefactor plate.

23.2.2 Electrical, thermostat.

23.2.3 Platinum or platinum and gold-alloy incineration crucibles (10 per 100 Pt, 90 per 100 Au), rectangular (60 x 40 x 25 mm) or round (diameter: 60 to 75 mm, height 20 to 25 mm) or crucibles of other material capable of withstand a temperature of 700 ° C.

23.3 Reassets.

23.3.1 Hydrochloric acid 3N.

23.3.2 Solution to 20 per 100 (w/v) trichloroacetic acid.

23.3.3 Solution to 1 per 100 (w/v) trichloroacetic acid.

23.4 Procedure.

23.4.1 Procedure A. Incinerate the sample according to the official method described for the determination of the gross ashes. The ashes obtained may also be used when making such determination. Transfer the ashes to a glass of 250 to 400 ml with 75 ml of 3N hydrochloric acid (23.3.1). Bring the liquid carefully to a gentle boil and keep it for fifteen minutes. Filter the hot solution onto a filter paper without ashes and wash the residue with hot water until the acid reaction disappears. Dry the filter containing the residue and incinerate in a tarado crucible at a temperature of at least 550 ° C and not more than 700 ° C. Refrigerate in desiccator and weigh.

23.4.2 Procedure B. Pesar, with precision of 1 mg, 5 g of the sample and introduce them into a glass of 250 to 400 ml. Add successively 25 ml of water and 25 ml of 3N hydrochloric acid (23.3.1), mix and wait at the end of the effervescence.

Add 50 ml of 3N hydrochloric acid (23.3.1). If there is a new gas release, wait for its end and then place the glass in a boiling water bath and keep it there for thirty minutes or more, if necessary, in order to completely hydrolyze the starch that can be present. Filter on hot filter without ash and wash the filter by 50 ml of hot water (23.7), place the filter containing the residue in a melting pot, dry and incinerate at a temperature of at least 550 °C and not more than 700 °C. Continue as indicated in the second paragraph of 23.4.1.

23.5 Calculations. Calculate the weight of the residue by deducting the weight of the empty crucible. Express the result as a percentage of the sample.

23.6 Remarks. If the leak is difficult, the determination shall be re-started by replacing the 50 ml of the 3N hydrochloric acid by 50 ml of trichloroacetic acid at 20 per 100 (23.3.2) and by washing the filter with the aid of a hot acid solution. 1 per 100 trichloroacetic acid (23.3.3).

23.7 References. First Commission Directive of 15 June 1971 (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

24. Mustard essence

24.1 Principle. The sample is put into suspension in water. The mustard essences are released under the ferment action, dragged by distillation in the presence of ethanol and collected in the diluted ammonia. The solution is hot with a specific volume of silver nitrate solution, chilled and filtered. The excess silver nitrate is titled by an ammonium thiocyanate solution.

The method allows the determination of the content in essence of mustard-drivable mustard, expressed in allyl isothiocyanate, of the cake of the Brassica and Sinapis species and of the compound feed containing it.

24.2 Material and apparatus.

24.2.1 Half-bottom 500 ml Matres and grinding plug.

24.2.2 Distill apparatus provided with a refrigerant and a device to prevent liquid drag.

24.3 Reassets.

24.3.1 White Mostaza (Sinapis alba).

24.3.2 Ethanol, 95 to 96 per 100 (v/v).

24.3.3 Antifoam Emulsion (silicone, for example).

24.3.4 Ammonia, d = 0.958.

24.3.5 Silver nitrate solution 0.1N.

24.3.6 0.1N ammonium thiocyanate solution.

24.3.7 Nitric acid, d = 1.40.

24.3.8 saturated solution of ammonium ferric sulfate.

24.4 Procedure. Weigh, with precision of 1 mg, 10 g of the sample, enter them in a flask of 500 ml flat bottom and add 2 g of crushed white mustard (ferment source) (24.3.1) and 200 ml of water at 20 ° C. Cap the flask and keep it for two hours at approximately 20 ° C stirring frequently. Add 40 ml of ethanol (24.3.2) and one drop of anti-foam emulsion (24.3.3). Distill approximately 150 ml and collect the distillate in a 250 ml volumetric flask containing 20 ml of ammonia (24.3.4) with attention to the end of the refrigerant being immersed in the liquid. Add 50 ml of the silver nitrate solution 0.1N (24.3.5) to the ammoniacal solution (or more if necessary), place a small funnel on the flask and heat the mixture for an hour in a boiling water bath. Leave to cool, complete the volume with water, shake and filter. Take 100 ml of the limpid filtrate, add 5 ml of nitric acid (24.3.7) and 5 ml approximately of ammonium ferric sulphate solution (24.3.8). Rate the excess of silver nitrate with the ammonium thiocyanate solution 0.1N (24.3.6).

Perform a blank test by applying the same method to 2 g of crushed white mustard finally.

24.5 Calculations. Subtract the volume of silver nitrate solution 0.1N consumed in the blank test consumed by the sample solution. The value obtained gives the number of ml of silver nitrate solution 0.1N consumed by the mustard essence of the sample analysed. 1 ml of silver nitrate 0.1N corresponds to 4,956 mg of allyl isothiocyanate. Express the result as a percentage of the sample.

24.6 References. First Commission Directive of 15 June 1971 (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

25. Lactose

25.1 Principle. The method makes it possible to determine the lactose content of feed containing more than 0,5 per 100 of this product.

Sugars dissolve in the water. The solution is subjected to fermentation by the yeast Saccharomyces cerevisiae, which leaves the lactose intact. After defecation and filtration, the lactose content of the filtrate is determined by the Luff-Schoorl method.

25.2 Material and apparatus. Water bath provided with thermostat, regulated from 38 to 40 ° C.

25.3 Reassets.

25.3.1 Suspension of Saccharomyces cerevisiae. Put 25 g of fresh yeast into suspension in 100 ml of water. The suspension is kept for a maximum of one week in the refrigerator.

25.3.2 Carrez solution I. Dissolve in water 24 g of zinc acetate dihydrate Zn (CHOOO)-2HO and 3 ml of glacial acetic acid. Complete 100 ml with water.

25.3.3 Carrez Solution II. Dissolve in water 10.6 g of potassium hexacianoferde (II) KFe (CN)-3HO. Complete 100 ml with water.

25.3.4 Citric acid solution. Dissolve 50 g of citric acid (CHOHO) in 50 ml of water.

25.3.5 Sodium carbonate solution. Dissolve 143.8 g of anhydrous sodium carbonate in 300 ml of hot water. Leave to cool and make up to 300 ml.

25.3.6 Copper sulfate solution. Dissolve 25 g of copper sulphate pentahydrate (CuSO-5HO), free of iron, in 100 ml of water.

25.3.7 Reactive of Luff-Schoorl. Carefully pour the citric acid solution (25.3.4) on the sodium carbonate solution (25.3.5). Then add the copper sulphate solution (25.3.6) and complete 1 litre with water. Leave to rest one night and filter. Control the normal of the reagent obtained (Cu 0,1 N; NaCO 2N). The pH of the solution should be approximately 9.4.

25.3.8 Grains of pumice stone boiled with hydrochloric acid, washed with water and dried.

25.3.9 Solution to 30 per 100 (w/v) potassium iodide.

25.3.10 sulphuric acid solution 6N.

25.3.11 Sodium thiosulfate solution 0.1N.

25.3.12 Starch solution. Mix 5 g of starch with 30 ml of water and add 1 litre of boiling water. Boil for three minutes, let cool. Add 10 mg of mercury iodide (II) as a preservative.

25.4 Procedure. Weigh, with precision of 1 mg, 1 gram of sample in a 100 ml graduated flask. Add 25 to 30 ml of water. Place the flask for thirty minutes in a boiling water bath and refrigerate up to approximately 35 ° C. Add 5 ml of yeast suspension (25.3.1) and homogenise. Let the flask stand for two hours in a water bath, at the temperature of 38 to 40 ° C. Refrigerate below up to approximately 20 ° C.

Add 2.5 ml of Carrez solution I (25.3.2) and shake for thirty seconds, add 2.5 ml of Carrez II solution and shake, complete with water at 100 ml, mix and filter. Pipette a volume of the filtrate that does not exceed 25 ml and contains between 40 to 80 mg of lactose and to be inserted into a 300 ml erlenmeyer. If it were necessary to complete 25 ml with water.

Perform a blank test with 5 ml of yeast suspension in the same way.

Add 25 ml exactly, from the Luff-Schoorl reagent (25.3.7), two pumice granules (25.2.8). Heat, manually agitating, on a medium-height free flame and bring the liquid to a boil for approximately two minutes, immediately placing the erlenmeyer on a metal fabric provided with a screen of asbestos with a hole of Approximately 6 cm in diameter, under which a flame has been previously lit. This is regulated so that only the bottom of the erlenmeyer is heated. Then adapt a coolant to reflux over the erlenmeyer. From this moment on, boil for ten minutes exactly, refrigerate immediately in cold water and, after approximately five minutes, value as follows:

Add 10 ml of potassium iodide solution (25.3.9) and immediately thereafter and carefully (due to the risk of abundant foam formation) 25 ml 6N sulphuric acid (25.2.10). Then assess the solution of sodium thiosulfate 0.1N (25.3.11) until the appearance of a clear yellow colour, add the indicator (25.3.12) and finish the assessment.

Make the same assessment of a measured mixture of 25 ml of Luff-Schoorl reagent (25.3.7) and 25 ml of water, having added 10 ml of potassium iodide solution (25.3.9) and 25 ml of 6N sulphuric acid. (25.3.10) without boiling.

25.5 Calculations. The amount of lactose in mg corresponding to the difference between the results of the two valuations expressed in ml of sodium thiosulfate 0.1N is determined by the attached table.

Express the results in anhydrous lactose parts per 100 sample.

25.6 Remarks.

25.6.1 For products containing more than 40 percent of fermented sugars, use more than 5 ml of yeast suspension (25.3.1).

25.6.2 Carry out a test with a known quantity of lactose monohydrate.

Values table for 25 ml Luff-Schoorl reagent

Na2S2O30.1N: Ml/Glucose fructose sugars inverted C6H12O6: mg/Difference/Lactose C12H22O11: mg/Difference/Maltose C12H22O11: mg/Difference

1/2.4/2.4/3.6/3.7/3.9/3.9

2/4.8/2.4/7.3/3.7/7.8/3.9

3/7.2/2.5/11.0/3.7/11.7/3.9

4/9.7/2.5/14.7/3.7/15.6/4.0

5/12.2/2.5/18.4/3.7/19.6/3.9

6/14.7/2.5/22.1/3.7/23.5/4.0

7/17.2/2.6/25.8/3.7/27.5/4.0

8/29.8/2.6/29.5/3.7/31.5/4.0

9/22.4/2.6/33.2/3.8/35.5/4.0

10/25.0/2.6/37.0/3.8/39.5/4.0

11/27.6/2.7/40.8/3.8/43.5/4.0

12/30.3/2.7/44.6/3.8/47.5/4.1

13/33.0/2.7/48.4/3.8/51.6/4.1

14/35.7/2.8/52.2/3.8/55.7/4.1

15/38.5/2.8/56.0/3.9/59.8/4.1

16/41.3/2.9/59.9/3.9/63.9/4.1

17/44.2/2.9/63.8/3.9/68.0/4.2

18/47.1/2.9/67.7/4.0/72.2/4.3

19/50.0/3.0/71.7/4.0/76.5/4.4

20/53.0/3.0/75.7/4.1/80.9/4.5

21/56.0/3.1/79.8/4.1/85.4/4.6

22/59.1/3.1/83.9/4.1/90.0/4.6

23/62.2//88.0//94.6/

25.7 References. First Commission Directive of 15 June 1971 (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

26. Potassium

26.1 Principle. The sample is incinerated and the ashes are collected in hydrochloric acid solution. The potassium content of the solution is determined by flame photometry in the presence of caesium chloride and aluminium nitrate. The addition of these substances eliminates, in a wide measure, the interference of disturbing elements.

The method allows for the determination of the content of potassium in feed.

26.2 Material and apparatus.

26.2.1 Platinum, quartz or porcelain incineration Crisols, if any, provided with tapestries.

26.2.2 Electrical, thermostat.

26.2.3 Flame Fotometer.

26.3 Reassets.

26.3.1 Hydrochloric acid p.a.; d = 1.12.

26.3.2 Cesium chloride p.a.

26.3.3 Non- hydrate Nitrate Al (NO)-9HO chemically pure.

26.3.4 Potassium chloride p.a. anhydrous.

26.3.5 Buffer solution. Dissolve in water 50 g of cesium chloride (26.3.2) and 250 g of aluminium nitrate (26.3.3), complete 1 litre with water and homogenise. Store in bottles of plastic material.

26.3.6 Potassium pattern solution. Dissolve in water 1,907 g of potassium chloride (26.3.4), adding 5 ml of hydrochloric acid (26.3.1), complete 1 litre with water and homogenise. Store in bottles of plastic material. 1 ml of this solution contains 1.00 mg of potassium.

26.4 Procedure.

26.4.1 Determination. Weigh to 10 mg precisely, approximately 10 g of the sample in an incineration crucible and incinerate at 450 ° C for three hours. After cooling, quantitatively transfer the incineration residue with the aid of 250 to 300 ml of water, and then with 50 ml of hydrochloric acid (26.3.1) to a 500 ml graduated flask. After the possible release of carbon gas, heat the solution and keep it for two hours at a temperature close to 90 ° C, stirring from time to time. Leave to cool to room temperature, make up, shake and filter. Insert an aliquot portion of the filtrate containing at least 1.0 mg of potassium into a 100 ml volumetric flask, add 10,0 ml of buffer (26.3.5) to make up to the water and homogenise. For higher potassium content, dilute the solution to be analysed in the appropriate proportion, prior to the addition of the buffer solution. The following table is given as an indication for a sampling of approximately 10 g.

Suspected content of the potassium sample Percentage K/dilution factor/aliquot part of the ml solution

Up To 0.1/-/50

0.1 to 0.5/-/10

0.5 to 1.0/-/5

1.0 to 5.0/1:10/10

5.0 to 10.0/1:10/5

10.0 to 20.0/1:20/5

Perform the measurement by flame photometry at the wavelength of 768 nm. Calculate the result by the calibration curve.

26.4.2 Calibration curve. Insert exactly 10 ml of the standard solution (26.3.6) into a 250 ml volumetric flask, make up to scratch with water and homogenise. Exactly: 0, 5, 10, 15, 20 and 25 ml of said solution, corresponding to potassium quantities of 0; 0.2; 0.4; 0.6; 0.8; and 1.0 mg, in 100 ml volumetric flasks. Add 10,0 ml of buffer solution (26.3.5) in each flask to make up to the water and homogenise. Carry out the measures as indicated in 26.4.1. The layout of the calibration curve is generally linear up to a potassium concentration of 1 mg in 100 ml of solution.

26.5 Calculations. The potassium content shall be expressed as a percentage of sample.

26.6 Observations. The addition of buffer solution (26.3.5) to eliminate interference from disruptive elements is not always necessary.

26.7 References. First Commission Directive of 15 June 1971 (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

27. Sodium

27.1 Principle. The sample is incinerated and the ashes are collected in hydrochloric acid solution. The sodium content of the solution is determined by flame photometry in the presence of caesium chloride and aluminium nitrate. The addition of these substances eliminates, to a large extent, the interference of disturbing elements.

The method allows the determination of the sodium content in feed.

27.2 Material and apparatus.

27.2.1 Platinum, quartz or porcelain incineration Crisols, if any, provided with tapestries.

27.2.2 Electrical, thermostat.

27.2.3 Flame Fotometer.

27.3 Reassets.

27.3.1 Hydrochloric acid p.a., d = 1.12.

27.3.2 Cesium chloride, p.a.

27.3.3 Aluminium Nitrate Nonahydrate, Al (NO)-9HO, chemically pure.

27.3.4 Sodium chloride p.a., anhydrous.

27.3.5 Buffer solution. Dissolve in water 50 g of cesium chloride (27.3.2) and 250 g of aluminium nitrate (27.3.3), complete with 1 litre with water and homogenise. Store in bottles of plastic material.

27.3.6 Sodium standard solution. Dissolve in water 2,542 g sodium chloride (27.3.4) by adding 5 ml of hydrochloric acid (27.3.1), complete 1 litre with water and homogenise. Store in bottles of plastic material. 1 ml of this solution contains 1.00 mg of sodium.

27.4 Procedure.

27.4.1 Determination. Weigh, with precision of 10 mg, approximately 10 g of the sample in an incineration crucible (27.2.1.) and incinerate at 450 ° C for three hours. Avoid projection and inflammation. After cooling, transfer the incineration residue quantitatively with the aid of 250 to 300 ml of water, and then with 50 ml of hydrochloric acid (27.3.1) to a 500 ml graduated flask.

After the possible release of carbon gas, heat the solution and keep it for two hours at a temperature close to 90 ° C, stirring from time to time. Leave to cool to room temperature, make up to scratch with water, stir and filter. Introduce into a 100 ml volumetric flask an aliquot of the filtrate, containing at most 1.0 mg of sodium, add 10,0 ml of buffer solution (27.3.5), make up to scratch with water and homogenise. For higher sodium content, dilute the solution to be analysed in the appropriate proportion, prior to the addition of the buffer solution. The following table is given for information purposes, for a sampling of approximately 10 g.

Suspected sample content in solution Percentage Na/dilution factor/aliquot part of solution ml Up to 0.1/-/50

0.1 to 0.5/-/10

0.5 to 1.0/-/5

1.0 to 5.0/1:10/10

5.0 to 10.0/1:10/5

10.0 to 20.0/1:20/5

Make the measurement using flame photometry at a wavelength of 589 nm.

Calculate the result by the calibration curve.

27.4.2 Calibration curve. Introduce exactly 10 ml of the standard solution (27.3.6) into a 250 ml volumetric flask, make up to scratch with water and homogenise. In 100 ml volumetric flasks, enter exactly 0, 5, 10, 15, 20 and 25 ml of the solution corresponding to amounts of sodium of 0; 0.2; 0.4; 0.6; 0.8; and 1.0 mg. Add 10,0 ml of buffer solution (27.3.5) in each flask to make up to the water and homogenise. Carry out the measures as indicated in 27.4.1. The layout of the calibration curve is generally linear up to a sodium concentration of 1 mg in 100 ml of solution.

27.5 Calculations. The sodium content shall be expressed as a percentage of sample.

27.6 Remarks.

27.6.1 For products whose sodium content is greater than 4 per 100 it is preferable to incinerate the substance for two hours in a crucible provided with a cover. After cooling, add water, put a residue in suspension with the aid of a platinum thread, dry and incinerate again for two hours in the crucible provided with its cover.

27.6.2 If the sample consists solely of mineral materials, proceed to dissolution without prior incineration.

27.7 References. First Commission Directive of 15 June 1971 (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

28. Magnesium

28.1 Principle. Incineration of the sample and treatment of ashes with dilute hydrochloric acid. If the sample does not contain organic substances, it dissolves directly into hydrochloric acid. The solution is diluted and the magnesium content is determined by atomic absorption spectrophotometry at 285.2 nm, by comparison with the calibration solutions.

It is especially suitable for determining the contents of magnesium below 5 per 100 in the

feed.

28.2 Material and apparatus.

28.2.1 Platinum, quartz or porcelain incineration Crisols.

28.2.2 Horno or mufla in thermostat.

28.3 Reassets.

28.3.1 Hydrochloric acid d = 1,16, p.a.

28.3.2 Hydrochloric acid from d = 1.19, p.a.

28.3.3 Magnesium, in tape or yarn, or magnesium sulfate heptahydrate, p.a. (Mg SO7HO) dry drying at room temperature.

28.3.4 Strontium salt solution (chloride or nitrate) p.a. to 2,5 per 100 (w/v) strontium, p.a. (76.08 g of ClSr 6 HO/litre, or 60.38 g of (NO) Sr/litre).

28.3.5 Magnesium calibration solution: weigh with precision of 1 mg, 1 g of magnesium (28.3.3), previously and carefully separating the oxide film, or a corresponding amount of magnesium sulfate (28.3.3). Introduce it into a 1000 ml flask add 80 ml of ClH (28.3.1) dissolve and complete 1000 ml with deionised HO. 1 ml of this solution contains 1,000 mg of magnesium.

28.4 Procedure.

28.4.1 Sample Preparation.

28.4.1.1 Pires made exclusively of mineral substances:

a) Pesar with precision of 1 mg, 5 g of the sample and introduce them into a 500 ml graduated flask with 250-300 ml of deionised water. Add 40 ml of hydrochloric acid (28.3.1), bring to a slow boil for thirty minutes. Cool, make up with deionised water, homogenise and filter in beaker with fold filter. Remove the first 30 ml of the filtrate.

b) In the presence of silica, treat 5 g of the sample with a sufficient quantity (15 to 30 ml) of hydrochloric acid (28.3.2) and evaporate to dryness over a water bath. To continue as indicated in (28.4.1.2) from the third stage.

28.4.1.2 Piuses consisting essentially of mineral substances. Weigh precisely 1 mg, 5 g of the sample in a crucible and incinerate at 550 ° C in the muffle oven until the ashes are obtained free of carbonaceous particles.

To remove the silica add a sufficient quantity (15 to 30 ml) of hydrochloric acid (28.3.2) to the ashes and evaporate to dryness over a water bath. Dry then one hour in the stove at 105 ° C. Collect the residue with 10 ml of hydrochloric acid (28.3.1) and transfer with the aid of hot deionised water to a 500 ml graduated flask. Bring to the boil, leave to cool and make up with deionized water. Homogenize and filter in glass through a fold filter. Remove the first 30 ml of the filtrate.

28.4.1.3 Piuses consisting essentially of organic substances. Weigh precisely 1 mg, 5 g of the sample in crucible (28.2.1) and incinerate in a muffle oven until the extraction of ash free from carbonaceous particles. Treat the ashes with 5 ml of hydrochloric acid (28.3.2), evaporate dry in water bath and then dry for an hour in stove at 105 ° C, to insolubilize the silica.

Collect the residue with 5 ml of hydrochloric acid (28.3.1), transfer with the aid of hot deionised water to a 250 ml graduated flask. Bring to the boil, leave to cool and make up with deionized water. Homogenize and filter in a glass through a fold filter. Remove the first 30 ml of the filtrate.

28.4.2 Measure of atomic absorption.

28.4.2.1 Calibration curve. Prepare, diluting the calibration solution (28.3.5) with deionized water, at least five solutions of increasing concentrations, chosen according to the optimal measurement zone of the spectrophotometer. Add to each solution 10 ml strontium salt solution (28.3.4) and complete the volume of 100 ml with deionised water.

28.4.2.2 Sample Preparation. Dilute with deionised water an aliquot portion of the filtrate obtained in 28.4.1.1, 28.4.1.2 and 28.4.1.3 so as to obtain a magnesium concentration within the concentration limits of the reference solutions. The hydrochloric acid concentration of that solution should not exceed 0,4N. Add 10 ml of the strontium salt solution (28.3.4) and complete the volume of 100 ml with distilled water.

Measure the absorption of the problem solution and that of the reference solutions at a wavelength of 285.2 nm.

28.5 Calculations. Calculate the amount of magnesium in the sample from the reference solutions. Express the result as a percentage of the sample.

28.6 Remarks. The difference between the results of two parallel determinations carried out on the same sample must not exceed 5 per 100 in relative value.

28.7 References. Fourth Commission Directive of 5 December 1972 (73/46/EEC). 'Official Journal of the European Communities' number L 83 of 30 March 1973.

29. Iron

29.1 Principle. Determination of the iron oligoelement in feed and its raw materials. The lower limit of determination is 20 mg/kg. The sample is dissolved in hydrochloric acid solution, after destruction of the organic matter. Iron is determined, after appropriate dilution, by atomic absorption spectrophotometry.

29.2 Material and apparatus.

29.2.1 Mifla, adjustable temperature.

29.2.2 Resistant borosilicate glass material. It will be recommended to use a material that serves exclusively for the dosage of the trace element.

29.2.3 Platinum and, eventually, quartz capsules.

29.2.4 Atomic absorption spectrophotometer, which responds to the requirements of the method, with regard to the sensitivity and precision required within the limits of utility used.

29.3 Reassets.

29.3.1 Hydrochloric acid p.a., d = 1.19.

29.3.2 Hydrochloric acid 6N.

29.3.3 Hydrochloric acid 0.5N.

29.3.4 Hydrochloric acid from 38-40 per 100 (v/v), with an iron content of less than 1 mg/l and whose evaporation residue is less than 10 mg/l (expressed in sulphates).

29.3.5 sulphuric acid p.a. (d = 1.84).

29.3.6 oxygenated water of about 100 volumes of oxygen (30 per 100 in weight).

29.3.7 Iron pattern solution (1,000 micrograms iron/ml): Dissolve 1 g of iron in p.a. threads, in 200 ml 6N hydrochloric acid (29.3.2) add 16 ml of oxygenated water (29.3.6) and complete with water up to 1 litre.

29.3.8 Working pattern solution (100 micrograms iron/ml): Take 10 ml of the standard solution (29.3.7) and take 100 ml with water.

29.3.9 lanthanum chloride solution: Dissolve 12 g of lanthanum oxide in 150 ml of water, add 100 ml of 6N hydrochloric acid (29.3.2) and complete with water up to 1 litre.

29.4 Procedure.

29.4.1 Sample containing organic compounds.

29.4.1.1 Incineration and preparation of the sample to be analyzed.

i) Place 5 to 10 g of the heavy sample with a precision of 0.2 mg, in a quartz or platinum capsule (29.2.3) (see 29.6.6), dry on a stove at 105 ° C and enter the capsule in the cold mufla oven (29.2.1). Close the oven (see 29.6.7) and progressively raise the temperature to reach 450 to 475 ° C in approximately ninety minutes. Maintain this temperature for four to six hours (e.g. overnight) to remove the carbonaceous matter, then open the oven and allow to cool (see 29.6.8).

Moisten the ashes with water, transfer them to a 250 ml beaker. Rinse the capsule with 5 ml of hydrochloric acid (29.3.1) and transfer, slowly and with caution, the rinse solution to the beaker (a violent reaction may occur by carbon dioxide formation). Add then drop to drop hydrochloric acid (29.3.1), while stirring the contents of the beaker, until the effervescence ceases. Evaporate dry by periodically stirring with a glass rod.

Add to the residue 15 ml of 6N hydrochloric acid (29.3.2) and about 120 ml of water. Mix with the aid of a glass rod, leave the same in the beaker and cover with a watch glass. Put the liquid in a gentle boil and keep the boil until it apparently no longer dissolves the ashes. Filter using an ash-free filter paper and collect the filtrate in a 250 ml volumetric flask. Wash the beaker and filter with 5 ml of 6N hydrochloric acid (29.3.2.) hot and twice with boiling water. Complete with water up to the level (the concentration in hydrochloric acid will be approximately 0.5N).

ii) If the residue remaining in the filter appears black (carbonaceous), place it back in the oven and incinerate from 450 to 475 ° C. This incineration, which will require only a few hours (approximately three to five hours), will be completed when the ashes appear white or almost white. Dissolve the residue in about 2 ml of hydrochloric acid (29.3.1). Evaporate to dryness and add 5 ml of 6N hydrochloric acid (29.3.2). Heat, filter the solution in the volumetric flask and complete with water (the concentration in hydrochloric acid will be approximately 0.5N).

29.4.1.2 Preparation of the standard solutions. Prepare a range of standard solutions from the working standard solution (29.3.8) so that each standard solution has a concentration of approximately 0,5N hydrochloric acid and a concentration of lanthanum chloride corresponding to 0.1 per 100 of lanthanum (w/v) (29.3.9). The chosen concentrations of the trace element must be in the sensitivity zone of the spectrophotometer used. The following table shall, by way of example, give the types of standard solution composition; depending on the type and sensitivity of the spectrophotometer used, it may be necessary to choose other concentrations:

Iron

xg of Fe/ml/0/0.5/1/2/3/4/5

ml of working pattern solution (29.3.8). /0/0.5/1/2/3/4/5

(1 ml = 100 xg Fe) + ml ClH 6N (29.3.2). /7/7/7/7/7/7/7

+ 10 ml of lanthanum chloride solution (29.3.9)

Complete with water up to 100 ml

If the sample had a weight ratio

Ca + Mg

------ T 2, the addition of the P solution can be omitted

of lanthanum chloride (29.3.9).

29.4.1.2.2 Preparing the solution to be analyzed. Pipette an aliquot of the prepared solution according to (29.4.1.1) into a 100 ml graduated flask; add 10 ml of lanthanum chloride solution (29.3.9). Complete with 0.5N hydrochloric acid (29.3.3).

If the sample had a weight ratio

Ca + Mg

------ T 2, the addition of the P solution can be omitted

of lanthanum chloride (29.3.9).

29.4.1.2.3 Test blank. Perform a blank test comprising all the prescribed stages of the operating procedure, without the presence of the sample. The standard solution 0 must not be used for the blank test.

29.4.1.2.4 Measure by atomic absorption. Measure the absorbance of the standard solutions and the solution to be analyzed, using an oxidant flame of air-acetylene at the wavelength of: 248.3 nm. Perform four times each measure.

29.4.2 Mineral compounds. In the absence of organic matter, prior incineration will be useless. Implement the operational procedure from point (29.4.1.1.i), second paragraph (see 29.6.6).

29.5 Calculations. Calculate the concentration of the trace element in the solution to be analysed by a calibration curve and express the result in mg of trace element per kg of sample (ppm).

The difference between the results of two parallel determinations performed on the same sample by the same analyst must not exceed:

-5 mg/kg in absolute value, for the content of the trace element up to 50 mg/kg.

-10 per 100 of the highest result for contents above 50 and up to 100 mg/kg.

-10 mg/kg, in absolute value, for contents greater than 100 and up to 200 mg/kg.

-5 per 100 of the highest result for contents greater than 200 mg/kg.

29.6 Remarks.

29.6.1 Water used for the preparation of reagents and the solutions required during the analysis must be exempt from the cation to be determined. It shall be obtained either by double distillation in a borosilicate or quartz apparatus or by double permutation in ion exchange resin.

29.6.2 The reagents must be at least the quality for analysis (p.a.). The absence of the element to be determined shall be controlled by a blank test. If necessary, the reagents will be subjected to a deeper purification.

29.6.3 The standard solutions described by commercial standard solutions may be replaced with such solutions that they are guaranteed and controlled prior to their use.

29.6.4 Green fodder (fresh or dehydrated) may contain large quantities of plant silica which can be retained by the trace element and which must be removed. The samples of such feed shall be subjected to the following treatment: carry out the operation (29.4.1.1) until the filtration phase. Wash the filter paper, which contains the insoluble residue, twice with boiling water and place it in a platinum capsule (29.2.3). Incinerate in the mufla furnace (29.2.1) at a temperature below 550 ° C until all the carbonaceous material has completely disappeared. Allow to cool, add some drops of water and 10 to 15 ml of hydrofluoric acid (29.3.4). Evaporate to dry at approximately 150 ° C. If the residue contains still silica, dissolve it in some ml of hydrofluoric acid (29.3.4) and evaporate to dryness. Add five drops of sulphuric acid (29.3.5) and heat up to the disappearance of the white fumes. Add 5 ml of 6N hydrochloric acid (29.3.2) and approximately 30 ml of water, heat, filter the solution in a 250 ml volumetric flask and complete up to scratch (the hydrochloric acid concentration will be approximately 0.5N). Continue the procedure from the point of view (29.4.1.2).

29.6.5 In determining the trace element it will be appropriate to draw attention to the risks of contamination. Therefore, the instruments used for the preparation of the samples must be free of such metal.

To reduce pollution risks, work on dust-free atmospheres, with rigorously clean material and carefully washed glass appliances.

29.6.6 Calculate the weight of the sample based on the approximate content of the trace element to be assessed and the sensitivity of the spectrophotometer used. For certain poor feed in the trace element it may be necessary to extract a sample of 10 to 20 g and limit the volume of the final solution to 100 ml.

29.6.7 Incinerate in a closed furnace without injection of air or oxygen.

29.6.8 The temperature must not exceed 475 ºC.

29.7 References. Eighth Commission Directive of 15 June 1978 (78/633/EEC). 'Official Journal of the European Communities' number L 206 of 29 July 1978.

30. Copper

30.1 Principle. Determination of the copper element in feed and raw materials. The lower limit of determination is 10 mg/kg. The sample is dissolved in hydrochloric acid solution, after destruction of the organic matter. Copper is determined, after appropriate dilution, by atomic absorption spectrometry.

30.2 Material and apparatus. Idem 29.2.

30.3 Reassets. Idem 29.3. up to 29.3.6. inclusive.

30.3.7 Copper standard solution (100 micrograms copper/ml). Dissolve 1 g of copper powder p.a. in 25 ml of 6N hydrochloric acid (29.3.2), add 5 ml of oxygenated water (29.3.6) and complete with water up to 1 l.

30.3.8 Work pattern solution (10 micrograms copper/ml). Take 10 ml of the standard solution (30.3.7) and bring to 1,000 ml with water.

30.4 Procedure. Idem 29.4. up to 29.4.1.2.

30.4.1.2 Spectrophotometric determination.

30.4.1.2.1 Preparation of the standard solutions from the worksolution (30.3.8) so that each standard solution has a hydrochloric acid concentration of about 0.5N. The chosen concentrations of the trace element must be in the sensitivity zone of the spectrophotometer used.

The following table shall, for information purposes, give the types of composition of the standard solution; depending on the type and sensitivity of the spectrophotometer used, it may be necessary to choose other concentrations:

Copper

xg Cu/ml/0/0.1/0.2/0.4/0.6/0.8/1.0

ml of working pattern solution (30.3.8). /0/1/2/4/6/8/10

(1 ml = 100 xg Cu) + ml ClH 6N (29.3.2). /8/8/8/8/8/8/8

Complete with water up to 100 ml

30.4.1.2.2 Preparing the solution to be analyzed. The solution prepared according to (29.4.1.1) may, as a general rule, be used directly. If it is necessary to bring its concentration to the range of the concentrations of the standard solutions, an aliquot may be introduced with pipette into a 100 ml graduated flask and complete with 0,5N hydrochloric acid (29.3.3) up to the level.

30.4.1.2.3 Test blank. Idem 29.4.1.2.3.

30.4.1.2.4 Measure by atomic absorption.

Measure the absorbance of the standard solutions and the solution to be analyzed, using an oxidant flame of air-acetylene, with a wavelength of 324.8 nm. Perform four times each measure.

30.4.2 Mineral compounds. Idem 29.4.2.

30.5 Calculations. Idem 29.5.

30.6 Observations. Idem 29.6.

30.7 References. Idem 29.

31. Manganese

31.1 Principle. Determination of the manganese oligoelement in feed and its raw materials. The lower limit of determination shall be 20 mg/kg. The sample is dissolved in hydrochloric acid solution, after destruction of the organic matter. Manganese is determined, after appropriate dilution, by atomic absorption spectrophotometry.

31.2 Material and apparatus. Idem 29.2.

31.3 Reassets. Idem 29.3. up to 29.3.6. inclusive.

31.3.7 manganese standard solution (1,000 micrograms Mn/ml): dissolve 1 g of manganese powder p.a. in 25 ml of 6N hydrochloric acid (29.3.2) and complete with water up to 1 litre.

31.3.8 Work pattern solution (10 micrograms Nm/ml): take 10 ml of the standard solution (31.3.7) and take 1,000 ml with water.

31.3.9 Idem 29.3.9.

31.4 Procedure. Idem 29.4 up to 29.4.1.2.3, including, with the exception of the table of the composition types of the standard solution.

Manganese

xG Mn/ml/0/0.1/0.2/0.4/0.6/0.8/1.0

ml of working pattern solution (31.3.8). /0/1/2/4/8/8/10

(1 ml = 10 xg Mn) + ml ClH 6N( 29.3.2). /7/7/7/7/7/7/7

+ 10 ml of lanthanum chloride solution (29.3.9).

Complete with water up to 100 ml

31.4.1 Measure by atomic absorption. Measure the absorbance of the standard solutions and the solution to be analyzed using an air-acetylene oxidant flame with a wavelength of 279.5 nm. Perform four times each measure.

31.4.2 Idem 29.4.2.

31.5 Calculations. Idem 29.5.

31.6 Remarks. Idem 29.6.

31.7 References. Idem 29.7.

32. Zinc

32.1 Principle. Determination of the zinc trace element in feed and its raw materials. The lower limit of determination shall be 20 mg/kg. The sample is dissolved in hydrochloric acid solution, after destruction of the organic matter. Zinc is determined, after appropriate dilution, by atomic absorption spectrophotometry.

32.2 Material and apparatus. Idem 29.2.

32.3 Reassets. Idem 29.3 up to 29.3.6 inclusive.

32.3.7 Zinc-standard solution (100 micrograms Zn/ml): dissolve 1 g of zinc in tape or plaque, p.a., in 25 ml of 6N hydrochloric acid (29.3.2) and complete with water up to 1 litre.

32.3.8 Work pattern solution (10 micrograms Zn/ml): take 10 ml of the standard solution (32.3.7) and take 100 ml with water.

32.3.9 Idem 29.3.9.

32.4 Procedure. Idem 29.4 up to 29.4.1.2.3, including, with the exception of the table of the composition types of the standard solution.

Cinc

xG Zn/ml/0/0.05/0.1/0.2/0.4/0.6/0.8

ml of working pattern solution (32.3.8). /0/0.05/1/2/4/6/8

(1 ml = 10 xg Zn) + ml ClH 6N( 29.3.2). /7/7/7/7/7/7/7

+ 10 ml of lanthanum chloride solution (29.3.9). Complete with water up to 100 ml

32.4.1 Measure by atomic absorption. Measure the absorbance of the standard solutions and the solution to be analyzed using an air-acetylene oxidant flame with a wavelength of 213.8 nm. Perform four times each measure.

32.4.2 Idem 29.4.2.

32.5 Calculations. Idem 29.5.

32.6 Remarks. Idem 29.6.

In addition, account (29.6.5) of the determination of zinc is particularly sensitive to contamination from glass appliances, reagents, dust and other disturbing elements.

32.7 References. Idem 29.7.

33. Moisture from animal fats and oils

and vegetables

33.1 Principle. The sample is subjected to drying at 103 ° C to constant weight.

This method enables the determination of moisture content (water and other volatile materials) of animal and vegetable fats and oils.

33.2 Material and apparatus.

33.2.1 Flat-bottom, corrosion-resistant material with diameter of 8-9 cm and height of approximately 3 cm.

33.2.2 Mercury thermometer, reinforced bulb and dilation chamber at the upper end, graduated from 80 to 110 ° C at least and approximately 10 cm in length.

33.2.3 Bath of sand or electric heating plate.

33.2.4 Desecator containing an effective dehydrator.

33.2.5 Analytical Balanza.

33.3 Procedure. Weigh, with precision of 1 mg, 20 g approximately of the homogenized sample, in the container (33.2.1), dry and tarado, containing the thermometer (33.2). Heat on sand bath or heating plate (33.2.3.), constantly stirring with the aid of the thermometer, so that the temperature reaches 90 ° C in about seven minutes.

Raise the temperature slowly, without exceeding 105 ° C, agitating with the thermometer until the cessation of bubbles.

To ensure complete removal of moisture, repeat several times the heating at 103 ° C ± 2 ° C, then cool in desiccator (33.2.4) to room temperature and weigh. Repeat this operation until the difference between two successive weightings does not exceed more than 2 mg.

33.4 Calculations. The moisture content of the sample, as a percentage, is given by the formula:

(P-P) x 100

(P-P) x 100

(P-P) x P

Being:

P = Weight, in grams, of the sample.

P = Weight, in grams, of the container with its contents, before heating.

P = Weight, in grams, of the container with its contents, after heating.

Results below 0.05 per 100 should be expressed as less than 0.05 per 100.

33.5 Remarks.

33.5.1 An increase in the weight of the sample after repeated heating indicates an oxidation of the fat. In this case, make the calculation from the heavy one made immediately before this weight increase appears.

33.5.2 The difference between the results of two parallel determinations carried out on the same sample must not exceed 0,05 per 100 in absolute value.

33.6 References. Fourth Commission Directive of 5 December 1972. (73/46/EEC). 'Official Journal of the European Communities' number L 83 of 30 March 1973.

34 (a). Starch (polarimetric method)

34 (a) .1 Principle. The method comprises a double determination. In the first, the sample is treated as hot by diluted hydrochloric acid. After defecation and filtration, the rotating power of the solution shall be measured by a polarimeter.

In the second, the sample is extracted by ethanol at 40 per 100. After acidification of the filtrate by hydrochloric acid, defecation and filtration, the rotating power is measured in the same conditions as in the first determination.

The difference between the two multiplied by a common factor results in the starch content of the sample.

The method makes it possible to determine the starch content and its high molecular weight degradation products in feed, except for those containing peelings, pulps, leaves or dried beet necks, potato pulp, Dehydrated yeast, products rich in inulin (e.g., peelings and chufas flour) or chicharrones.

34 (a) .2 Material and apparatus.

34 (a) .2.1 Erlenmeyer 250 ml of frosted mouth, with reflux coolant.

34 (a) .2.2 Polarimeter or saccharimeter.

34 (a) .3 Reassets.

34 (a) .3.1 Hydrochloric acid at 25 per 100 (w/w), d = 1,126.

34 (a) .3.2 Hydrochloric acid at 1,128 per 100 (w/v). The concentration should be verified by titration with the aid of a 0.1N sodium hydroxide solution in the presence of methyl red at 0.1 per 100 (w/v) in ethanol at 94 per 100 (v/v) (10 ml = 30.94 ml NaOH 0.1N).

34 (a) .3.3 Carrez solution I: Dissolve in water 21.9 g of zinc acetate dihydrate (Zn (CH-COO). 2HO) and 3 g of glacial acetic acid. Complete 100 ml with water.

34 (a) .3.4 Carrez solution II: Dissolve in water 10.6 g of potassium ferrocyanide trihydrate (KFe (CN). 3HO). Complete with water.

34 (a) .3.5. Ethanol at 40 per 100 (v/v), d = 0.948 at 20 ° C.

34 (a) .4 Procedure.

34 (a) .4.1 Sample preparation. Grind the sample so that it passes in its entirety through a mesh sieve of 0.5 mm.

34 (a) .4.2 Determination of total rotating power (P or S) (v. observations (34. (a) .6.1).

weigh, with precision of 1 mg, 2.5 g of the ground sample and enter into a 100 ml graduated flask. Add 25 ml of hydrochloric acid (34 (a) .3.2), shake to obtain a good distribution of the sample and add 25 ml of hydrochloric acid again (34 (a) .3.2). Submerge the flask into a boiling water bath and, during the first three minutes, shake vigorously and regularly to prevent the formation of agglomerates. The amount of water in the bath must be sufficient to allow the boiling bath to be maintained when the flask is submerged in the bath. This will not be able to be removed from the bathroom throughout the agitation. After fifteen minutes exactly, remove the flask from the bath, add 30 ml of cold water and cool immediately to 20 ° C.

Add 5 ml of Carrez solution I (34 (a) .3.3) and shake for one minute. Add 5 ml of Carrez II solution (34 (a) .3.4) and shake again for one minute. Complete the volume with water, homogenize and filter. If the filtering is not perfectly clear (which is rare), the analysis should be started again using a larger quantity of the solutions of Carrez I and II, for example 10 ml.

Measure the rotating power of the solution in a 200 mm tube by means of a polarimeter or a saccharimeter.

34 (a) .4.3 Determination of the rotating power (P ' or S') of the soluble substances in ethanol at 40 per 100.

weigh, with precision of 1 mg, 5 g of the sample, into a 100 ml graduated flask and add approximately 80 ml of ethanol (34 (a) .3.5) (v. observations 34 (a) .6.2). Leave the flask at rest for one hour at room temperature, over this period of time, proceed five times to energetic agitation so that the sampling is well mixed with the ethanol. Then carry to volume with ethanol (34 (a) .3.5), homogenize and filter.

Introduce by pipette 50 ml of the filtrate (=2.5 g of the sample) into a 250 ml erlenmeyer. Add 2.1 ml. Add 2.1 ml of hydrochloric acid (34 (a) .3.1) and shake vigorously. Adjust a coolant to reflux to the erlenmeyer and dip it into a boiling water bath. After fifteen minutes exactly, remove the erlenmeyer from the bath, transfer its contents to a 100 ml graduated flask washing with a little cold water and cool to 20 ° C. Defecate below with the help of the solutions of Carrez I (34 (a) .3.3) and Carrez II (34 (a) .3.4), complete with water volume, homogenize filter and measure the rotating power as indicated in 34 (a) .4.2, second and third paragraphs.

34 (a) .5 Calculations. The starch content in sample percentage is obtained by the following formulas:

34 (a) .5.1 Measurements made with polarimeter.

% starch = 2000 (P-P ')

% starch = 2000 (P-P ')

% starch = (u)

Being:

P = Total rotating power in arc degrees.

P ' = Rotating power in arc degrees for ethanol-soluble substances at 40 per 100.

(u) = Specific rotating power of pure starch. The supported conventional values for that factor are as follows:

+ 185.9º: Rice starch.

+ 185.4º: potato starch.

+ 184.6th: maize starch.

+ 182.7th: wheat starch.

+ 181.5º: barley starch.

+ 181.3º: Oat starch.

+ 184.0º: other types of starch, as well as mixtures of starches of compound feed.

34 (a) .5.2 Measurements made by means of saccharimeter.

% starch = 2000-(2N x 0.665) (S-S') =

% starch = 2000-2N x 0.665) (S-S') =

% starch = (u)-100 =

= 26.6N (-S')

= 26.6N (-S')

= (u)

Being:

S = Total rotating power in saccharimeters degrees.

S' = rotating power in saccharimetric degrees given by the soluble substances in ethanol at 40 per 100.

N = Weight, in g, of sucrose in 100 ml of water giving, under a thickness of 200 mm, a rotating power of 100º saccharimetric.

16.29 for French saccharimeters.

26.00 for German saccharimeters.

20.00 for mixed saccharimeters.

(u) = specific rotary power of pure starch (34 (a) .5.1).

The difference between the results of two parallel determinations carried out on the same sample must not exceed 0,4 in absolute value for the starch content of less than 40 per 100; 1,1 per 100 in relative value for starch content equal to or greater than 40 per 100.

34 (a) .6 Remarks.

34 (a) .6.1 When the sample contains more than 6 per 100 carbonates calculated in calcium carbonate, these must be destroyed by a treatment with the aid of an exact amount of diluted sulphuric acid, before the determination of the total rotating power.

34 (a) .6.2 In the case of products of high lactose content, such as whey powder or skim milk powder, proceed as follows after the addition of 80 ml of ethanol (34 (a) .3.5). Adjust the flask to a reflux coolant, submerge the flask for thirty minutes in a water bath at 50 ° C. Leave below to cool and continue with the analysis as indicated in (34 (a) .3.5).

34 (a) .7 References. Third Commission Directive of 27 April 1972. (72/199/EEC). 'Official Journal of the European Communities' number L 123 of 29 May 1972.

34 (b). Starch (Pancreatic method)

34 (b) .1 Principle. The sugars present in the sample are eliminated by extraction with ethanol. The starch from the extraction residue is saccharified by the pancreas. The sugars are hydrolysed by hydrochloric acid and the glucose formed is valued by the Luff-Schoorl method. The amount of glucose obtained multiplied by a constant factor gives the starch content of the sample.

The method makes it possible to determine the starch content and products of its high molecular weight degradation in foods containing pellets, pulp, leaves or dried beet necks, potato pulp, yeast dehydrated, products rich in inulin (e.g. fractions and flour or potatoes). The assessment should be performed only when the microscopic examination indicates the presence in the sample of appreciable amounts of starch.

34 (b) .2 Material and apparatus.

34 (b) .2.1 Extractor (see figure 34 (b) .1) consisting of:

34 (b) .2.1.1 Long-necked 500ml Matraz erlenmeyer.

34 (b) .2.1.2 Reflow coolant adapted to the erlenmeyer flask.

34 (b) .2.1.3 Sliding variant located in the central tube of the coolant provided with a hook on its lower end and a clamp for fixing the rod.

34 (b) .2.1.4 Metal Cestillo, intended to be suspended from the hook of the rod (34 (b) .2.1.3) and to support the filter crucible (34 (b) .2.1.5).

34 (b) .2.1.5 Filter plate crucible, maximum pore dimension: 90-150 micrometers (e.g., G1), approximately 30 ml capacity.

34 (b) .2.1.6 Format paper filters suitable for filter crucible (34 (b) .2.1.5).

34 (b) .2.2 Incubation study regulated at 38 ° C.

34 (b) .2.3 200 ml graduated matrices with normalised frosted mouth and reflux coolant.

34 (b) .2.4 100 ml graduated matrices with normalised frosted mouth and reflux refrigerant.

34 (b) .3 Reassets.

34 (b) .3.1 Ethanol 90 per 100 (V/V) neutral to phenolphthalein.

34 (b) .3.2 Alcohol n-amyl p.a.

34 (b) .3.3 Toluene p.a.

34 (b) .3.4 buffer solution. Dissolve in water 9,078 g of monopotassium phosphate KPOH and 11,876 g of disodium phosphate dihydrate NaPOH. 2HO. Complete one litre with water.

34 (b) .3.5 Sodium chloride solution: 0.2N.

34 (b) .3.6 Carrez solution I. Dissolve in water 21.9 g zinc acetate dihydrate Zn (CHCOO). 2HO and 3 g of glacial acetic acid. Complete 100 ml with water.

34 (b) .3.7 Carrez solution II. Dissolve in water 10.6 g of potassium ferrocyanide trihydrate K4 (Fe (CN)). 3HO. Complete 100 ml with water.

34 (b) .3.8 Hydrochloric acid 1N.

34 (b) .3.9 Hydrochloric acid p.a. approximately 8N (d = 1,125).

34 (b) .3.10 Sodium hydroxide solution p.a. approximately 10N (d = 1.33).

34 (b) .3.11. Indicator. Methyl orange solution at 0.1 per 100 (P/V).

34 (b) .3.12 Pancreatine pulverulent in response to the requirements given in (34 (b) .6.3). Store in closed containers, protected from light and moisture.

34 (b) .3.13 Reactive of Luff-Schoorl. Carefully agitating the citric acid solution (34 (b) .3.13.2) in the sodium carbonate solution (34 (b) .3.13.3). Then add the copper sulphate solution (34 (b) .3.13.1) and complete up to one litre with water. Leave to rest one night and filter. Control the normality of the reagent thus obtained (0.1N Cu, 2N NaCO). The pH shall be approximately 9.4.

34 (b) .3.13.1 Copper sulfate solution. Dissolve 25 g of copper sulphate pentahydrate p.a. CuSO. 5HO in 100 ml of water.

34 (b) .3.13.2 Citric acid solution. Dissolve 50 g of citric acid monohydrate p.a. CHO. HO in 50 ml of water.

34 (b) .3.13.3 Sodium carbonate solution. Dissolve 143.8 g of sodium carbonate anhydrous p.a. in 300 ml of hot water. Leave to cool.

34 (b) .3.14 pumice granules boiled with hydrochloric acid washed with water and dried.

34 (b) .3.15 Solution at 30 per 100 (w/v) potassium iodide p.a.

34 (b) .3.16 sulphuric acid approximately 6N (d = 1.18).

34 (b) .3.17 Sodium thiosulfate solution 0.1N.

34 (b) .3.18 Starch solution. Add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes and then leave to cool. Prepare immediately before your employment.

34 (b) .4 Procedure.

34 (b) .4.1 Sample preparation. Grind the sample so that it passes totally through a mesh sieve of 0.5 mm.

34 (b) .4.2 Extraction. Weigh, with the accuracy of 1 mg, 2 g of sample and introduce them into a filter crucible (34 (b) .2.1.5), the bottom of which has been previously coated with filter paper (34 (b) .2.1.6) wetted with ethanol ((34 (b) .3.1). Introduce in the flask erlenmeyer (34 (b) .2.1.1)) 55 ml of ethanol (35 (b) .3.1) and some pumice granules (34 (b) .3.14). Place the filter crucible on the metal stle (34 (b) .2.1.4) and suspend the rod hook (34 (b) .2.1.3). Place the coolant on the flask erlenmeyer and lower the rod so that the crucible bottom protrudes from the surface of the ethanol. Fix the rod at this height with the help of the clamp. Bring the ethanol to the boil and keep it for three hours. Leave to cool below and raise the rod (34 (b) .2.1.3) so as to raise the crucible as high as possible in the erlenmeyer flask. Watch it carefully and drop 45 ml of water along the wall of the erlenmeyer. Place the coolant again on the flask and keep the crucible filter at 10 cm from the liquid level. Bring this liquid to a boil and keep it three hours. Cool immediately, open the flask and remove the crucible from the cestillo.

34 (b) .4.3 Sacarification or hydrolysis. Place the filter crucible in an empty container and dry by aspiration. Deposit the extraction residue in a mortar and finely crush. Quantitatively transfer the powder with the aid of 60 ml of water approximately to a standard 200 ml graduated round flask and add some drops of amyl alcohol (34 (b) .3.2). Adjust a reflux coolant to the container. Heat to the boil and keep that for an hour. Then cool and disconnect the coolant. Add 25 ml of buffer solution (34 (b) .3.4) and 250 mg of pancreatitis (34 (b) .3.12) 2.5 ml of sodium chloride solution (34 (b) .3.5) of 10 drops of toluene. Shake for two minutes, place the container in the hatchery (34 (b) .2.2) and keep it for twenty-one hours, stirring occasionally. Then leave to cool to room temperature.

Add 5 ml of Carrez solution I (34 (b) .3.6) and shake for one minute. Add 5 ml of solution Carrez II (34 (b) .3.7) shake again for one minute. Complete the volume with water, homogenize and filter. Take 50 ml of filtrate with pipette and take them to a 100 ml container. Add a few drops of the indicator (34 (b) .3.11) and acidify with 8N hydrochloric acid (34 (b) .3.9) until it turns red. Add 6.25 ml of 8N hydrochloric acid, in excess (12.5 ml if 100 ml of filtering is performed). Attach the reflux coolant to the container, bring the solution to the boil, and keep it for an hour. Allow to cool, neutralise with the sodium hydroxide solution 10N (34 (b) .3.10) until the indicator turns to yellow. Acidify then slightly by adding a bit of 1N hydrochloric acid (34 (b) .3.3), complete the volume with water and homogenise. Determine the glucose content with the Luff-Schoorl method as indicated in (34 (b) .4.4).

34 (b) .4.4 Valuation according to Luff Schoorl. Take with pipette, 25 ml of Luff-Schoorl reagent (34 (b) .3.13) and take it to a 300 ml erlenmeyer; add 25 ml, exactly measured, of the solution obtained in (34 (b) .4.3) containing a maximum of 60 mg glucose. Add two pieces of pumice stone (34 (b) .3.14), heat, shake by hand, on a free flame of a medium height, until the liquid is boiling in approximately two minutes. Immediately place the erlenmeyer on a metal fabric with an asbestos disc where an opening of approximately 6 cm in diameter has been made, under which a flame has been previously lit. This is regulated so that only the bottom of the flask is heated. Then adapt a reflux coolant to the flask. From this moment I will bring it to a boil for ten minutes exactly. Cool immediately in cold water and after five minutes, approximately, rate as follows:

Add 10 ml of potassium iodide solution (34 (b) .3.15) and immediately with caution (due to the risk of abundant foam formation), add 25 ml of 6N sulphuric acid (34 (b) .3.16). Then assess with sodium thiosulfate solution 0.1N (34 (b) .3.17), until the appearance of a mild yellow coloration; add as an indicator the starch solution (34 (b) .3.18) and perform the assessment to the end.

Make the same assessment of a mixture of exactly 25 ml of Luff-Schoorl reagent (34 (b) .3.13) and 25 ml of water, then add 10 ml of potassium iodide solution (34 (b) .3.15) and 25 ml of 6N sulphuric acid (34 (b) .3.16) without boiling.

34 (b) .4.5 Blank test. Perform a blank test using the operating mode described in 34 (b) .4.3 and 34 (b) .4.4 in the absence of the sample.

34 (b) .5 Calculations. The amount of glucose in mg corresponding to the difference between the results of the two assessments (expressed in ml of sodium thiosulfate 0.1N) which refer to both the sample and the test in the table is provided with the help of the attached table. white.

The starch content expressed as a percentage of the sample is given by the following formula:

0.72 (a-b)

In which:

a = mg of glucose in the sample.

b = mg of glucose in the blank test (see observation 34 (b) .6.2.).

34 (b) .6 Remarks.

34 (b) .6.1 The simultaneous presence in the partially dextinated starch sample to lactose may result in a result with an excess of 0.5 to 3 per 100 starch. In this case, the actual starch content is obtained as follows:

a) Determine the content in reducing sugars of the ethanolic extract obtained at (34 (b) .4.2) and express the result as a percentage of glucose.

b) Determine the contents of the sample of reducing sugars, soluble in water and express the result as a percentage of glucose.

c) Be the result obtained in a) of the obtained in b) and multiply the difference by 0.9.

d) To be the value obtained in c) of the starch content obtained by the application of the method and to calculate it as indicated in (34 (b) .5).

34 (b) .6.2 The amount of glucose in the blank test is normally 0.25 mg and cannot be greater than 0.50 mg.

34 (b) .6.3 Pancreatic-related Prescripciones:

Physical appearance: Yellow, amorphous white powder.

Glucose content: The amount of glucose in the blank test (see 34 (b) .4.5) is usually 0.25 mg. A result greater than 0.50 indicates that the pancreas cannot be used.

Control of iodine consumption: Put in suspension 62.5 mg of pancreatitis in 50 ml of water and bring to 25-30 ° C. Add 1 ml of 0.1N iodine solution. Stir for two minutes. Titrate with a sodium thiosulfate solution 0.1N (34 (b) .3.17) in the presence of the starch indicator. The consumption of iodine solution by the pancreas should not pass from 0.5 ml.

Control of amylolytic activity: Mix 100 ml of starch solution (34 (b) .3.18), 5 ml of buffer solution (34 (b) .3.4), 0.5 ml of sodium chloride solution (34 (b) .3.5) and 62.5 mg of pancreatitis. The mixture is heated to 25-30 ° C and is removed for two minutes. Then 1 ml of 0.1N iodine solution is added. The blue coloring must disappear before the fifteen minutes that follow the addition of the iodine solution.

34 (b) .7 References. Fifth Commission Directive of 25 March 1974. (74/203/EEC). 'Official Journal of the European Communities' number L 108/7 of 22 April 1974.

Value table for 25 ml of reagent

according to Luff-Schoorl

ml of Na2S20301N, two minutes of heating, ten minutes of boiling

NaS2030.1N :ml/Glucose, fructose, invert sugars C6H1206: mg/Difference/Lactose C12H22011: mg/Difference/Maltose C12H22011: mg/Difference

1/2.4/2.4/3.6/3.7/3.9/3.9

2/4.8/2.4/7.3/3.7/7.8/3.9

3/7.2/2.5/11.0/3.7/11.7/3.9

4/9.7/2.5/14.7/3.7/15.6/4.0

5/12.2/2.5/18.4/3.7/19.6/3.9

6/14.7/2.5/22.1/3.7/23.5/4.0

7/17.2/2.6/25.8/3.7/27.5/4.0

8/19.8/2.6/29.5/3.7/31.5/4.0

9/22.4/2.6/33.2/3.8/35.5/4.0

10/25.0/2.6/37.0/3.8/39.5/4.0

11/27.6/2.7/40.8/3.8/43.5/4.0

12/30.3/2.7/44.6/3.8/47.5/4.1

13/33.0/2.7/48.4/3.8/51.6/4.1

14/35.7/2.8/52.2/3.8/55.7/4.1

15/38.5/2.8/56.0/3.9/59.8/4.1

16/41.3/2.9/59.9/3.9/63.9/4.1

17/44.2/2.9/63.8/3.9/68.0/4.2

18/47.1/2.9/67.7/4.0/72.2/4.3

19/50.0/3.0/71.7/4.0/76.5/4.4

20/53.0/3.0/75.7/4.1/80.9/4.5

21/56.0/3.1/79.8/4.1/85.4/4.6

22/59.1/3.1/83.9/4.1/90.0/4.6

23/62.2//88.0//94.6/

(FIGURE OMITTED)

35. Ureasic activity of soy-derived products

35.1 Principle. The test makes it possible to determine the urease activity of the products derived from soya and to make it clear that these products are being cooked under the same conditions.

The ureasic activity is determined by the amount of ammoniacal nitrogen released by 1 g of product in one minute, at 30 ° C, from a urea solution.

35.2 Material and apparatus.

35.2.1 Very sensitive (0.02 pH) potentiometric or pH-meter valuation apparatus, with magnetic stirrer.

35.2.2 Bath of water provided with a thermostat regulated at 30 ° C exactly.

35.2.3 150 x 18 mm test tubes with grinding plugs.

35.3 Reassets.

35.3.1 Hydrochloric acid 0.1N.

35.3.2 Sodium hydroxide solution 0.1N.

35.3.3 phosphate buffer 0.05 M containing 4.45 g sodium hydrogen phosphate dihydrate (NaHPO-2HO) and 3.40 g potassium dihydrogen phosphate (KHPO) in 1,000 ml.

35.3.4. Recently prepared urea buffered solution containing 30.0 g of urea per 1,000 ml of buffer solution; pH 6,9-7.0.

35.4 Procedure. Grind 10 g of the sample, so that it passes through a mesh sieve of 0.2 mm. In a test tube (35.2.3) weigh, with precision of 1 mg, 0.2 g of the ground sample and add 10 ml of the solution (35.3.4). Cap immediately and shake vigorously. Take the tube to the water bath (35.2.2) and leave it for thirty minutes exactly. Immediately afterwards, add 10 ml of 0.1N hydrochloric acid (35.3.1), quickly cool to 20 ° C by quantitatively transfer the contents of the tube to a valuation vessel, washing twice with 5 ml of water. Measure immediately and quickly by means of a 0.1N sodium hydroxide solution (35.3.2) per potentiometer, using glass electrode up to pH 4.7. To perform a blank test, operating as follows:

Introduce quickly and successively into a tube (35.2.3) an aliquot of the 0.2 g sample, heavy with precision of 1 mg, 10 ml of 0.1N hydrochloric acid (35.3.1) and 10 ml of urea buffered solution (35.3.4). Immediately cool the tube in ice water and leave it for thirty minutes. Transfer the contents of the tube to the container and value by means of the sodium hydroxide solution 0.1N (35.3.2) to a pH 4,7 below, under the conditions indicated above.

35.5 Calculations. The ureasic activity is given by the formula:

mg N at 30 ° C = 1.4 (V-V)

mg N at 30 ° C = 1.4 (V-V)

g minute at 30 ° C = 30 x P

Being:

V = Volume, in ml, of 0.1N sodium hydroxide solution consumed in the analysis.

V = Volume, in ml, of 0.1N sodium hydroxide solution consumed by the blank test.

P = Sample weight in g.

35.6 Remarks.

35.6.1 The method is suitable for a ureasic activity that can reach 1 mg of N/g per minute at 30 °C. For more active products, the aliquot of the sample may be reduced to 50 mg.

35.6.2 Products with a gross fat content of more than 10% must have been degreased previously in cold.

35.7 References. First Commission Directive of 15 June 1971. (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

36. Free and total gosipol

36.1 Principle. The method allows the determination of free gosipol, total gossypol and chemically similar substances of constitution derived from gossypol, present in the seeds, flours and cakes of cotton, as well as in the feed containing them. The lower limit of the determination is 20 mg/kg.

The gosipol is extracted in the presence of 3-amino-1-propanol, either by a mixture of isopropanol and hexane for the determination of free gosipol, either by dimethylformamide for the determination of total gosipol. Gosipol is transformed by aniline into gosipol-dianiline, whose absorbance is measured at 440 nm.

36.2 Material and apparatus.

36.2.1 Agitator (vaiven). Around 35 swings per minute.

36.2.2 Spectrophotometer.

36.3 Reassets.

36.3.1 Isopropanol-hexane Mix. Mix 60 parts in isopropanol volume with 40 parts in n-hexane volume.

36.3.2 Solvent A. Deposit in a 1 litre, 500 ml graduated flask of the isopropanol-hexane mixture (36.3.1), 2 ml of 3-amino-1-propanol, 8 ml of glacial acetic acid and 50 ml of water. Complete the volume by mixing isopropanol-hexane (36.3.1). Said reagent is stable for one week.

36.3.3 Solvent B. Deposit by pipette into a flask of 100 ml, 2 ml of 3-amino-1-propanol and 10 ml of glacial acetic acid. Cool to room temperature and complete the volume by N, N-dimethylformamide. Said reagent is stable for one week.

36.3.4 Aniline. If the absorbance of the blank test exceeds 0,022, distill the aniline on zinc powder by eliminating the first and last fractions of 10 per 100 of the distillate. This reagent in bottle covered with topaz glass and in the refrigerator is preserved for several months.

36.3.5 Gosipol Pattern A Solution. Place in a 250 ml volumetric flask, 27.9 mg of gossypol acetate. Dissolve and complete the volume by solvent A (36.3.2). Pipette 50 ml of said solution into a 250 ml graduated flask and complete with solvent A. The concentration in gosipol of the solution is 0.02 mg/ml. Leave to rest for an hour at room temperature, before your employment.

36.3.6 Gosipol pattern B solution. Place in a 50 ml graduated flask, 27.9 mg of gossypol acetate. Dissolve and complete by volume using solvent B (36.3.3). The concentration in gosipol of this solution is 0.5 mg/ml.

The pattern A and B solutions of gossypol are kept stable for twenty-four hours.

36.4 Procedure.

36.4.1 Sample Take. The sampling is in relation to the alleged gosipol content of the sample. It is preferable to operate on a small sampling and on an aliquot portion of the relatively important filtrate, so that a sufficient amount of gosipol is obtained to perform a precise photometric measurement. For the determination of free gosipol in cotton seeds, flours and cakes, sampling must not exceed 1 g; for compound feed, it may be 5 g. An aliquot part of 10 ml of filtrate is sufficient in most cases; it must contain 50 to 100 micrograms of

gosipol. For the determination of total gossypol, sampling may vary from 0,5 to 5 g in order for an aliquot part of 2 ml of filtrate to contain 40 to 200 micrograms of gossypol.

The analysis must be carried out at an ambient temperature close to 20 ° C.

36.4.2 Determination of free gosipol. Insert the sample into a 250 ml frosted mouth flask, the bottom of which is covered with glass beads. Add 50 ml of solvent A (36.3.2.) with pipette, cover the flask and mix for one hour in the agitator. Filter dry and collect the filtrate in a small, frosted neck flask. Along the leak, coat the funnel with a watch glass. Pipette, respectively, into two 25 ml graduated flasks (A and B) with identical aliquot parts of filtration containing 50 to 100 micrograms of gossypol. Complete if necessary, the volume to 10 ml with the aid of solvent A (36.3.2), then complete the volume of the flask (A) by the isopropanol-hexane mixture (36.3.1). This solution shall be used as a reference solution for the measurement of the sample solution.

Enter with pipette 10 ml of solvent A (36.3.2) respectively in two other 25 ml graduated flasks (C and D). Complete the volume of the flask (C) by the isopropanol-hexane mixture (36.3.1). This solution shall be used as a reference solution for the measurement of the blank test solution.

Add 2 ml of aniline (36.3.4) respectively in the flasks (D) and (B). Heat for thirty minutes on a boiling water bath to develop the color. Refrigerate at room temperature, complete by volume using the isopropanol-hexane mixture (36.3.1), homogenise and leave to rest for an hour.

Determine by the spectrophotometer at 440 nm in 1 cm buckets the absorbance of the blank test solution (D) compared to the reference solution (C) and the absorbance of the sample solution (B) in comparison with the reference solution (A).

Subtract the value of the solution absorbance from the blank test solution of the sample (corrected absorbance). Calculate from that value the free gosipol content as indicated in 36.5.

36.4.3 Determination of total gosipol. Insert a sample containing 1 to 5 mg of gosipol into a 50 ml graduated flask and add 10 ml of solvent B (36.3.3). Simultaneously prepare a blank test by introducing 10 ml of solvent B (36.3.3) into another 50 ml graduated flask. Heat both flasks for thirty minutes over a boiling water bath. Refrigerate at room temperature and complete the contents of each flask by volume with the isopropanol-hexane mixture (36.3.1). Homogenise and leave to rest for ten to fifteen minutes; filter then and collect the filtrates in frosted mouth flasks.

Carry 2 ml of filtrate from the sample respectively to two 25 ml and 2 ml graduated flasks from the white test filter respectively to two other 25 ml flasks. Take a flask of each series and complete the respective contents to 25 ml by the isopropanol-hexane mixture (36.3.1). These solutions will be used as reference solutions.

Add 2 ml of aniline (36.3.4) respectively in the other two flasks. Heat for thirty minutes in boiling water bath to develop the color. Refrigerate at room temperature, complete to 25 ml by mixing isopropanol-hexane (36.3.1), homogenise and leave to rest for one hour.

Determine the absorbance as indicated in 36.4.2 for free gosipol. Calculate from that value the total gossypol content as indicated in 36.5.

36.5 Calculations. The calculation of the results can be done from the specific absorbance (36.5.1) or referring to a calibration curve (36.5.2).

36.5.1 From the specific absorbances. Under the conditions described, the specific absorbances are as follows:

Free Gosipol: E

Free Gosipol: E = 625.

Total Gosipol: E

Total Gosipol: E = 600.

The free or total gosipol content of the sample is given by the following formula:

A-1250

Gosipol% = A-1250

E

E-p-a

Being:

A = Corrected absorbance, determined as shown in 36.4.2.

p = Sample weight in g.

a = aliquot portion of the filtrate in ml.

36.5.2 From a calibration curve.

36.5.2.1 Gosipol free. Prepare two series of five 25 ml graduated flasks. In each series, insert a respective volume of 2,0-4,0-6,0-8,0 and 10,0 ml of the standard solution A of gosipol (36.3.5) into the flask by pipette, complete 10 ml with the solvent A. Complete each series by a blank consisting of a 25 ml graduated flask containing only 10 ml of solvent A (36.3.2).

Complete to 25 ml the volume of the flasks of the first series (including white) by the isopropanol-hexane mixture (36.3.1) (reference series).

Add 2 ml of aniline (36.3.4) in each flask in the second series (including white). Heat for thirty minutes in a boiling water bath to develop the color. Refrigerate at room temperature, complete the volume by means of the isopropanol-hexane mixture (36.3.1), homogenise and leave to rest for one hour (standard series).

Determine in the conditions indicated in 36.4.2 the absorbance of the solutions of the standard series in comparison with the corresponding solutions of the reference series. Graphically plot the calibration curve by putting the absorbances in relation to the amounts of gosipol (in micrograms).

36.5.2.2. Total gossypol. Prepare six 50 ml graduated flasks. Introduce in the first flask 10 ml of solvent B (36.3.3) and in the others, respectively 2,0-4,0-6,0-8,0 and 10,0 ml of the pattern B solution of gosipol (36.3.5). Complete the contents of each flask up to 10 ml with the help of solvent B (36.3.3). Heat for thirty minutes in a boiling water bath. Refrigerate at room temperature, complete by volume using isopropanol-hexane mixture (36.3.1) and homogenize.

Introduce 2.0 ml of these solutions respectively into two series of six 25 ml graduated flasks. Complete to 25 ml the content of the first series flasks by mixing isopropanol-hexane (36.3.1) (reference series).

Add 2 ml of aniline (36.3.4) in each flask in the second series. Heat for thirty minutes in a boiling water bath. Refrigerate at room temperature, complete by volume using the isopropanol-hexane mixture (36.3.1), homogenise and leave to rest for one hour (standard series).

Determine in the conditions indicated in 36.4.2 the absorbance of the solutions of the standard series in comparison with the corresponding solutions of the reference series. Graphically plot the calibration curve by putting the absorbances in relation to the amounts of gosipol in micrograms.

The difference between the results of two parallel determinations performed on the same sample must not exceed:

-Of 15 per 100, in relative value, for gosipol contents of less than 500 mg/kg.

-75 mg/kg in absolute value, for contents between 500 and 750 mg/kg.

-10 per 100, in relative value, for contents greater than 750 mg/kg.

36.6 References. Third Commission Directive of 27 April 1972. (72/199/EEC). 'Official Journal of the European Communities' number L 123 of 29 May 1972.

37. Theobromine

37.1 Principle. The method makes it possible to determine the teobromine content of the cocoa seed transformation byproducts. Theobromine is extracted by chloroform. The extract evaporates to dryness, is placed in aqueous solution and is treated with a determined volume of silver nitrate solution. The released nitric acid is titled by a sodium hydroxide solution.

37.2 Material and apparatus.

37.2.1 Half-bottom 500 ml Matres and grinding plug.

37.3 Reassets.

37.3.1 Chloroform p.a.

37.3.2 Ammonia, d: 0.958.

37.3.3 Sodium sulphate p.a., anhydrous.

37.3.4 Sodium hydroxide solution 0.1N.

37.3.5 Silver nitrate solution 0.1N.

37.3.6 A 1 per 100 (w/v) ethanolic solution of phenol red.

37.3.7 Ether diethyl.

37.4 Procedure. Weigh, with precision of 1 mg, an aliquot of 10 g at most, which does not contain more than 80 mg of theobromine. Introduce it into a 500 ml flask of flat bottom and frosted stopper, add 270 ml of chloroform (37.3.1) and 10 ml of ammonia (37.3.2). Cap the flask and shake vigorously for five minutes. Add 12 g of anhydrous sodium sulfate (37.3.3), shake again and leave to rest until the next day. Filter in a 500 ml erlenmeyer and wash the residue with 100 ml of chloroform (37.3.1). Distill the solvent and remove the last remaining in a boiling water bath. Collect the extract in 50 ml of water and bring to the boil.

Refrigerate, neutralise exactly with the sodium hydroxide solution (37.3.4) in the presence of 0.5 ml of phenol red solution (37.3.6). Assess the nitric acid released with the sodium hydroxide solution (37.3.4) until the indicator turns (pH 7, 4).

37.5 Calcles. 1 ml of NaOH 0.1N corresponds to 18 mg of theobromine. Express the result as a percentage of the sample.

37.6 Remarks. Products whose gross fat content exceeds 8 per 100 must be pre-defatted by extraction for six hours with petroleum ether (Eb. 40 ° C).

37.7 References. First Commission Directive of 15 June 1971. (71/250/EEC). 'Official Journal of the European Communities' number L 155 of 12 July 1971.

38. Retinol (vitamin A)

38.1 Principle. The method allows the determination of the content in retinol (vitamin A) in feed, concentrates and premixtures. The lower limit of detection is 10,000 IU/kg (1 IU equal to 0.3 micrograms retinol) for heavily pigmented feed and 4,000 IU/kg for other products. The products are classified according to their assumed content in retinol in two groups:

Group A: contents less than 200,000 IU/kg.

Group B: contents equal to or greater than 200,000 IU/kg.

The sample is hot hydrolyzed by potassium hydroxide in an ethanolic medium and in the presence of an antioxidant or nitrogen atmosphere. The mixture is subjected to extraction with 1,2-dichloroethane. The extract evaporates to dryness and is redeemed in petroleum ether. The solution is chromatography in aluminium oxide column (for group B products, chromatography is only necessary in certain cases). The retinol is determined by spectrophotometry at 610 nm after obtaining a complex coloured according to the CarrPrice reaction, in the case of the products of group A; by spectrophotometry U.V. at 325 nm in the case of group B products.

38.2 Material and apparatus.

38.2.1 Bath of Water.

38.2.2 A rotary vacuum evaporator with round flasks of different capacities.

38.2.3 Glass columns for chromatography (length: 300 mm; inner diameter: approximately 13 mm).

38.2.4 Spectrophotometer with 10 mm thick buckets (quartz for measurements in U.V.).

38.2.5 Lamp U.V. 365 nm.

38.3 Reassets.

38.3.a) Used for analysis of product groups A and B.

38.3.a.1 Ethanol of 96 per 100 (v/v).

38.3.a.2 Solution to 10 per 100 (w/v) sodium ascorbate p.a. or (38.3.a.3).

38.3.a.3 Purified Nitrogen.

38.3.a.4 Solution to 50 per 100 (w/v) potassium hydroxide p.a.

38.3.a.5 1N potassium hydroxide solution.

38.3.a.6 0.5N potassium hydroxide solution.

38.3.a.7 1,2-dichloroethane p.a.

38.3.a.8 Oil Ether, pure, boiling range 30-50 ° C. If necessary, purify it as follows:

Shake 1,000 ml of petroleum ether with 20 ml portions of concentrated sulphuric acid until the acid remains colourless.

Remove the acid and wash the ether successively with 500 ml of water, twice with 250 ml of a solution at 10 per 100 of sodium hydroxide and three times with 500 ml of water. Remove the aqueous layer, dry the ether for an hour on activated carbon and anhydrous sodium sulfate, filter and distill.

38.3.a.9 Standardized aluminum oxide according to Brockmann. Calcined for eight hours at 750 °C, cool in desiccator and store in amber glass bottle, screw cap. Before using it in chromatography, moisten as follows: introduce in an amber glass flask 10 g of aluminium oxide and 0.7 ml of water, cover hermetically, heat five minutes in a boiling water bath stirring energetically. Leave to cool agitating. Check the activity of the aluminium oxide so prepared, analyzing according to 38.4.2 and 38.4.3 a known quantity of retinol pattern (38.3.b.4).

38.3.a.10 Basic aluminium oxide, activity grade 1.

38.3.a.11 Pure diethyl ether. Remove peroxides and traces of water by column chromatography of basic aluminium oxide (38.3.a.10) (25 g of aluminium oxide for 250 ml of diethyl ether).

38.3.a.12. Petroleum ether solutions (38.3.a.8) with 4, 8, 12, 16 and 20 per 100 (v/v) diethyl ether.

38.3.a.13 Sodium sulphide solution 0.5 M in 70 per 100 glycerin, prepared from sodium sulfide p.a.

38.3.b) Used exclusively for the analysis of group A products.

38.3.b.1 benzene p.a. crystallizable.

38.3.b.2 Chloroform p.a. Eliminate ethanol, phosgene and water traces by column chromatography of basic aluminium oxide (38.3.a.10) (50 g of aluminium oxide per 200 ml of chloroform); chromatography should be second time the first 50 ml of the eluid.

38.3.b.3 Reactive according to Carr-Price. Stir approximately 25 g of antimony trichloride p.a. (preserved in desiccator) with 100 ml of chloroform (38.3.b.2) until solution saturation. A small deposit of antimony trichloride does not alter. Add 2 ml of acetic anhydride p.a. Store in a freezer in an amber glass bottle with a screw cap. This reactant thus preserved is stable for several weeks.

38.3.b.4 Retinol pattern controlled by spectrophotometry.

38.3.c) Used exclusively for the analysis of group B. products.

38.3.c.1 Isopropanol for chromatography.

38.4 Procedure.

38.4.1 Hydrolysis and extraction. Take a portion of the finely divided sample, proportional to the supposed vitamin A content:

-0,1 to 1.0 g for concentrates (contents greater than 20,000 IU/g).

-3,0 to 5,0 g for premixtures (contents ranging from 400 to 20,000 IU/g).

-10 to 20 g for mineral mixtures.

-30 g for group A products

Immediately introduce it into a 500 ml flask of a frosted stopper.

Add successively 40 ml of ethanol (38.3.a.1), 2 ml of sodium ascorbate solution (38.3.a.2) 10 ml of potassium hydroxide solution at 50 per 100 (38.3.a.4) and 2 ml of 0.5 M sodium sulfide solution (38.3.a.13). Heat for thirty minutes at 70-80 ° C under reflux coolant and cool to the tap. Add 50 ml of ethanol (38.3.a.1) and 100 ml (taken with pipette) of 1,2-dichloroethanol (38.3.a.7).

Shake vigorously and decant the supernatant liquid into decantation funnel and add 150 ml of the 1N potassium hydroxide solution (38.3.a.5), shake for ten seconds, and leave to rest until the phases are separated. Collect the lower phase of dichloroethane in another decanting funnel, add 40 ml of 0,5N potassium hydroxide solution (38.3.a.6), shake for ten seconds and leave to rest until the phases are separated. Collect the dichloroethane phase in another decantation funnel and wash six or eight times with portions of 40 ml of water until the absence of alkali in the washing water (phenolphthalein assay). Collect the dichloroethane phase and remove the last traces of water by means of filter paper bands.

Evaporate dry-dry and water-bath at 40 ° C, an aliquot of the solution. Redissolve the residue rapidly with 5 ml of petroleum ether (38.3.a.8).

For group A products, chromatography as indicated in 38.4.2.1.

For group B products, transfer the solution to a 50 ml volumetric flask, complete with oil ether volume (38.3.a.8), homogenise and measure the absorbance as indicated in 38.4.3.

38.4.2 Cromatography.

38.4.2.1 Group A products A. Fill the column for chromatography (38.2.3) up to a height of 200 mm aluminium oxide (38.3.a.9), previously impregnated with petroleum ether (38.3.a.8). Insert the solution obtained in 38.4.1 into the column and immediately add 20 ml of petroleum ether (38.3.a.8). Elude successively with 10 ml portions of the petroleum ether solutions with 4, 8, 12, 16 and 20 per 100 diethyl ether (38.3.a.12) either partially or under pressure; the elution speed must be two to three drops per second.

The carotene is eluted in the first place. (The carotene content of this fraction can be determined by the measure of the absorbance at 450 nm; in = 2. 600).

38.4.2.2 Group B. Products chromatography must not be carried out unless the absorbance measurements obtained in 38.4.3.2 are not in conformity with the requirements set out in 38.4.3.2.

If chromatography was required, enter into the chromatographic column an aliquot of the petroleum ether solution obtained in 38.4.1 containing approximately 500 IU retinol and chromatography as indicated in 38.4.2.1.

38.4.3 Measure of absorbance.

38.4.3.1 Evaporar to dry, empty, the chromatographic fraction containing the retinol obtained in 38.4.2.1. Redissolve the residue with 2 ml of benzene (38.3.b.1). Take 0.3 ml of this solution and add 3 ml of the Carr-Price reagent (38.3.b.3). A blue coloring is developed. Measure the absorbance in the spectrophotometer at 610 nm exactly within thirty seconds of the beginning of the reaction.

Determine the retinol content using a pattern curve obtained from the benzenic solutions of increasing concentrations in retinol pattern treated by the Carr-Price reagent (from 2 to 16 IU of vitamin A pattern) (38.3.b.4) with 0.3 ml of benzene (38.3.b.1) plus 3 ml of the Carr-Price reagent (38.3.b.3).

The pattern curve should be checked regularly and in brief time intervals by a newly prepared solution of the Carr-Price reagent.

38.4.3.2 Group B. Products Take an aliquot of the petroleum ether solution obtained at 38.4.1, containing approximately 200 IU retinol. Evaporate dry dry and dissolve the residue with 25 ml of isopropanol (38.3.c.1). Measure the absorbance in the spectrophotometer at 334, 325 and 310 nm. The maximum absorption is located at 325 nm. The retinol content of the solution is calculated as follows:

A x 18.30 = retinol UI

However, the relationships between absorbances A/A and A/A must be 6/7 = 0.857.

If one of these relationships is significantly separated from this value (less than 0,830 or greater than 0,880), the measurement of the absorbances must be preceded by a chromatography according to the procedure indicated in 38.4.2. If the measurement of the absorbances carried out after chromatography indicates that the aforementioned relationships are still significantly separated from the value 0.857 (less than 0,830 or greater than 0,880), the determination must be carried out according to the the procedure indicated for group A products.

38.5 Calculations. Calculate the retinol content of the sample taking into account the starting weight and the dilutions performed throughout the analysis.

Express the result in IU of retinol per kg. The difference between the results of two parallel determinations made on the same sample must not pass from:

-20 per 100 in relative value for retinol contents less than 75,000 IU/kg.

-15,000 IU for the retinol contents between 75,000 and 150,000 IU/kg.

-10 per 100 in relative value for contents between 150,000 and 250,000 IU/kg.

-25,000 for contents between 250,000 and 500,000 IU/kg.

-5 per 100 in relative value for contents greater than 500,000 IU/kg.

38.6 Remarks.

38.6.1 All manipulations must be done in the absence of direct light and if possible in amber glass material.

38.6.2 The addition of ascorbate is not necessary if the hydrolysis is performed in nitrogen current (38.4.1).

38.6.3 For lactation feed and products with a tendency to swell, fold the amount of reagents indicated in 38.4.1, ethanol (38.3.a.1), sodium ascorbate solution (38.3.a.2), potassium hydroxide solution (38.3.a.4) and sodium sulfide solution (38.3.a.13).

38.6.4 Save precautions due to the hazard of reagents (especially benzene).

38.7 References. Fourth Commission Directive of 5 December 1972. 73 /46/EEC. 'Official Journal of the European Communities' number L 83/21 of 30 March 1973.

39. Thiamine (aneurysm, vitamin B)

39.1 Principle. The sample is treated as hot with dilute sulphuric acid and is then hydrolysed by enzyme.

The solution obtained is subjected to alkaline oxidation. The formed thiocromo is extracted with isobutanol and is determined by fluorimetry.

The method allows the determination of thiamine (aneurysm, vitamin B) in feed, concentrates and premixtures. The detection limit is 5 ppm.

39.2 Material and apparatus.

39.2.1 Water Bath.

39.2.2 Centrileak (3,500 rpm) with 30 to 50 ml tubes, fitted with a screw cap.

39.2.3 Fluorimeter.

39.3 Reassets.

39.3.1 100 microgram/ml tiamine standard solution

Dissolve 112.3 mg of thiamine hydrochloride, standard quality, previously desiccated to a constant weight, in 1,000 ml of sulphuric acid 0.2N (39.3.2). In cold and in darkness this solution is stable for a month.

39.3.2 sulphuric acid 0.2N.

39.3.3 Sodium Metabisulfite, Pure NaSO.

39.3.4 Solution to 20 per 100 (w/v) potassium hexacianoferde (III) p.a.

39.3.5 Solution to 25 per 100 (w/v) potassium hydroxide p.a.

39.3.6 Oxidizing Mixture: Mix 2 ml of potassium hexacianoferde (III) solution (39.3.4) with 48 ml of potassium hydroxide solution (39.3.5). This mixture is not preserved for more than four hours.

39.3.7 Isobutanol p.a.

39.3.8 Sodium acetate solution 2.5N.

39.3.9 Multienzimatic preparation containing protease, phosphatase and amylase (e.g. clarase).

39.3.10 Ethanol from 96 per 100 (v/v).

39.4 Procedure.

39.4.1 enzymatic Hydrolysis. Enter into two 250 ml graduated flask A and B, identical amounts of finely ground sample, containing 100 micrograms of thiamine and 125 ml of sulphuric acid (39.3.2). Add only to the flask A 1.0 ml of standard solution (39.3.1) (internal standard).

Shake vigorously, bring the flasks into a boiling water bath and keep them for fifteen minutes stirring occasionally. Allow to cool to approximately 45 ° C. Add to each flask 20 ml of sodium acetate solution (39.3.8) and 0.5 g of multifylimatic preparation (39.3.9), then leave to rest for 20 minutes at room temperature. Add 20 ml of sodium acetate solution and complete volume with water, homogenise and filter. Collect the filtered A and B after having eliminated the first 15 ml. Prepare the following solutions:

39.4.1.1 Witness solution T. Introduce into a centrifuge tube (39.2.2), 5 ml of filtering A and 10 mg approximately of sodium metabisulfite (39.3.3). Dip the tube for fifteen minutes in a boiling water bath and leave to cool below to room temperature.

39.4.1.2 Solution A (internal pattern) and B (sample). Introduce 5 ml of A filtrate into a centrifuge tube (39.2.2) and 5 ml of the B filtrate into another centrifuge tube (39.2.2).

39.4.2 Oxidation. Add to the solutions T, A and B, 5 ml of the oxidizing mixture (39.3.6) and after one minute, 10 ml of isobutanol (39.3.7). Cap the tubes and shake vigorously for five seconds. Leave to rest for one minute and centrifuge to separate the phases. Take, from each tube, 5 ml of the supernatant isobutanolic phase, enter them respectively in the 25 ml graduated flasks, complete with ethanol (39.3.10) and homogenise. This results in T, A and B. extracts.

39.4.3 Measurement of fluorescence. Perform the measurements at the wavelength to which the fluorimeter gives an optimal response for the fluorescence of the tiocrome. Radiate at approximately 365 nm.

Adjust the instrument to zero with the T extract Measure the fluorescence intensity of extracts A and B.

39.5 Calculations. The microgram content of thiamine per kg of sample is given by the formula:

d x b

(a-b) x c

Being:

a = The fluorescence intensity of the extract A (internal standard).

b = The fluorescence intensity of the B extract (sample).

c = Weight, in grams, of the sample.

d = The amount of thiamine in micrograms added to the sample (internal pattern).

The difference between the results of two parallel determinations performed on the same sample must not be greater than:

-10 per 100 in relative value for contents less than 500 ppm and

-5 per 100 in relative value for contents equal to or greater than 500 ppm.

39.6 References. Fourth Commission Directive of 5 December 1972. 73 /46/EEC. 'Official Journal of the European Communities' number L 83/21 of 30 March 1973.

40. Ascorbic acid and dehydroascorbic acid

(vitamin C)

40.1 Principle. The method makes it possible to determine the sum of ascorbic and dehydroascorbic acid (vitamin C) in feed, concentrates and premixtures. The detection limit is 5 ppm. The products are classified into two groups according to their supposed vitamin C content:

Group A: Contents less than 10 g/kg.

Group B: Contents equal to or greater than 10 g/kg.

The sample, placed in suspension in a diluted solution of metafosforic acid, is subjected to extraction with chloroform. The aqueous phase is treated with a 2,6-dichlorophenol-indophenol solution to transform ascorbic acid into dehydroascorbic acid, and then a 2,4-dinitrophenylhydrazine solution. The formed hydrazone is extracted by a mixture of ethyl acetate, glacial acetic acid and acetone. The solution is chromatography in silica gel column, the eluid evaporates to dryness and the residue is dissolved in dilute sulphuric acid. The absorbance of the solution is measured in the spectrophotometer at 509 nm.

For group A products, the eluid from the column chromatography is subjected to a thin layer chromatography to isolate the hydrazone.

40.2 Material and apparatus.

40.2.1 Bath of water regulated at temperature of 20 ° C by thermostat.

40.2.2 Centrileak (3,500 r.p.m.) with 40 to 50 ml tubes provided with screw plugs.

40.2.3 Empty rotary evaporator with 250 ml flasks.

40.2.4 Glass columns for chromatography (length: 100 mm inside diameter: 20 mm) with fritted glass plate (for example, Allihn tubes).

40.2.5 Spectrophotometer or colorimeter of filters with buckets of 10 mm thick.

40.2.6 Material for thin layer chromatography with silica gel plates H (40.3.12), layer thickness: 0.5 to 0.6 mm. Dry the two-hour plates thirty minutes to three hours in a stove at 120-130 ° C. Allow to cool and store in desiccator for twenty-four hours before use.

40.2.7 Regulated to 120-130 ° C.

40.3 Reassets.

40.3.1 Standard solution of 0.05 per 100 L-ascorbic acid: dissolve 50 mg L-ascorbic acid p.a. in 20 ml approximately of metaphosforic acid (40.3.2) and complete 100 ml with water. Prepare immediately before your employment.

40.3.2 Solution to 10 per 100 of metafosforic acid: dissolve in water 200 g of metafosforic acid p.a. crushed in mortar, and complete 2,000 ml with water. Store at 4 °C. Renew after one week.

40.3.3 Chloroform p.a.

40.3.4 Solution to 0.5 per 100 (w/v) of 2,6-dichlorophenol-indophenol p.a. Prepare immediately prior to use.

40.3.5 Leak aid (S and S number 121 or equivalent).

40.3.6 Acid solution at 2 per 100 (w/v) 2,4-dinitroenylhydrazine: dissolve 2 g of 2,4-dinitrophenylhydrazine in 100 ml diluted sulphuric acid (25 ml sulphuric acid p.a. d = 1.84, diluted to 100 ml with water). In cold this solution is preserved for a week.

40.3.7 Nitrogen or (40.3.8).

40.3.8 Carbon Anhydride.

40.3.9 Ethyl acetate mixture p.a ./acetic acid glacial/acetone p.a.: 96/2/2 in volume.

40.3.10 Mix of dichloromethanol p.a ./glacial acetic acid: 97/3 in volume.

40.3.11 silica gel, particle size: 0,05 to 0,2 mm.

40.3.12 silica gel H according to Stahl, for thin layer chromatography.

40.3.13 Diluted sulphuric acid: introduce 105 ml of water into a 200 ml volumetric flask, complete with volume with sulphuric acid p.a., d = 1.84.

40.3.14 Elution solvent for thin-layer chromatography: Mix 75 ml of diethyl ether p.a., 25 ml of ethyl acetate p.a., and 4.0 ml of 96 acetic acid per 100 w/v, p.a. Refresh after two or three chromatography.

40.4 Procedure.

40.4.1. Extraction. Insert in two graduated 250 ml flasks A and B, identical amounts of the finely ground sample containing 200 micrograms of vitamin C. Add only to the flask A, 0.4 ml of standard solution (40.3.1) and homogenise gently stirring (internal pattern). Add 30 ml of chloroform (40.3.3) and 25 ml of metafosforic acid solution (40.3.2) to each flask at 4 ° C. Shake slightly and leave to stand for ten to fifteen minutes. Add 25 ml of water, cap the flasks, shake vigorously for ten seconds and leave to rest from ten to fifteen minutes in water bath (40.2.1). Centrifuge to separate the aqueous phase from the chloroformic phase. Perform the following operations simultaneously on the aqueous extracts A (internal pattern) and B.

40.4.2 Oxidation. Take 40 ml of the aqueous supernatant (slightly cloudy) solution obtained in (40.4.1) by pipette, insert it into a screw cap reaction tube, add 0.5 to 1 ml of 2,6-dichlorophenol-indophenol solution (40.3.4) and homogenise. A colouring is formed which must persist for fifteen minutes at least. Add approximately 300 mg of filtration aid (40.3.5), shake and filter on a dry-fold filter. Filtering should not necessarily be transparent.

40.4.3 Reaction with 2,4-dinitrophenylhydrazine and extraction of the hydrazone. Take 10 ml of the filtrate obtained in (40.4.2) by pipette, insert them into a centrifuge tube (40.2.2), add 2 ml of 2,4-dinitrophenyl-hydrazine solution (40.3.6) and homogenise. Rapidly pass through the tube a stream of nitrogen (40.3.7) or carbon dioxide (40.3.8), plug the tube and immerse it for about fifteen hours (one night) in the water bath (40.2.1). Add 3 ml of water, 20 ml of the mixture of ethyl acetate/glacial acetic acid/acetone (40.3.9) and approximately 800 mg of filtration aid (40.3.5). Cap the tube, shake vigorously for thirty seconds, and centrifuge. Introduce 15 ml of the supernatant phase into an evaporative flask and evaporate at reduced pressure in the rotary evaporator (40.2.3) until a oily residue is obtained. Dissolve the residue in 2 ml of the mixture of ethyl acetate/glacial acetic acid/acetone (40.3.9) by heating to 50 ° C, allow to cool, add 10 ml of the dichloromethane/glacial acetic acid mixture (40.3.10) homogenise.

40.4.4 Column chromatography. Fill the chromatographic column (40.2.4) to a height of 30 mm with the mixture dichloromethane/glacial acetic acid (40.3.10). Put in suspension (vigorously stirring) 5 g of silica gel (40.3.11) in 30 ml of the dichloromethane/glacial acetic acid mixture (40.3.10) and pour the suspension into the column. Leave to deposit and compress under weak nitrogen pressure (40.3.7).

Transfer to the chromatographic column the solution obtained in 40.4.3, wash the flask with a small amount of dichloromethane mixture/glacial acetic acid (40.3.10) and transfer to the column, fill with the mixture (40.3.10) and continue the washing of the column with the same mixture (3 or 4 portions of approximately 5 ml), until a colourless eluid is obtained. Remove the coloured eluid fraction from yellow.

Elude the reddish area in the head of the column by mixing ethyl acetate/glacial acetic acid/acetone (40.3.9), collecting the eluid and evaporating to dryness.

40.4.4.1 For group A products (contents of vitamin C less than 10 g/kg), dissolve the residue in 2.0 ml of the ethyl acetate/acetic acid/acetone acetate mixture (40.3.9) and proceed immediately to chromatography in thin layer as indicated in 40.4.5.

40.4.4.2 For group B products (vitamin C content equal to or greater than 10 g/kg), redissolve the oily residue in 4.0 ml of diluted sulphuric acid (40.3.13), shake vigorously to completely dissolve the residue. and proceed to the extent of the absorbance as indicated in 40.4.6.

40.4.5 Fine-layer chromatography. Carry out the operations indicated from here in duplicate.

Deposit in the form of a band on the thin-layer chromatography plate (40.2.6) 0.5 ml of the solution obtained in 40.4.4.1. Develop for twenty minutes at least by the eluent (40.3.14) in a saturated chamber until clear separation of the zone of the hydrazone, colored in pink. Let air dry. Delimit the pink zone, tear it with a spatula and quantitatively transfer the pulverulent mass to a chromatography column (40.2.4).

elude successively once with 2 ml and twice with 1.5 ml of the ethyl acetate/acetic acid/acetone acetate mixture (40.3.9). Collect the eluid in a small flask (the last fraction must be colourless). Evaporate to dryness, redissolve the oily residue in 4.0 ml of dilute sulphuric acid (40.3.13), shake vigorously to completely dissolve the residue and proceed to the extent of the absorbance.

40.4.6 Measure of absorbance. Measure the absorbance in a spectrophotometer at 509 nm of twenty to thirty minutes since the residue was dissolved in diluted sulphuric acid (40.3.13).

Carry out the measures by comparison with diluted sulphuric acid (40.3.13).

40.4.7 Test blank.

Perform in parallel a blank test using the same operating method.

40.5 Calculations. The content in g of vitamin C per kg of sample is given by the formula:

(c-a) x 2

(b-c) x 10 d

Being:

a = Blank absorbance.

b = The absorbance of the internal pattern solution.

c = The absorbance of the sample solution.

d = Weight, in g, of the sample.

The difference between the results of two parallel determinations carried out on the same sample must not pass from 10 per 100 in relative value for the vitamin C content of less than 10 g/kg and 5 per 100 in relative value. for content equal to or greater than 10 g/kg.

40.6 References. Fourth Commission Directive of 5 December 1972. (73/46/EEC). 'Official Journal of the European Communities' number L 83/21 of 30 March 1973.

41. Menadione (vitamin K)

41.1 Principle. The sample is subjected to extraction by diluted ethanol. The mixture is defecated with tannin solution and centrifuged. The extract is treated with sodium carbonate solution. The released menadione is extracted with 1,2-dichloroethane. The extract in dichloroethane is treated according to its content in menadione, directly or after evaporation, with 2,4-dinitro-phenylhydrazine in solution in ethanol acidified by hydrochloric acid. The hydrazone obtained gives rise, when adding an excess of ammonia, to a complex of blue-greenish color whose absorbance is measured at 635 nm. The method enables the determination of menadione (vitamin K) in feed, concentrates and premixtures. The lower limit of detection is 1 ppm.

41.2 Material and apparatus.

41.2.1 Mechanical Agitator.

41.2.2 Centrileak (3,000 to 5,000 rpm).

41.2.3 100-ml and 250 ml decantation, with a grinding plug.

41.2.4 Empty rotary evaporator with 250 ml flasks.

41.2.5 Bath water.

41.2.6 Spectrophotometer with 10 mm thick buckets.

41.3 Reassets.

41.3.1 Ethanol at 96 per 100 (v/v).

41.3.2 Ethanol (41.3.1) diluted to 40 per 100 with water.

41.3.3 Solution to 10 per 100 (w/v) tannin, obtained from pure tannin powder.

41.3.4 1,2, dichloroethane p.a.

41.3.5 Solution to 10 per 100 (w/v) anhydrous sodium carbonate p.a.

41.3.6 Hydrochloric acid at 37 per 100 (w/w), of = 1.19.

41.3.7 Ethanol absolute.

41.3.8 Reactive 2,4-dinotrofenylhydrazine: dissolve 40 mg of 2,4-dinitrophenylhydrazine p.a. in 40 ml approximately of the absolute ethanol (41.3.7) boiling. Leave to cool and transfer to a 50 ml graduated flask. Add 1 ml of hydrochloric acid (41.3.6) and complete volume with absolute ethanol (41.3.7). Prepare immediately before your employment.

41.3.9 Ammonium hydroxide at 25 per 100 (w/w), d = 0.91.

41.3.10 Ammoniacal ethanol solution: mix a volume of ethanol (41.3.7) and an ammonium hydroxide volume (41.3.9).

41.3.11 Menadione standard solutions: dissolve 20 mg of menadione (vitamin K) in 1,2-dichloroethane (41.3.4) and complete 200 ml. Dilute aliquot parts of this solution by 1,2-dichloroethane (41.3.4) to obtain a series of standard solutions whose concentrations in menadione are between 2 and 10 micrograms per millilitre. Prepare immediately before your employment.

41.4 Procedure.

41.4.1 Extract. Take a finely ground and proportional sample portion to the presumed content of menadione:

-0.1 to 5.0 g for concentrates and premixtures.

-20 to 30 g for feed.

Immediately introduce it into a 250 ml flask with a frosted stopper. Add 96 ml of exactly diluted ethanol (41.3.2) and shake mechanically for fifteen minutes at room temperature. Add 4.0 ml of tannin solution (41.3.3), mix, transfer the extract to a centrifuge tube, centrifuge (3,000 to 5,000 rpm) and decant.

Introduce 20 to 40 ml exactly as measured from the extract into a 250 ml decantation funnel, pipette add 50 ml of 1,2-dichloroethane (41.3.4), mix and add 20 ml of sodium carbonate solution (41.3.5) by pipette. Shake vigorously for thirty seconds and then collect the dichloroethane phase in a 100 ml decantation funnel. Add 20 ml of water, stir for fifteen seconds, collect the phase of the dichloroethane and remove the traces of water by strips of filter paper.

For concentrates and premixtures, take an extract aliquot and dilute with 1,2-dichloroethane (41.3.4) to obtain a concentration in menadione of 2 to 10 micrograms per millilitre. For feed, evaporate dry at 40 ° C, under reduced pressure and in nitrogen atmosphere, an aliquot of the extract. Redissolve the residue in the appropriate amount of 1,2-dichloroethane (41.3.4) to obtain a solution containing 2 to 10 micrograms of menadione per ml.

41.4.2 hydrazone formation. Carry 2,0 ml of the extract in dichloroethane obtained in 41.4.1 to a 10 ml graduated flask and add 3,0 ml of 2,4-dinitroenylhydrazine reagent (41.3.8). Cap the flask hermetically so as to avoid any evaporation and heat for two hours at 70 ° C on water bath. Leave to cool, add 3.0 ml of ethanol solution of ethanol (41.3.10), mix, complete volume with absolute ethanol (41.3.7) and mix again.

41.4.3 Measure of absorbance. Measure the absorbance of the greenish blue colored complex in spectrophotometer at 635 nm by comparison with a target of reagents obtained by treating 2.0 ml of 1,2-dichloroethane (41.3.4) as indicated in 41.4.2. Determine the amount of menadione by the calibrated curve established for each analysis series.

41.4.4 Calibration curve. Treat 2.0 ml of each standard solution of menadione (41.3.11) as indicated in 41.4.2. Measure the absorbance as indicated in 41.4.3. Draw the calibration curve by taking the absorbance values and the corresponding amounts of menadione in microgram to order the values of the absorbance.

41.5 Calculations. Calculate the menadione content of the sample taking into account the weight of the sample and the dilutions made in the course of the analysis. Express the result in mg of menadione per kg.

The difference between the results of two parallel determinations performed on the same sample must not pass from:

-20 per 100 in relative value for menadione content less than 10 mg/kg.

-2 mg in absolute value for the content of menadione between 10 and 14 mg/kg.

-15 per 100 in relative value for contents between 14 and 100 mg/kg.

-15 mg/kg in absolute value for the contents between 100 and 150 mg/kg.

-10 per 100 in relative value for contents greater than 150 mg/kg.

41.6 Remarks. All manipulations should be done in the absence of direct light and if possible in amber glass material.

41.7 References. Fifth Commission Directive of 25 March 1974. 74 /203/EEC. 'Official Journal of the European Communities' number 108/7 of 22 April 1974.

42. Virginiamycin

(by broadcast on agar)

42.1 Principle. The method enables the determination of virginiamycin in feed and premixtures. The lower limit of the determination is 2 mg/kg. 1 mg of virginiamycin is equivalent to 1,000 UK units.

The sample is submitted to extraction by means of the Tween 80's methanolic solution. The extract is decanted or centrifuged, and diluted afterwards. Its antibiotic activity is determined by the measurement of the spread of virginiamycin in an agar medium sown with Micrococus luteus. The diffusion is manifested by the formation of zones of microorganism inhibition. The diameter of these zones is considered to be directly proportional to the logarithm of the antibiotic concentration for the range of concentrations used.

42.2 Reassets.

42.2.1 Microorganism: "Microfocus luteus" ATCC 9341 (NCTC 8340, NCIB 8553).

42.2.1.1 Maintenance of the strain. -Sembre the culture medium (42.2.2.1) in inclined tubes, with Microfocus luteus. Incubate for 24 hours at 30 ° C, store in a refrigerator at about 4 °C and renew the sowing every 15 days.

42.2.1.2 Preparation of bacterial suspension. Collect the germs from a recent preparation agar tube (42.2.1.1), with 2 to 3 ml of sodium chloride solution (42.2.2.3). Sow with this suspension 250 ml of the culture medium (42.2.2.1) in a vial of Roux and incubate for eighteen to twenty hours at 30 ° C. Collect the germs in 25 ml of sodium chloride solution (42.2.2.3) and homogenise.

Dilute the suspension to 1/10 with the help of the sodium chloride solution (42.2.2.3). The light transmission of the suspension, measured at 650 nm under a thickness of 1 cm, by comparison with the sodium chloride solution (42.2.2.3), should be around 75 per 100. This suspension may be kept for a week at about 4 °C. Other methods may be used, provided they are shown to produce similar bacterial suspensions.

42.2.2 Crop media and reagents.

42.2.2.1 Maintenance of strain and base for determination. Any commercial culture medium of a similar composition can be used and of the same results:

Meat Peptone: 6.0 g.

Triptone: 4.0 g.

yeast extract: 3.0 g.

Meat extract: 1.5 g.

Glucose: 1.0 g.

Agar: 10.0 to 20.0 g.

Water 1,000 ml.

pH: 6.5 (after sterilization).

42.2.2.2 Tampon phosphate pH 6:

KHPO potassium hydrogen phosphate: 2 g.

Potassium dihydrogen phosphate KHPO: 8 g.

Water up to 1,000 ml.

42.2.2.3 Solution to 0.8 per 100 (w/v) sodium chloride:

Dissolve 8 g of sodium chloride in water, dilute up to 1,000 ml and sterilize.

42.2.2.4 Methanol.

42.2.2.5 Phosphate buffer (42.2.2.2) and methanol (42.2.2.4): 80/20 (v/v).

42.2.2.6 Solution in methanol to 0.5 per 100 (w/v) Tween 80: dissolve 5 g of Tween 80 in methanol (42.2.2.4) and dilute up to 1,000 ml with methanol.

42.2.2.7 Reference substance: virginiamycin of known activity.

42.3 Procedure.

42.3.1 Reference solutions. Dissolve a heavy quantity of exactly the reference substance (42.2.2.7) in methanol (42.2.2.4) and dilute with methanol (42.2.2.4) to obtain a 1,000 microgram of virginiamycina/ml. Preserved in a capped bottle at 4 ° C, this solution is stable for five days. Prepare from this solution and by successive dilutions with the mixture (42.2.2.5), the following solutions:

S: 1 microgram/ml.

S: 0.5 microgram/ml.

S: 0.25 microgram/ml.

S: 0.125 microgram/ml.

42.3.2 Preparation of the extract and solutions.

42.3.2.1 Extract.

42.3.2.1.1 Products whose virginiamycin content does not exceed 100 mg/kg. Weigh a sample amount of 50 g. Add 200 ml of solution (42.2.2.6), shake for thirty minutes and then desettle or centrifuge. Collect 20 ml of the supernatant solution and evaporate up to 5 ml in a rotary evaporator at a temperature not exceeding 40 ° C. Dissolve the residue with the help of the mixture (42.2.2.5) to obtain an assumed concentration of virginiamycin of 1 microgram/ml (= U).

42.3.2.1.2 Products whose virginiamycin content is greater than 100 mg/kg. Weigh a quantity of sample not exceeding 10,0 g and containing 1 to 50 mg of virginiamycin. Add 100 ml of solution (42.2.2.6), shake for thirty minutes, and leave to settle after or centrifuge. Dilute the supernatant solution with the help of the mixture (42.2.2.5) to obtain a concentration of virginiamycin of 1 microgram/ml (= U).

42.3.2.2 Extract solutions. Prepare, from the U8 solution and by successive dilutions (1 + 1) with the help of the mixture (42.2.2.5), the U solutions (assumed concentration = 0.5 microgram/ml), U (assumed concentration = 0.25 microgram/ml) and U (assumed concentration = 0.125 microgram/ml).

42.3.3 Modes of Determination.

42.3.3.1 Inoculation of the culture medium. Sow at about 50 ° C the base of the determination (42.2.2.1) with the bacterial suspension (42.2.1.2). By preliminary tests on plates with the medium (42.2.2.1), determine the amount of bacterial suspension that allows obtaining for the different concentrations of virginiamycin areas inhibition as wide as possible without ceasing to be (d)

42.3.3.2 Preparing the boxes. The agar diffusion is carried out in boxes with the four concentrations of the reference solution (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each box must necessarily receive the four reference concentrations and the extract. To this end, the dimensions of the boxes must be chosen so that they can be excavated in the middle of the agar at least 8 cavities 10 to 13 mm in diameter, the centers of which are not less than 30 mm. They can be used as flat glass plates, topped by an aluminium or plastic ring of 200 mm in diameter and 20 mm in height.

Insert into the boxes an amount of the medium (42.2.2.1), sown as indicated in (42.3.3.1), which allows to obtain a layer of about 2 mm thickness (60 ml for a box of 200 mm in diameter). Allow to solidify, excavate the cavities and place in them exactly measured volumes of the reference solutions and the extract (0.10 to 0.15 ml per cavity according to diameter). Repeat at least 4 times each concentration so that each determination comprises the assessment of 32 inhibition zones.

42.3.3.3 Incubation. Incubate the boxes for sixteen to eighteen hours at 30 ° C ± 2 ° C.

42.4 Calculations. Measure the diameter of the inhibition zones with an error of not more than 0,1 mm. For each concentration, record the mean measurements in semi-logarithmic paper, moving the logarithm of the concentrations relative to the diameters of the inhibition zones. Draw the tightest straight lines for the reference solution and for the extract, for example, using the following procedure:

Determine the most appropriate point of the lowest level of the reference solution (SL) with the help of the formula:

7S + 4S + S-2S

(a) SL = -----------------------

10

Determine the most appropriate point of the highest level of the reference solution (SH) with the help of the formula:

7S + 4S + S-2S

(a) SL = -----------------------

10

Determine the most appropriate points of the extract for the lowest level (UL) and the highest level (UH) by replacing S, S, S, and S in the above formulas with U, U, U, and U.

To record the SL and SH values in the same chart. When the two points are joined together, the correct line is obtained for the reference solution. The same method is followed for UL and UH, and the correct line is obtained for the extract.

If there is no interference, the straight lines are considered parallel when (SH-SL) and (UH-UL) do not differ by more than 10 per 100 of their average.

If the straight lines are not parallel, U and S or U and S. can be removed. The values SL, SH, UL, and UH that allow to obtain the tightest straight are then calculated with the help of the following formulas:

5S + 2S-S 5S + 2S-S

(a) SL = ------------------- or -------------------

6 6

5S + 2S-S 5S + 2S-S

(b) SH = ------------------- or -------------------

6 6

and analogous formulas for UL and UH. The use of this alternative requires the same criteria of parallelism to be respected.

In the analytical bulletin, a result from three levels should be mentioned.

When the straights are considered parallel, there are

calculate the logarithm of the relative activity (log A), using one of the following formulas, depending on the number of levels (4 or 3) used for the parallelism assessment.

For 4 levels:

(c) log A = (U + U + U + U-S-S-S-S) x 0.602

U + U + S + S-U-U-S-S

For 3 levels:

(d) log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

(d) log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

Sample extract activity = activity of the corresponding reference A, i.e. = S x A.

If the relative activity is not in the range of values between 0,5 and 2.0, the determination shall be repeated by proceeding to appropriate adjustments of the concentrations of the extract or, where appropriate, of the reference. Where this activity cannot be covered by the range of the required values, the result shall be considered to be approximate and shall be entered in the analysis sheet.

When the straights are not considered parallel, the determination will be repeated. If parallelism is not achieved now, then the determination will be considered unsatisfactory.

Express the result in milligrams of virginiamycin per kilogram of feed.

The difference between the results of two determinations made with the same sample by the same analyst should not exceed:

2 mg/kg, in absolute value, for the contents of virginiamycin less than 10 mg/kg.

20 per 100 of the highest result, for contents of 10 to 25 mg/kg.

5 mg/kg, in absolute value, for the contents of 25 to 50 mg/kg.

10 per 100 of the highest result, for contents greater than 50 mg/kg.

42.5 References.

Commission Directive of 20 December 1983 amending Directives 71/393, 72/199 and 7 8/6 3 3 (84/4/EEC). 'Official Journal of the European Communities' of 18 January 1984, number L 15/28, Annex II.

43. Bacitracin-zinc

(By Broadcast on agar)

43.1 Principle. The method enables the determination of bacitracin zinc in feed and premixtures. The lower limit of the determination is 5 mg/kg. 1 mg bacitracin-zinc (feed quality) is equivalent to 42 international units (IU).

The sample is extracted at pH 2 by a mixture of ethanol, water and hydrochloric acid and a sodium sulfide solution. The sodium sulfide allows to precipitate the soluble copper salts that could hinder the determination. The extract, carried at pH 6.5 is concentrated (if necessary) and dilutes. Its antibiotic activity is determined by the diffusion of the bacitracin-zinc in an agar medium, seeded with Micrococus luteus (flavus). The diffusion is manifested by formation of inhibition zones of the microorganism. The diameter of these zones is considered to be directly proportional to the logarithm of the antibiotic concentration for the range of concentrations used.

43.2 Reassets.

43.2.1 Microorganism: Microcosm luteus (flavus) ATCC 10240.

43.2.1.1 Maintenance of the strain.

Sow the culture medium (43.2.2.1) in inclined tubes, with Micrococcus luteus (flavus). Incubate for 24 hours at 30 ° C, store in a refrigerator at about 4 °C and renew the sowing every 15 days.

43.2.1.2 Preparation of bacterial suspension. Collect the germs from a recent preparation agar tube (43.2.1.1) with the help of 2 to 3 ml of the sodium chloride solution (43.2.2.3). Sow with this suspension 250 ml of the culture medium (43.2.2.1) in a vial of Roux and incubate for eighteen to twenty hours at 30 ° C. Collect the germs in 25 ml of sodium chloride solution (43.2.2.3) and homogenise.

Dilute the suspension to 1/10 with the help of the sodium chloride solution (43.2.2.3). The light transmission of the solution, measured at 650 nm under a thickness of 1 cm by comparison with the sodium chloride solution (43.2.2.3), should be around 75 per 100. This suspension may be kept for a week at about 4 °C. Other methods may be used provided they are shown to produce similar bacterial suspensions.

43.2.2. Culture media and reagents.

43.2.2.1 Strain maintenance medium.

Meat Peptone: 6.0 g.

Triptone: 4.0 g.

yeast extract: 3.0 g.

Meat extract: 1.5 g.

Glucose: 1.0 g.

Agar: 10.0 to 20.0 g.

Water: 1,000 ml.

pH: 6.5 to 6.6 (after sterilization).

Any commercial culture medium of analog composition can be used, which of the same results.

43.2.2.2 Base of determination.

Triptone: 10.0 g.

yeast extract: 3.0 g.

Meat extract: 1.5 g.

Glucose: 1.0 g.

Agar: 10.0 to 20.0 g.

Tween 80: 1 ml.

Water: 1,000 ml.

pH: 6.5 (after sterilization).

Any commercial culture medium of analog composition can be used, which of the same results.

43.2.2.3 Solution to 0.8 per 100 w/v sodium chloride: dissolve 8 g of sodium chloride in water, dilute to 1,000 ml and sterilize.

43.2.2.4 Methanol/water/hydrochloric acid mixture (43.2.2.6): 80/17 ,5/2, 5 (v/v/v).

43.2.2.5 Tampon phosphate pH 6.5.

Potassium hydrogen phosphate KHPO: 22.15 g.

KHPO potassium phosphate Kihydrogen: 27.85 g.

Water up to 1,000 ml.

43.2.2.6 Hydrochloric acid, d= 1.18-1.19.

43.2.2.7 Hydrochloric acid, 0.1M.

43.2.2.8 Sodium hydroxide solution 1 M.

43.2.2.9 Sodium sulphide solution 0.5 M approximately.

43.2.2.10 Bromoresol purple solution at 0.04 per 100 (w/v)

Dissolve 0.1 g of bromoresol purpura in 18.5 ml of 0.01 M solution of sodium hydroxide. Complete up to 250 ml with water and homogenise.

43.2.2.11 Reference substance: bacitracin-zinc of known activity (in IU).

43.3 Procedure.

43.3.1 Reference solutions. Weigh a quantity of reference substance (43.2.2.11) corresponding to 1050 IU (depending on the activity indicated). Add 5 ml of 0.1 M hydrochloric acid (43.2.2.7) and leave to rest for fifteen minutes. Add 30 ml of water, adjust the pH to 4.5 with phosphate buffer (43.2.2.5) (about 4 ml), complete up to 50 ml with water and homogenise (1 ml = 21 IU).

Prepare from this solution and by successive dilutions with the help of buffer pH 6.5 (43.2.2.5), the following solutions:

S: 0.42 IU/ml.

S: 0.21 IU/ml.

S: 0.105 IU/ml.

S: 0.0525 IU/ml.

43.3.2 Preparation of the extract.

43.3.2.1 Extract.

43.3.2.1.1 Mineral premixtures and correctors. Weigh a sample amount of 2.0 to 5.0 g, add 29.0 ml of the mixture (43.2.2.4) and 0.1 ml of the sodium sulfide solution (43.2.2.9); shake briefly. Check that the pH is about 2. Shake for ten minutes, add 30 ml of phosphate buffer (43.2.2.5), shake for fifteen minutes and centrifuge. Take an aliquot part of the supernatant solution and adjust the pH to 6.5 with sodium hydroxide solution 1 M (43.2.2.8) using a pH-meter or bromoresol purple solution as an indicator (43.2.2.10).

Dilute with phosphate buffer (43.2.2.5) to obtain with phosphate buffer (42.2.2.5) to obtain an assumed concentration in bacitracin-zinc of 0.42 IU/ml (= U).

43.3.2.1.2 Protein Concensates. Weigh a sample amount of 10.0 g, add 49.0 ml of mixture (43.2.2.4) and 1.0 ml of the sodium sulfide solution (43.2.2.9); shake briefly. Check that the pH is about 2. Shake for ten minutes, add 50 ml of phosphate buffer (43.2.2.5), shake for fifteen minutes and centrifuge. Take an aliquot part of the supernatant solution and adjust the pH to 6.5 with the bromoresol hydroxide solution as an indicator (43.2.2.10).

Evaporate about half of the volume in a rotating evaporator at a temperature not exceeding 35 ° C. Dilute with phosphate buffer (43.2.2.5) to obtain an assumed concentration of zinc bacitracin from 0.42 IU/ml (= U).

43.3.2.1.3 Other feed. Weigh a sample quantity of 10 g (20,0 or for an assumed concentration of bacitracin-zinc of 5 mg/kg). Add 24.0 ml of mixture (43.2.2.4) and 1.0 ml of the sodium sulfide solution (43.2.2.9); homogenise for ten minutes. Add 25 ml of phosphate buffer (43.2.2.5), shake for fifteen minutes, and centrifuge. Take 20 ml of the supernatant solution and adjust the pH to 6.5 with sodium hydroxide solution (43.2.2.8), using a neutral pH or bromoresol purple solution (43.2.2.10) as an indicator. Evaporate up to 4 ml in a rotary evaporator at a temperature not exceeding 35 ° C. Dilute the residue with phosphate buffer (43.2.2.5) to obtain an assumed concentration of bacitracin-zinc of 0.42 IU/ml (= U).

43.3.2.2 Extract solutions. Prepare, from solution U and by successive dilutions (1 + 1) with phosphate buffer (43.2.2.5) solutions U (assumed concentration = 0.21 IU/ml), U (assumed concentration = 0.105 IU/ml) and U (assumed concentration = 0.0525 IU/ml).

43.3.3 Modes of Determination.

43.3.3.1 Inoculation of the culture medium. Sow at about 50 ° C the base of the determination (43.2.2.2) with the bacterial suspension (43.2.1.2). By means of preliminary tests on plates with the medium (43.2.2.2), determine the amount of bacterial suspension it allows to obtain for the different concentrations of bacitracin-zinc inhibition zones as wide as possible without ceasing to be (d)

43.3.3.2 Preparation of the boxes. The agar diffusion is carried out in boxes with the four concentrations of the reference solution (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each box must necessarily receive the four reference concentrations and the extract. To this end, it is necessary to choose the size of the boxes in such a way that they can be excavated in the middle of agar at least 8 cavities 10 to 13 mm in diameter, the centers of which are not less than 30 mm. They can be used as boxes, flat glass plates, topped by an aluminium or plastic ring of 200 mm in diameter and 20 mm in height.

Introduce into the boxes an amount the medium (43.2.2.2), sown as indicated in 43.3.3.1, which allows to obtain a layer of about 2 mm thickness (60 ml for a box of 200 mm in diameter). Allow to solidify, excavate the cavities, and place in them exactly measured volumes of the reference solutions and the extract (0.10 to 0.15 ml per cavity, according to the diameter). Repeat at least four times each concentration so that each determination is the evaluation of 32 inhibition zones.

43.3.3.3 Incubation. Incubate the boxes for sixteen to eighteen hours at 30 ° C ± 2 ° C.

43.4 Calculations.

Measure the diameter of the inhibition zones with an error of not more than 0,1 mm. For each concentration, record the mean measurements in semi-logarithmic paper, moving the logarithm of the concentrations relative to the diameters of the inhibition zones. Draw the tightest straight lines for the reference solution and for the extract, for example, using the following procedure:

Determine the most appropriate point of the lowest level of the reference solution (SL) with the help of the formula:

(a) SL = 7S + 4S + S-2S

10

Determine the most appropriate point of the highest level of the reference solution (SH) with the help of the formula:

(b) SH = 7S + 4S + S-2S

10

Determine the most appropriate points of the extract for the lowest level (UL) and the highest level (UH) by replacing S, S, S, and S in the above formulas with U, U, U, and U.

To record the SL and SH values in the same chart. When the two points are joined together, the correct line is obtained for the reference solution. If you follow the same method for UL and UH you get the tightest straight for the extract.

If there is no interference, the straight lines would be parallel. In practice they are considered parallel when (SH-SL) and (UH-SL) do not differ more than 10 per 100 from their average.

If the straight lines are not parallel, U and S or U and S. can be removed. The values SL, SH, UL and UH that allow to obtain more adjusted straight lines, are then calculated with the help of the following formulas:

5S + 2S-S 5S + 2S-S

(a) SL = ------------------- or -------------------

6 6

5S + 2S-S 5S + 2S-S

(b) SH = ------------------- or -------------------

6 6

and analogous formulas for UL and UH. The use of this alternative requires the same criteria of parallelism to be respected. The analytical bulletin should mention the production of a result from three levels.

When the straights are considered parallel, the logarithm of the relative activity (log A) must be calculated using one of the following formulas, depending on the number of levels (4 or 3) used for the parallelism assessment.

For 4 levels:

(c) log A = (U + U + U + U-S-S-S-S) x 0.602

U + U + S + S-U-U-S-S

For 3 levels:

(d) log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

(d) log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

Sample extract activity = activity of the corresponding reference x A, i.e. U = S x A.

If the relative activity is not in the range of values between 0,5 and 2.0, the determination shall be repeated by proceeding to appropriate adjustments of the concentrations of the extract or, where appropriate, of the reference. Where this activity cannot be covered by the range of the required values, the result shall be considered to be approximate and shall be entered in the analysis sheet.

When the straights are not considered parallel, the determination will be repeated. If the parallelism is not achieved now, the determination will be considered unsatisfactory.

Express the result in milligrams of bacitracin-zinc per kilogram of feed.

The difference between the results of two determinations made with the same sample by the same analyst should not exceed:

2 mg/kg, in absolute value, for the contents of bacitracin-zinc less than 10 mg/kg.

20 per 100 of the highest result, for contents of 10 to 25 mg/kg.

5 mg/kg, in absolute value, for the contents of 25 to 50 mg/kg.

10 per 100 of the highest result, for contents greater than 50 mg/kg.

43.5 References. Commission Directive of 20 December 1983 amending Directives 71/393, 72/199 and 78/633 (Directive 84 /4/EEC). 'Official Journal of the European Communities' of 18 January 1984, number L 15/28, Annex III.

44. Flavophospholipol

(By Broadcast on agar)

44.1 Principle. The method allows the dosing of flavophospholipol in feed, concentrates and premixtures. The lower dose limit is 1 mg/kg.

The sample is subjected to extraction by diluted methanol, by heating to reflux. The extract is centrifuged, purified, if necessary, in ion exchange resins, and diluted. The antibiotic activity is determined by the diffusion of flavophospholipol in an agar medium, seeded with Staphylococcus aureus. The diffusion is revealed by the formation of areas of inhibition of microorganisms. The diameter of these zones is considered to be directly proportional to the logarithm of the concentrations used.

44.2 Reassets.

44.2.1 Microorganism: Staphylococcus aureus ATCC 6538 P.

44.2.1.1 Maintenance of the strain. Sow the culture medium (44.2.2.1) in inclined tubes, with Staphylococcus aureus. Incubate for twenty-four hours at 37 ° C, store in refrigerator at approximately 4 ° C and renew the sowing every month.

44.2.1.2 Preparation of bacterial suspension. Take two tubes containing the mother crop (4.2.2.1) and renew the sowing each week. Incubate 24 hours at 37 ° C and store in a refrigerator at approximately 4 °C.

Twenty-four hours before dosing, sowing by means of these crops, two to four inclined tubes containing culture medium (44.2.2.1).

Incubate from sixteen to eighteen hours at 37 ° C. Then put the germs in suspension in the sodium chloride solution (44.2.2.3). The light transmission of the suspension, measured at 578 nm under a thickness of 1 cm, by comparison with the sodium chloride solution, shall be approximately 40 per 100. Other methods may be used if they show that they produce a similar bacterial suspension.

44.2.2 Crop media and reagents.

44.2.2.1 Strain maintenance medium.

Meat Peptone: 6.0 g.

Triptone: 4.0 g.

yeast extract: 3.0 g.

Meat extract: 1.5 g.

Glucose: 1.0 g.

Agar: 15.0 g.

Water: 1,000 ml.

pH = 6.5 after sterilization.

Any commercial culture medium of analog composition may be used and of the same results, for example Oxoid Antibiotic Medium (CM 327) added to agar Oxoid number 3 (L 13).

44.2.2.2 Basis of dosing.

44.2.2.2.1 Lower Layer.

Meat Peptone: 6.0 g.

yeast extract: 3.0 g.

Meat extract: 1.5 g.

Agar: 10.0 g.

Water: 1,000 ml.

pH = 6.5 after sterilization.

Any commercial culture medium of analog composition may be used and of the same results, for example Oxoid Antibiotic Medium 2 (CM 335) added to agar Oxoid number 3 (L 13).

44.2.2.2.2 Layer to seed. Measure (4.2.2.1) with 2 g of silicone anti-foaming emulsion. For example SE 2 of Wacker Chemie Gmon, Munich.

44.2.2.3 Solution to 0.4 per 100 (w/v) sodium chloride:

Dissolve 4 g of sodium chloride p.a. in water, dilute up to 1,000 ml and sterilize.

44.2.2.4 Pure Methanol.

44.2.2.5 Methanol to 50 per 100 (v/v). Dilute 500 ml of methanol with 500 ml of water.

44.2.2.6 Methanol to 80 per 100 (v/v). Dilute 800 ml of methanol 44.2.2.4 with 200 ml of water.

44.2.2.7 Tris (hydroxymethyl) aminomethane p.a.

44.2.2.8 Methanolic solution at 1.5 per 100 (w/v) potassium chloride. Dissolve 1.5 g of potassium chloride p.a. in 20 ml of water, complete up to 100 ml with methanol (44.2.2.4).

44.2.2.9 Cation exchanger: Dowex 50 WX8, 20-25 mesh, form Na (cat, Serva number 41600) or equivalent.

44.2.2.10 Anion Exchanger: Dowex 1X2, 50 100 mesh, form Cl/cat. Serva number 41010) or equivalent. Before use, keep the product for twelve to fourteen hours in methanol at 80 per 100 (44.2.2.6).

44.2.2.11 Glass wool.

44.2.2.12 pH indicator paper (pH 6,6-8, 1).

44.2.2.13 Ascorbic acid.

44.2.2.14 Pattern substance. Flavophospholipol of known activity.

44.3 Material and apparatus.

44.3.1 Tubo for chromatography, glass, internal diameter: 9 mm, length 150 to 200 mm, fitted with a key in the lower narrowing and standard grinding (for splicing of the funnel) at the top.

44.3.2 Embudo with 250 ml deposit, with a frosted key, normalized.

44.3.3 Erlenmeyer 250 ml, standard grinding.

44.3.4 Refrigerant reflux, normalised.

44.4 Procedure.

44.4.1 Pattern solutions. Dissolve a quantity of exactly heavy standard substance (44.2.2.14) in methanol at 50 per 100 (44.2.2.5) and dilute to obtain a 100 microgram/ml flavophospholipol stock solution. Preserved in a capped bottle at 4 ° C, this solution is stable for two months.

Prepare from such solution, and by successive dilutions, by methanol at 50 per 100 (44.2.2.5) the following solutions:

S: 0.2 microgram/ml.

S: 0.1 microgram/ml.

S: 0.05 microgram/ml.

S: 0.025 microgram/ml.

44.4.2 Preparation of the extract.

44.4.2.1 Extract.

44.4.2.1.1 Concentric, premixtures and mineral correctors. Weigh a sample quantity of 2 to 5 g and add about 150 mg ascorbic acid (44.2.2.13). Mix with 150 ml of methanol at 50 per 100 (44.2.2.5) in an erlenmeyer (44.2.3.3) and adjust the pH to 8.1-8.2 by about 400 mg tris (hydroxymethyl) aminomethane (44.2.2.7). Control of pH by indicator paper (44.2.2.12). Leave to macerate fifteen minutes, adjust the pH again to 8,1-8.2, by tris (hydroxymethyl) aminomethane (44.2.2.7), then boil for ten minutes with coolant to reflux (44.2.3.4), stirring constantly. Leave to cool, centrifuge and decant the extract.

44.4.2.1.2 Other feed. Weigh a sample quantity of 5 to 30 g containing at least 30 micrograms of flavophospholipol. Mix 150 ml of methanol to 50 per 100 (44.2.2.5) in an erlenmeyer (44.2.3.3) and adjust the pH to 8.1-8.2 with approximately 400 mg of tris (hydroxymethyl) aminomethane (4.2.2.7). Control pH by indicator paper. Leave to macerate fifteen minutes, adjust the pH again to 8.1-8.2 with tris (hydroxymethyl) aminomethane and then boil for ten minutes with reflux coolant (44.2.3.4) stirring constantly. Leave to cool, centrifuge and decant the extract.

44.4.2.2 Purification (such modality may be omitted for concentrates, premixtures and mineral correctors).

Mix 110 ml of the extract with 11 g of cations exchanger (44.2.2.9), boil for one minute with reflux coolant (44.2.3.4) constantly stirring. Separate the cation exchanger by centrifugation or filtration. Mix 100 ml of the extract with 150 ml of methanol (44.2.2.4) and allow the solution of twelve to fifteen hours at 4 ° C. Remove the flocculated matter by cold filtration.

Put in the lower end of a tube (44.2.3.1) a glass wool stopper (44.2.2.11), pour in the tube 5 ml anion exchanger (44.2.2.10) and wash the column with 100 ml of methanol at 80 per 100 (44.2.2.6). Then transfer to the column through the funnel (44.2.3.2) a filter volume of 100 ml that is supposed to contain at least 16 micrograms of flavophospholipol (200 ml for a sample of 30 g of feed at 1 ppm). If necessary, dilute the filtrate before transferring it to the column with methanol at 80 per 100 (44.2.2.6) to obtain a suspected concentration of 16 micrograms flavophospholipol in 100 ml. Regulate the flow rate of liquid to 2 ml approximately per minute. Remove the entire filtering. Then wash the column with 50 ml of methanol at 80 per 100 (44.2.2.6) and remove the filtrate.

Elude flavophospholipol by means of the potassium chloride methanolic solution (44.2.2.8) by maintaining the drip flow rate of approximately 2 ml per minute. Collect 50 ml of the elution in a graduated flask, add 30 ml of water and homogenise. This solution should have a content in flavophospholipol of 0.2 micrograms/ml (= U).

44.4.2.3 Extract solutions. If necessary, (in particular in cases where purification has been omitted) dilute the extract obtained in 44.4.2.1.1 with methanol to 50 per 100 (44.2.2.5) to obtain an assumed concentration in flavophospholipol of 0.2 microgram/ml (= (u).

Prepare from solution U for successive dilutions (1 + 1) by methanol at 50 per 100 (44.2.2.5) solutions U (assumed concentration = 0.1 microgram/ml), U (assumed concentration = 0.05 microgram/ml) and U (assumed concentration = 0.025 microgram/ml).

44.4.3 Dose Modes.

44.4.3.1 Inoculation of the culture medium. Sow at 50 ° C approximately the base of the dosage (44.2.2.2.2) with the bacterial suspension (44.2.1.2). By means of preliminary layered tests with the medium (44.2.2.2.2) determine the amount of bacterial suspension to be obtained for the different concentrations in flavophospholipol, as extensive inhibition zones as possible and that remain clear (about 30 ml per litre).

44.4.3.2 Preparation of the boxes. The agar diffusion is performed in boxes with the four concentrations of the standard solution (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each box must necessarily receive the four concentrations of the pattern and the extract. To this end, choose the size of the boxes so that they can be hollowed out in the middle of agar at least 8 cavities of 10 to 13 mm in diameter, the centers of which are not separated by less than 30 mm. It may be used as boxes, flat glass plates, topped by an aluminium or plastic ring of 200 mm in diameter and 20 mm in height.

Introduce into the boxes an amount of the medium (44.2.2.2.1) that allows to obtain a layer of 1.5 mm approximately thickness (45 ml for a box of 200 mm in diameter). Allow to solidify and add a quantity of the medium (44.2.2.2.2), sown as indicated in point 44.3.3.1, which allows to obtain a layer of 1 mm thick (30 ml for a box of 200 mm in diameter). Leave to solidify, hollowed out the cavities and place in the same the exact measured volumes of the standard solutions and the extract (0.10 to 0.15 ml per cavity, according to diameter).

Make at least four repetitions of each concentration, so that each determination is the subject of an evaluation of 32 inhibition zones.

43.4.3.3 Incubation. Incubate the boxes from sixteen to eighteen hours at 29-30 ° C.

44.5 Calculations. Measure the diameter of the inhibition zones with a 0.1 mm approach. For each concentration, record the measurements in semi-logarithmic paper by scoring the logarithm of the concentrations against the diameters of the inhibition zones. Draw the best-fitting straights for the standard solution and for the extract by proceeding, for example, as follows:

Determine the most appropriate point of the lowest level of the standard solution (SL) using the formula:

(a) SL = 7S + 4S + S-2S

10

Determine the most appropriate point of the highest level of the standard solution (SH) using the formula:

(b) SH = 7S + 4S + S-2S

10

Determine the most appropriate points of the extract for the lowest level (UL) and the highest level (UH) by replacing S, S, S, and S in the formulas mentioned above by U, U U, and U.

Represent the SL and SH values in the same chart. By joining the two points, you will get the tightest straight for the standard solution. By proceeding in the same way for UL and UH, the correct line is obtained for the extract.

In the absence of any interference, the straights should be parallel. In practice, they shall be treated as if they were parallel when (SH-SL) and (UH-UL) do not differ by more than 10 per 100 from their average.

If the straight lines are not parallel, they may be removed either U and S or U and S. The values SL, SH, UL and UH which allow to obtain the most fitting straight lines shall then be calculated by the following formulae:

5S + 2S-S 5S + 2S-S

(a ') SL = ------------------- or -------------------

6 6

5S + 2S-S 5S + 2S-S

(b) SH = ------------------- or -------------------

6 6

and similar formulas for UL and UH. The use of this alternative shall also be the subject of a verification as to the parallelism of the straight lines indicated above. It should be mentioned in the analysis bulletin that a result from three levels is obtained.

When the straight lines are considered parallel, the logarithm of the relative activity (log A) will be calculated using one of the following formulas:

(c) log A = (U + U + U + U-S-S-S-S) x 0.602

U + U + S + S-U-U-S-S

For 3 levels:

(d) log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

(d ') log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

Actual activity = assumed activity x relative activity.

When the straights are not considered as non-parallel, repeat the determination. If the same will continue without allowing parallelism to be reached, calculate the logarithm of the relative activity (log. (a) by means of formula (c). However, the result will be considered as approximate and it will be appropriate to mention it in the analysis bulletin.

The difference between the results of two determinations made in the same sample by the same analyst must not exceed:

-0,5 mg/kg, in absolute value, for the contents of flavophospholipol from 1 to 2 mg/kg.

-25 per 100 of the highest result for contents greater than 2 mg/kg and up to 10 mg/kg.

-20 per 100 of the highest result, for contents greater than 10 mg/kg and up to 25 mg/kg.

-5 mg/kg, in absolute value, for contents greater than 25 mg/kg and up to 50 mg/kg.

-10 per 100 of the highest result, for contents greater than 50 mg/kg.

44.6 References. Eighth Commission Directive of 15 June 1978 (78/633/EEC). 'Official Journal of the European Communities' number L 206 of 29 July 1978, page 43, pages 5 to 9 of the Annex.

45. Tylosin

(By Broadcast on agar)

45.1 Principle. The method makes it possible to determine tylosin in feed, concentrates and premixtures. The lower limit of the determination is 2 mg/kg.

The sample is treated by buffer solution pH 8, previously carried at 80 ° C, and then subjected to extraction by methanol. After centrifugation, the extract is diluted and its antibiotic activity is determined by the measurement of the diffusion of tylosin in the medium of agar sown with Sarcina lutea. The diffusion is indicated by the formation of inhibition zones in the presence of the microorganism. The diameter of these areas is directly proportional to the logarithm of the antibiotic concentration.

45.2 Reassets.

45.2.1 Microorganism: Sarcina lutea ATCC number 9341.

45.2.1.1 Maintenance of the strain. Seed Sarcina lutea in agar tube inclined with the culture medium (45.2.2.1) adjusted at pH 7.0. Incubate for one night at approximately 35 ° C. Preserve the culture in refrigerator and resembrate every month on the inclined agar.

45.2.1.2 Preparation of the germ suspension. Collect the germs from a newly prepared inclined agar tube (45.2.1.1), with 2 to 3 ml of physiological serum (45.2.2.4). Sow with said suspension a vial of Roux containing 250 ml of the culture medium (45.2.2.1), adjusted at pH 7.0. Incubate for twenty-four hours at 35 ° C, collect the germs with 25 ml of physiological serum (45.2.2.4). Homogenise and dilute said suspension to obtain a luminous transmission of 75 per 100 at approximately 650 nm.

Conservated in refrigerator, said suspension is usable for a week.

By means of preliminary tests on plates with the base of the determination (45.2.2.1), determine the quantity of inoculum to be obtained, for the different concentrations of tylosin used, areas of inhibition as extensive as possible that are also sharp. The inoculation of the culture medium is 48-50 ° C.

45.2.2 Crop and reagent media.

45.2.2.1 Base for determination.

Glucose: 1.0 g.

Peptona trypsica: 10.0 g.

Meat extract: 1.5 g.

yeast extract: 3.0 g.

Agar according to quality: 10.0 to 20.0 g.

Distilled water up to 1,000 ml.

Adjust at the time of use at pH 7.0 for maintenance of the strain and preparation of the germ suspension and at pH 2.0 for determination.

Any commercial culture medium of analogous composition and which of the same results, can be employed.

45.2.2.2 Tampon phosphate pH 8.

Potassium dihydrogen phosphate KHPO p.a.: 0,523 g.

Potassium hydrogen phosphate KHPO p.a.: 16,730 g.

Distilled water up to 1,000 ml.

45.2.2.3 Tampon phosphate pH 7.

Potassium dihydrogen phosphate KHPO p.a.: 5.5 g.

Potassium hydrogen phosphate KHPO p.a.: 13.6 g.

Distilled water up to 1,000 ml.

45.2.2.4 Sterile physiological suero.

45.2.2.5 Pure Methanol.

45.2.2.6 Methanol at 10 per 100 (v/v).

45.2.2.7 phosphate buffer (45.2.2.2 )/pure methanol (60/40 v/v).

45.2.2.8 Reference substance: known tylosin.

45.3 Procedure.

45.3.1 Reference solutions. Dry the reference solution (45.2.2.8) for three hours at 60 ° C in a vacuum stove (5 mm mercury). Weigh 10 to 50 mg to a graduated flask, dissolve them in 5 ml of methanol (45.2.2.5) and dilute the solution using phosphate buffer pH 7 (45.2.2.3) to obtain a base tylosine concentration of 1,000 micrograms/ml. From said solution-mother, prepare by mixing (45.2.2.7) a reference work solution containing 2 micrograms of tylosin base per ml.

Prepare next for successive dilutions (1 + 1) with the help of the mixture (45.2.2.7), the following concentrations:

S: 1 micrograms/ml.

S: 0.5 micrograms/ml.

S: 0.25 micrograms/ml.

45.3.2 Extract. Take for products highly concentrated in tylosin a sample portion of 10 g; for premixtures and feed a sample portion of 20 g. Add 60 ml of buffer pH 8 (45.2.2.2), previously heated to 80 ° C, and homogenise for two minutes (Ultra-turrax appliances etc.)

Let stand for ten minutes, add 40 ml of methanol (45.2.2.5) and homogenise for five minutes. Centrifuge, take an aliquot part of extract and dilute by solution (45.2.2.7) to obtain an assumed concentration in tylosin of 2 micrograms/ml (= U). Prepare the U, U and U concentrations by successive dilutions (1 + 1) with the aid of the solution (45.2.2.7).

For contents less than 10 mg/kg evaporate the dry extract in a rotary evaporator at 35 ° C and collect the residue by methanol at 40 per 100 (45.2.2.6).

45.3.3 Modes of Determination.

45.3.3.1 Siembra of the culture medium. Sow at 48-50 ° C the base of the determination (45.2.2.1), adjusted to pH 8, with the suspension of germs (45.2.1.2).

45.3.3.2 Preparation of the boxes. The agar diffusion is carried out in boxes with the four concentrations of the reference solution (S, S, S and S) and the 4 concentrations of the extract (U, U, U and U). Each box must necessarily receive the four reference concentrations and the extract.

To this end, choose the dimensions of the boxes so that they can be practiced in the middle of agar at least eight cavities of 10 to 13 mm in diameter. Calculate the quantity of the seed growing medium (45.3.3.1) to be used so that a uniform coating of approximately 2 mm thickness can be obtained. It is preferable to use as boxes, flat glass plates, with an aluminium ring or a perfectly flat plastic material of 200 mm in diameter and 20 mm in height.

Introduce into the cavities by pipette amounts exactly measures of antibiotic solution comprised between 0.10 and 0.15 ml, according to diameter.

For each sample, make at least four diffusion repetitions with each concentration, so that each determination is the subject of an evaluation of 32 inhibition zones.

45.3.3.3 Incubation. Incubate the boxes for one night at 35 ± 37 ° C.

45.4 Calculations. Measure the diameter of the inhibition zones, preferably by projection. Represent the measures on semi-logarithmic paper, taking the logarithm of the concentrations in relation to the diameters of the inhibition zones. Draw the reference solution and extract straight lines. In the absence of interference, the two straight lines shall be parallel.

The logarithm of the relative activity is calculated by the following formula:

(U + U + U + U-S-S-S-S) x 0.602

U + U + S + S-U-U 4 S-Actual activity = assumed activity x relative activity.

The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 per 100, in relative value.

45.5 References. Third Commission Directive of 27 April 1972 (72/199/EEC). 'Official Journal of the European Communities' number L 123/6 of 29 May 1972.

46. Spiramycin

(Broadcast method on agar)

46.1 Principle. The method allows for the determination of spiramycin in feed and premixtures. The lower limit of detection is 1 ppm (1 mg spiramycin is equivalent to 3,200 international units). The sample is subjected to extraction with a mixture of methanol and phosphate-hydrogen carbonate at pH 8. The extract is decanted or centrifuged, and then diluted. The antibiotic activity of the extract is determined by measuring the diffusion of the spiramycin in an agar medium, seeded with Micrococus luteus. The diffusion is manifested by the formation of zones of inhibition of the microorganism. The diameter of these zones is considered to be directly proportional to the logarithm of the antibiotic concentration for the range of concentrations used.

46.2 Reassets.

46.2.1 Microorganism: Microfocus luteus ATCC 9341 (NCTC 8340, NCIB 8553).

46.2.2 Maintenance of the strain. Sow the culture medium (46.2.4) in inclined tubes, with Microfocus luteus. Incubate for 24 hours at 30 ° C, store in refrigerator at approximately 4 ° C and renew the sowing every fortnight.

46.2.3 Preparation of bacterial suspension (46.5.1). Collect the germs in a newly prepared agar tube (46.2.2), with 2 to 3 ml of sodium chloride solution (46.2.6). Sow 250 ml of culture medium (46.2.4) with this suspension in a Roux flask and incubate for eighteen to twenty hours at 30 ° C. Collect the germs in 25 ml of sodium chloride solution (46.2.6) and homogenise. Dilute the suspension to 1/10 with the help of the sodium chloride solution (46.2.6). The light transmission of the suspension, measured at 650 nm, under a thickness of 1 cm by comparison with the sodium chloride solution (46.2.6), shall be approximately 75 per 100. This suspension may be stored for approximately one week at 4 °C.

46.2.4 strain maintenance culture media (46.5.2).

Meat Peptone: 6.0 g.

Triptone: 4.0 g.

yeast extract: 3.0 g.

Meat extract: 1.5 g.

Glucose: 1.0 g.

Agar: 10.0 to 20.0 g.

Water: 1,000 ml.

pH 6.5-6.6 (after sterilization).

46.2.5 Base culture medium of determination (46.5.2).

Triptone: 5.0 g.

yeast extract: 4.0 g.

Meat extract: 3.0 g. Agar: 10,0 to 20,0 g.

Water: 1,000 ml.

pH: 8.0 (after sterilization).

46.2.6 Solution to 0.8 per 100 (w/v) sodium chloride. Dissolve in water 8 g of sodium chloride, dilute in 1,000 ml and sterilise.

46.2.7 Tampon phosphate-hydrogencarbonate, pH 8.0.

KHPO potassium hydrogen phosphate: 16.7 g.

KHPO potassium dihydrogen phosphate: 0.5 g.

Hydrogen Sodium carbonate NaHCO: 20.0 g.

Water up to 1,000 ml.

46.2.8 Methanol and phosphate-bicarbonate buffer (46.2.7). 50/50 (v/v).

46.2.9 Pattern Substance. Known activity spiramycin (IU).

46.2.10 Pattern solution. Dissolve an exact amount of the standard substance (46.2.9) in the mixture (46.2.8) and dilute with the same mixture to obtain a 1000 IU solution of spiramicine/ml.

Conservated in a bottle with a 4 ° merilylated cap, this solution is stable for five days.

Prepare from this solution and by successive dilutions with the help of the mixture (46.2.8) the following solutions:

S: 1 IU/ml.

S: 0.5 IU/ml.

S: 0.25 IU/ml.

S: 0.125 IU/ml.

46.3 Procedure.

46.3.1 Extraction. Weigh a sample quantity of 20.0 g for feed and 1.0 to 20.0 g for premixtures. Add 100 ml of mixture (46.2.8) and shake for thirty minutes.

Centrifuge or decantar, then dilute the solution that floats on the surface with the help of the mixture (46.2.8) to obtain an assumed concentration in spiramycin of 1 IU/ml (U).

For spiramycin contents below 2,5 mg/kg of feed, perform the extraction as follows. Weigh a sample amount of 20.0 g. Add 100 ml of mixture (46.2.8), shake for thirty minutes, then centrifuge for a few minutes. Take 50 ml of the supernatant solution and evaporate up to 4 ml approximately under reduced pressure in a rotary at a temperature not exceeding 40 °C. Dilute the residue with the help of the mixture (46.2.8) to obtain an assumed concentration in spiramycin 1 IU/ml (U).

Starting from the U solution, prepare for successive dilutions (1 + 1), with the help of the mixture (46.2.8), the solutions whose concentrations are: U (0.5 IU/ml) U (0.25 IU/ml) and U (0.125 IU/ml).

46.3.2 Inoculation of the culture medium. Sow at about 50 ° C approximately the base of the determination (46.2.5) with the bacterial suspension (46.2.3). By means of preliminary tests on plates with the medium (46.2.4), determine the amount of bacterial suspension to be obtained for the different concentrations of spiramycin, the most extensive inhibition zones that are still possible. Clean.

46.3.3 Preparation of the plates. The agar diffusion is carried out on plates with four concentrations of the standard solution (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each plate must necessarily receive the four concentrations of the pattern and the extract. To this end, the dimensions of the plates must be chosen so that at least eight cavities of 10 to 13 mm in diameter can be carried out in the agar, the centres of which are at least 30 mm. Flat glass plates, capped by an aluminium or plastic ring of 200 mm in diameter and 20 mm in height, can be used as plates.

Introduce into the plates an amount of the medium (46.2.5), sown as indicated in (46.3.2), which allows to obtain a layer of 2 mm thick (60 ml for a plate of 200 mm in diameter). Allow to solidify, to effect the cavities and to deposit exactly measured volumes of solutions of the pattern and the extract (0.10 to 0.15 ml per cavity, according to diameter). Perform at least four repetitions of each concentration so that each determination is the subject of an evaluation of 32 inhibition zones. Incubate the plates from sixteen to eighteen hours at 30 ° C ± 2 ° C.

46.4 Calculations. Measure the diameter of the inhibition zones with a precision of 0,1 mm. For each concentration, record the mean values in semi-logarithmic paper, leading the logarithms of the concentrations on the diameters of the inhibition zones. Draw the tightest straight lines for the standard solution and for the extract by proceeding, for example, as follows:

Determine the most appropriate point of the lowest level of the pattern solution (SL) with the help of the formula:

(a) SL = 7s + 4s + s-2s

10

Determine the most appropriate point of the highest level of the standard solution (SH) using the formula:

(b) SH = 7s + 4s + s-2s

10

Determine in the same way the most appropriate points of the extract for the lowest level (UL) and the highest level (UH) replacing s, s, s and s in the formulas cited above by u, u, u and u. Being the lowercase letters "s" and "u" the diameters of the inhibition zones. Bring the SL and SH values to the same chart. By joining the two points you get the tightest straight for the standard solution. Proceeding the same way for UL and UH you get the tightest straight for the extract.

If there is no interference, the straights will be parallel. In practice, they are considered parallel when SH-SL and UH-UL do not differ by more than 10 per 100 of their means.

If the straights are not parallel, they can be removed either u and s or u and s. The values SL, SH, UL, and UH that allow to get the tightest straight are then calculated with the help of the following formulas:

5s + 2s-s 5s + 2s-s

(a ') SL = ------------------- or -------------------

6 6

5s + 2s-s 5s + 2s-s

(b ') SH = ------------------- or -------------------

6 6

and similar formulas for UL and UH. The use of this alternative means that the same parallelism criteria are respected. Obtaining a result from three levels should be mentioned in the analysis bulletin.

When the straights are considered parallel, calculate the logarithm of the relative activity (log. A) using one of the following formulas, depending on the number of levels (four or three) used for the evaluation of parallelism.

For four levels:

(c) log A = (u + u + u + u-s-s-s) x 0.602

u + u + s + s-u-u-s-s

For 3 levels:

(d) log A = (u + u + u-s-s-s) x 0.401

u + s-u-s

(d ') log A = (u + u + u-s-s-s) x 0.401

u + s-u-s

Sample extract activity = Activity of the corresponding pattern x A

(U-S x A)

If the relative activity is not in the range of values between 0.5 and 2.0 repeat the determination proceeding to appropriate adjustments of the concentrations of the extract or, eventually, of the standard solutions. Where this activity cannot be carried over to the range of the required values, the result shall be considered approximate and this indication shall be entered in the analysis sheet.

When the straights are considered non-parallel, repeat the determination. If the parallelism is never reached, the determination must be considered unsatisfactory.

Express the result in mg of spiramycin base per kg of feed.

The differences in the results of two parallel determinations made with the same sample by the same person must not exceed:

-2 mg/kg, in absolute value, for base spiramycin contents of less than 10 mg/kg.

-20 per 100 of the highest result for contents of 10 to 25 mg/kg.

-5 mg/kg, in absolute value, for the contents of 25 to 50 mg/kg.

-10 per 100 of the highest result for contents greater than 50 mg/kg.

46.5 Remarks.

46.5.1 Other methods may be used, provided they are shown to produce similar bacterial suspensions.

46.5.2 Any commercial culture medium of analogous composition can be used which of the same results.

46.6 References. 10th Community Directive of 25 July 1984, 84 /425/EEC. 'Official Journal of the European Communities' number L 238/34 of 6 September 1984.

47. Monensin sodium

(Broadcast method on agar)

47.1 Principle. The method enables the determination of sodium monensin in feed and premixtures. The lower limit of detection is 10 mg/kg (1 mg monensin sodium is equivalent to 1000 U, "UK").

The sample is extracted with methanol at 90 per 100. The extract is applied to the appropriate treatments according to the sodium monensin content of the sample. Its antibiotic activity is determined by the diffusion of monensin sodium in the medium of agar sown with Bacillus subtilis. The diffusion is manifested by the formation of zones of microorganism inhibition. The diameter of these areas is considered to be directly proportional to the logarithm of the antibiotic concentration for the range of concentrations used. The sensitivity of this method is reduced in the presence of sodium ions.

47.2 Material and apparatus.

47.2.1 Rotary Evaporator, with 250 ml round-bottom flask.

47.2.2 Glass columns for chromatography, inside diameter: 25 mm, length: 400 mm, with a diameter of 2 mm at the lower end.

47.2.3 Glass column for chromatography, inside diameter: 11 mm, length: approximately 300 mm, with a diameter of 2 mm at the lower end.

47.3 Reassets.

47.3.1 Microorganism: Bacillus subtilis ATCC 6633 (NCIB 8054).

47.3.2 Conservation of the strain.

Sow the culture medium (47.3.4), in inclined tubes and with Bacillus subtilis. Incubate for one night at 30 ° C, store in a refrigerator at 4 ° C and renew the sowing every month.

47.3.3 Preparation of spores suspension (47.6.1). Collect the germs from a newly prepared agar tube (47.3.2), with help of 2 to 3 ml of sterile water. Sow with this suspension 300 ml of culture medium (47.3.4) in a vial of Roux and incubate three to five days at 30 ° C. After controlling the sporulation under a microscope, collect the spores in 15 ml of ethanol (47.3.6) and homogenise. This suspension may be stored for five months at approximately 4 °C.

47.3.4 Strain maintenance culture medium (47.6.2).

Triptone: 10.0 g.

yeast extract: 3.0 g.

Meat extract: 1.5 g.

Glucose: 1.0 g.

Agar (as per quality): 10.0 to 20.0 g.

Water: 1,000 ml.

pH: 6.5 (after sterilization).

47.3.5 Base culture medium for determination.

Glucose: 10.0 g.

yeast extract: 2.5 g.

Potassium Hydrogen Phosphate (KHPO): 0.69 g.

Potassium Dihydrogen Phosphate (KHPO): 0.45 g.

Agar (as per quality): 10.0 to 20.0 g.

Water: 1,000 ml.

pH: 6.0 (after sterilization).

47.3.6 Ethanol at 20 per 100 (v/v). Dilute 200 ml of ethanol with 800 ml of water.

47.3.7 Methanol anhydrous.

47.3.8 Methanol to 90 per 100 (v/v). Dilute 900 ml of methanol (47.3.7) with 100 ml of water.

47.3.9 Methanol at 50 per 100 (v/v). Dilute 500 ml of methanol (47.3.7) with 500 ml of water.

47.3.10 Pellated Aluminium Oxide (Alcoa F, 20 mesh: Activated Alumina UG: F. Lancester and Co., or equivalent).

47.3.11 Pattern substance: monensin sodium of known activity (available in International Laboratory for Biological Standards, Central Veterinary Laboratory, Weybridge, Surrey Kt15 3 NB, Great Britain).

47.3.12 Pattern solutions. Dissolve an exactly heavy quantity of the standard substance (47.3.11) in methanol (47.3.7), dilute to obtain a sodium monensin solution of 800 micrograms/ml. Store in bottle capped at 4 °C, this solution is stable for two weeks.

Prepare from this solution and for successive dilutions with methanol at 50 per 100 (47.3.9), the following solutions:

S: 8 micrograms/ml.

S: 4 micrograms/ml.

S: 2 micrograms/ml.

S: 1 micrograms/ml.

47.4 Procedure.

47.4.1 Extraction. Premixtures. Weigh 2.0 g of sample, add 100 ml of methanol to 90 per 100 (47.3.8), homogenise and centrifuge below for a few minutes. Dilute the supernatant solution with methanol to 50 per 100 (47.3.9) to obtain an approximate concentration of 8 micrograms/ml (U) sodium monensin.

47.4.2 Extract. Feed in which the sodium monensin content is not less than 50 mg/kg.

weigh 10,0 to 20.0 g of sample, add 100 ml of methanol to 90 per 100 (47.3.8), homogenise for fifteen minutes and leave to rest.

Introduce a cotton stopper at the lower end of the glass column (47.2.2), add the aluminium oxide (47.3.10), give light shocks to the column, until the filling is 75 to 80 mm high.

Decant the extract in the aluminum oxide column and collect the filtrate. Dilute 30 ml of the filtrate to 50 ml with water.

Dilute immediately with methanol to 50 per 100 (47.3.9) to obtain an approximate concentration in sodium monensin

8 micrograms/ml (U).

47.4.3 Extraction. Feed in which the sodium monensin content is less than 50 mg/kg (up to the limit of 10 mg/kg).

weigh 10,0 to 20.0 g of sample, add 100 ml of methanol to 90 per 100 (47.3.8), homogenise for fifteen minutes. Centrifuge to obtain a clean extract.

Take 40 ml of supernatant liquid for a sample of which the sodium monensin content is 20 mg/kg; 80 ml for a sample of 10 mg/kg. Evaporate vacuum with rotavapor until dryness (47.2.1) at a temperature of not more than 40 ° C. Dissolve the residue with 10 ml of methanol at 90 per 100 (47.3.8).

Introduce a cotton stopper at the lower end of the glass column (47.2.3), add the aluminium oxide (47.3.10), give light shocks to the column, until the filling is 75 to 80 mm high.

Decant the methane solution of the residue on the aluminum oxide column and collect the filtrate. Wash the column with 10 ml of methanol at 90 per 100 (47.3.8) and join the previous filter.

Evaporate the vacuum solution in rotavaby dryness (47.2.1) a temperature below 40 ° C. Dissolve the residue with 10 ml of anhydrous methanol (47.3.7) and complete 20 ml with water.

Centrifuge at 4,000 rpm for at least five minutes. Dilute then with methanol at 50 per 100 (47.3.9) to obtain an approximate concentration in sodium monensin of 8 micrograms/ml (U).

47.4.4 Extract solutions. Prepare, from solution U and by successive dilutions (1 + 1) with methanol at 50 per 100 (47.3.9) the solutions whose assumed concentrations are U (4 micrograms/ml), U (2 micrograms/ml) and U (1 microgram/ml).

47.4.5 Modes of determination. Sow at 50-60 ° C the base culture medium for determination (47.3.5) with the suspension of spores (47.3.3). By means of preliminary tests on plates with the medium (47.3.5), determine the amount of inoculum that allows to obtain the most extensive inhibition zones and which are also sharp for the different concentrations of sodium monensin.

47.4.6 Preparation of the plates. The agar diffusion is carried out on the plates with the four concentrations of the standard solution (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each plate must necessarily receive the four pattern and extract concentrations. To this end, it is necessary to choose the dimension of the plates so that they can be practiced in the middle of agar at least 8 cavities of 10 to 13 mm in diameter, whose centers do not distinguish between less than 30 mm. They can be used as plates, flat sheets of glass, topped by an aluminium or plastic ring of 200 mm in diameter and 20 mm in height.

Introduce into the plates an amount of the medium (47.3.5), seeded as indicated (47.4.5), so that a layer of about 2 mm thick (60 ml for a 200 mm diameter plate) is obtained. Allow to solidify, open the cavities and deposit exactly measured volumes of standard solutions and extract (0.10 to 0.15 ml per cavity, according to diameter). Perform at least four repetitions of each concentration so that each determination is the subject of an assessment of 32 inhibition zones.

47.4.7 Incubation. Incubate the nine plates for eighteen hours at approximately 35-37 ° C.

47.5 Calculations.

Measure the diameter of the inhibition zones with a precision of 0.1 mm. For each concentration, the mean values in semi-logarithmic paper represent the logarithm of the concentrations against the diameter of the inhibition zones. Draw the tightest straight lines for the standard solution and for the extract by proceeding, for example, as follows:

Determine the most appropriate point of the lowest level of the standard solution (SL) using the formula:

(a) SL = 7S + 4S + S-2S

10

Determine the most appropriate point of the highest level of the reference solution (SH) with the help of the formula:

(b) SH = 7S + 4S + S-2S

10

Also determine the most appropriate points of the extract for the lowest level (UL) and the highest level (UH) substituting S, S, S, and S for U, U, U, and U in the above formulas.

Enroll the SL and SH values on the same chart. By joining the two points you get the tightest straight for the standard solution. Proceeding the same way for UL and UH you get the straightest straight for the extract.

In the absence of interference, the straights should be parallel. In practice they are considered parallel when SH-SL and UH-UL do not differ more than 10 per 100 from their average.

If the straight lines are not parallel, U and S or U and S. can be removed. The values SL, SH, UL, and UH that allow to obtain the tightest straight are calculated using the following formulas:

5S + 2S-S 5S + 2S-S

(a) SL = ------------------- or -------------------

6 6

5S + 2S-S 5S + 2S-S

(b) SH = ------------------- or -------------------

6 6

and with similar formulas for UL and UH. The use of this alternative means that the same parallelism criteria are respected. Obtaining a result from three levels should be mentioned in the analysis bulletin.

Once the parallel straights are considered, calculate the logarithm of the relative activity (log. A) using one of the following formulas:

For 4 levels:

(c) log A = (U + U + U + U-S-S-S-S) x 0.602

U + U + S + S-U-U-S-S

For 3 levels:

(d) log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

(d ') log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

Actual activity = assumed activity x relative activity.

If the relative activity is lower than the values between 0.5 and 2.0, repeat the determination by making appropriate adjustments of the concentrations of the extract or if necessary, of the standard solutions. If the activity cannot be carried out between the above two values, the result should be considered as approximate and this should be noted in the analysis bulletin.

If the straights are considered not to be parallel, repeat the determination. If parallelism is not achieved, the determination must be considered unsatisfactory.

The difference between results of two determinations made on the same sample by the same person should not exceed:

-20 per 100 of the highest result for sodium monensin contents between 10 and 25 mg/kg.

-5 mg/kg, in absolute value, for the contents between 25 and 50 mg/kg.

-10 per 100 of the highest result for contents greater than 50 mg/kg.

47.6 Remarks.

47.6.1 Other methods may be used to the extent that they are proven to produce similar spore suspensions.

47.6.2 Any commercial culture medium of similar composition can be used and of the same results.

47.7 References. Ninth Commission Directive of 31 July 1981, 81 /715/EEC. 'Official Journal of the European Communities' number L 257/9 of 10 September 1981.

48. Avoparcin

(By Broadcast on agar)

48.1 Principle. The method allows the determination of avoparcin in feed and premixtures. The lower limit of detection is 2 mg/kg. The antibiotic polyethers interfere in the determination.

The sample is subjected to an extraction with a mixture of acetone/water/hydrochloric acid. The antibiotic activity of the extract is determined by measuring the diffusion of the avoparcin in an agar medium, seeded with Bacillus subtilis. The diffusion is manifested by the formation of zones of microorganism inhibition. The diameter of these areas is considered to be directly proportional to the logarithm of the antibiotic concentration for the range of concentrations used.

48.2 Reassets.

48.2.1 Microorganism. Bacillus subtilis ATCC 6633 (NCIB 8054).

48.2.2 Conservation of the strain. Sow the culture medium (48.2.4), in inclined tubes, with Bacillus subtilis. Incubate for one night at 30 ° C, store in refrigerator at approximately 4 ° C and renew the sowing every month.

48.2.3 Preparation of spores suspension. (See 48.5.1). Collect the germs from a newly prepared agar tube (48.2.2), with 2 or 3 ml of sterile water. Sow with this suspension 300 ml of the culture medium (48.2.4) in a vial of Roux and incubate three to five days at 30 ° C. After controlling the sporulation under a microscope, collect the spores with 15 ml of ethanol (48.2.5) and homogenise. This suspension may be stored for up to five months at approximately 4 °C.

48.2.4 Strain maintenance medium (48.5.2).

Peptone: 6.0 g.

Triptone: 4.0 g.

yeast extract: 3.0 g.

Meat extract: 1.5 g.

Glucose: 1.0 g.

Agar: 15.0 g.

Water: 1,000 ml.

pH: 6.5 (after sterilizing).

48.2.5 Ethanol at 20 per 100 (v/v). Dilute 200 ml of ethanol with 800 ml of water.

48.2.6 Hydrochloric acid, d = 1.18-1.19.

48.2.7 Sodium hydroxide solution 2 M.

48.2.8 Tampon phosphate 0.1 M. Pesar 13.6 g potassium dihydrogen phosphate (KHPO) and dilute up to 1,000 ml with water and adjust to pH 4.5.

48.2.9 Mix acetone/water/hydrochloric acid (48.2.6): 65/32 ,5/2, 5 (v/v/v).

48.2.10 Pattern Substance. Avoparcin sulfate of known activity.

48.2.11 Pattern solution. Dissolve 10 mg, exactly heavy, of standard substance (48.2.10) in phosphate buffer (48.2.8) and dilute with this buffer to obtain an avoparcin solution of 100 micrograms/ml. Store in bottle capped at 4 °C for up to seven days.

48.2.12 Pattern solutions for premixtures. Prepare from the solution (48.2.11) and by successive dilutions with phosphate buffer (48.2.8) the following solutions:

S: 4 micrograms/ml.

S: 2 micrograms/ml.

S: 1 microgram/ml.

S: 0.5 microgram/ml.

48.2.13 Solutions for feed. Prepare from the solution (42.2.11) and by successive dilutions with phosphate buffer (48.2.8) the following solutions:

S: 2 micrograms/ml.

S: 1 microgram/ml.

S: 0.5 micrograms/ml.

S: 0.25 microgram/ml.

48.3 Procedure.

48.3.1 Preparation of the extract and solutions. Premixtures. Weigh to 10 mg precisely, a sample amount of 10 to 100 mg of avoparcin, transfer to 100 ml volumetric flask, add 60 ml of mixture (48.2.9) and shake for fifteen minutes with a mechanical agitator. Verify the pH and adjust it to pH = 2, if necessary, with hydrochloric acid (48.2.6). Complete the volume with the mixture (48.2.9) and homogenize. Filter a part on Whatman paper number 1 or similar and remove the first 5 ml. Take an aliquot and adjust the pH to 4.5 with sodium hydroxide solution (48.2.7). Dilute this phosphate buffer solution (48.2.8) to obtain a 4 microgram/ml (U) of avoparcin.

Prepare from this solution and for successive dilutions (1 + 1) with phosphate buffer (48.2.8), the supposed solutions U (2 micrograms/ml), U (1 microgram/ml) and U (0.5 micrograms/ml). 48.3.2 Preparation of the extract and solutions. Feed. Weigh a sample quantity of 50 g, add 100 ml of mixture (48.2.9) and shake for thirty minutes with mechanical agitator. Clarify by centrifugation the extract (in capped tubes), take an aliquot of the clean extract (see table) and adjust to pH 4.5 with sodium hydroxide solution (48.2.7). Dilute this phosphate buffer solution (48.4.8) to obtain the U solution (see table).

TABLE

Suspected content in avoparcin (mg/kg)/5/7.5/10/15/20/40

Sample weight in g (± 0.1 g)/50/50/50/50/50/50

Mix volume (ml) (48.2.9)/100/100/100/100/100

Clean extract volume (ml)/20/15/20/15/20/10

Final Volume (ml): U/25/25/50/50/100/100

Assumed concentration U in micrograms/ml. /2/2

approx. /2/2

approx. /2/2

Prepare from this solution and for successive dilutions (1 + 1) with phosphate buffer (48.2.8) the supposed solutions U (1.0 microgram/ml), U (0.5 micrograms/ml), U (0.25 micrograms/ml).

48.3.3 Determination. Inoculation of the culture medium. Sow at 50-60 ° C the base of the determination (48.2.4) with the suspension of spores (48.2.3). By means of preliminary tests on plates with the medium (48.2.4), determine the quantity of inoculum to obtain the most extensive and also sharp inhibition zones for the different concentrations of avoparcin.

48.3.4 Determination. Preparation of the plates. The agar diffusion is performed on plates with the four concentrations of the standard solution (S, S, S and S) and the four concentrations of the extract (U, U, U and U).

Each plate must necessarily receive the four pattern and extract concentrations. To this end, choose the dimensions of the plates so that at least eight cavities of 10 to 13 mm in diameter can be practiced in the medium, the centers of which do not distinguish between less than 30 mm. They can be used as plates, flat sheets of glass, topped with an aluminium ring or plastic material of 200 mm in diameter and 20 mm in height.

Introduce into the plates an amount of the medium (48.2.4), sown as indicated in (48.3.3), which allows to obtain a layer approximately 2 mm thick (60 ml for a 200 mm diameter plate). Allow to solidify, open the cavities and deposit exactly measured volumes of standard solutions and extract (0.10 to 0.15 ml per cavity, according to the diameter). Perform at least four repetitions of each concentration so that each determination is the subject of an assessment of 32 inhibition zones.

48.3.5 Incubation. Incubate plates between sixteen and eighteen hours at 30 ° C.

48.4 Calculations. Measure the diameter of the inhibition zones with a precision of 0,1 mm. For each concentration, record the mean measurements on semi-logarithmic paper, representing the logarithm of the concentrations against the diameters of the inhibition zones. Draw the tightest straight lines for the standard solution and for the extract by proceeding, for example, as follows:

Determine the most appropriate point of the lowest level of the standard solution (SL) using the formula:

(a) SL = 7S + 4S + S-2S

(a) SL =

10

Determine the most appropriate point of the highest level of the standard solution (SH) using the formula:

(b) SH = 7S + 4S + S-2S

(b) SH =

10

Determine the most appropriate points of the extract for the lowest level (UL) and the highest level (UH) by replacing S, S, S, and S in the formulas per U, U, U, and U.

Take the SL and SH values on the same chart. By joining the two points you get the tightest straight for the standard solution. Proceeding the same way for UL and UH you get the tightest straight for the extract.

In the absence of any interference, the straights should be parallel when SH-SL and UH-UL do not differ more than 10 per 100 from their average.

If the straight lines are not parallel, you can remove either U and S or U and S. The values SL, SH, UL and UH that allow you to get the tightest straight are calculated according to:

5S + 2S-S 5S + 2S-S

(a ') SL = ------------------- or -------------------

6 6

5S + 2S-S 5S + 2S-S

(b ') SH = ------------------- or -------------------

6 6

and the analogous formulas for UL and UH. The use of this alternative means that the same parallelism criteria are respected. Obtaining a result from three levels should be mentioned in the analysis bulletin.

When the straights are considered parallel, calculate the logarithm of the relative activity (log A) as follows:

For 4 levels:

(c) log A = (U + U + U + U-S-S-S-S) x 0.602

U + U + S + S-U-U-S-S

For 3 levels:

(d) log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

(d) log A = (U + U + U-S-S-S) x 0.401

U + S-U-S

Actual activity = assumed activity x relative activity.

If the relative activity is below the values between 0.5 and 2.0, repeat the determination proceeding to appropriate adjustments of the concentrations of the extract or, eventually, of the solutions patterns. If this activity cannot be included among these values, the result should be considered approximate and this should be included in the analysis bulletin.

When the straights are not considered parallel, repeat the determination. If parallelism is not achieved, the determination must be regarded as unsatisfactory.

The difference between the results of two determinations made on the same sample by the same person should not exceed:

-2 mg/kg, in absolute value, for the contents of avoparcin of 2 to 10 mg/kg.

-20 per 100 of the highest result for contents between 10 and 25 mg/kg.

-5 mg/kg in absolute value, for contents between 25 and 50 mg/kg.

-10 per 100 of the highest result for contents greater than 50 mg/kg.

48.5 Remarks.

48.5.1 Other methods may be used to the extent that they are proven to produce similar spore suspensions.

48.5.2 Another commercial culture medium may be used which has an analogous composition and the same results.

48.6 References. Ninth Commission Directive of 31 July 1981. 81 /715/EEC. 'Official Journal of the European Communities' number L 257/38 of 10 September 1981.

49. Antibiotics in the tetracycline group

49.1 Detection and identification.

49.1.1 Principle. The method makes it possible to detect and identify the antibiotics in the group of tetracyclines in feed containing at least 0,1 mg/kg of them, in concentrates and in premixtures.

The sample is subjected to extraction by a mixture of methanol and hydrochloric acid. The extract is chromatography on paper by way of a comparison to its reference solutions. Antibiotics are detected and identified in comparison with their Rf values with those of the standard substances, either by UV light fluorescence (high antibiotic content), either by means of bioautography on the basis of agar sowing with B. cereus.

49.1.2 Reassets.

49.1.2.1 Tampon, pH 3.5. citric acid monohydrate p.a.: 10,256 g.

Sodium monohydrogen phosphate dihydrate (NaHPO.2HO) p.a.: 7.45 g.

Acetone p.a.: 300 ml.

Distilled Water: Up to 1,000 ml.

49.1.2.2 Tampon phosphate: pH 5.5 potassium dihydrogen phosphate (KHPO) p.a.: 130.86 g.

Sodium monohydrogen phosphate dihydrate (NaHPO.2HO) p.a.: 6.947 g.

Distilled Water: Up to 1,000 ml.

49.1.2.3 Eluent I: nitroethane mixture p.a ./chloroform p.a ./alpha dichlorhydrin: 20/10/1, 5 in volume. Prepare at the time of your employment.

49.1.2.4 eluent II: Nitrometric mixture p.a ./ chloroform p.a ./alpha picolinium (20/10/3 in volume). Prepare at the time of your employment.

49.1.2.5 Mezcla netanol p.a ./hydrochloric acid (d = 1 ,98/2 in volume).

49.1.2.6 Hydrochloric acid 0.1N.

49.1.2.7 Ammonia, d = 0.91.

49.1.2.8 Pattern substances: chlortetracycline, oxytetracycline, tetracycline, the activity of which is expressed as hydrochloride.

49.1.2.9 Microorganism: B. cereus ATCC number 11,778. Maintenance of the strain, preparation of the suspension of spores and inoculation of the culture medium. Apply the provisions (49.2.2.1) and (49.2.2.2) of the method of determination of chlorotetracycline, oxytetracycline and tetracycline, by diffusion on agar, object of the part (49.2).

49.1.2.10 Crop medium. (See 41.15.1):

Glucose: 1 g.

Peptone trypsica: 10 g.

Meat extract: 1.5 g.

yeast extract: 3 g.

Agar: 20 g.

Distilled Water: Up to 1,000 ml.

Adjust the pH to 5.8 at the time of your job.

49.1.3 Material.

49.1.3.1 Equipment for ascending chromatography on paper (paper height 25 cm). Papers Schleicher and Schull 20040 b or 20043 b or equivalent.

49.1.3.2 Centrileak.

49.1.3.3 Incubation study, regulated at 30 ° C.

49.1.3.4 UV Lamp for fluorescence detection.

49.1.3.5 20 x 30 cm glass plates that allow the assembly of a flat box for bioautobiography.

49.1.4 Procedure.

49.1.4.1 Calibration solutions.

49.1.4.1.1 Pattern solutions. Prepare from the calibration substances (49.1.2.8) and with the help of hydrochloric acid (49.1.2.6) solutions whose concentration corresponds respectively to 500 micrograms of chlortetracine-HCl or of oxytetracine-HCl or of Tetracycline-HCl per ml.

49.1.4.1.2 Reference solutions for UV light detection. Dilute the solutions (49.1.4.1.1) with phosphate buffer (49.1.2.2) to obtain solutions whose concentration corresponds to 100 micrograms of chlortetracine-HCl or of oxytetracilin-HCl or tetracinclina-HCl per ml.

49.1.4.1.3 Reference solutions for bioautography detection. Dilute the solutions (49.1.4.1.1) with phosphate buffer (49.1.2.2) to obtain solutions whose concentration corresponds to 5 micrograms of chlortetracine-HCl, or of oxytetracine-HCl or tetracycline-HCl per ml.

49.1.4.2 Extract. Where the assumed content of the antibiotic is less than 10 mg/kg, the homogenised sample or the finer fraction separated by sifting may be used, as the antibiotics shall preferably be found in that fraction.

Put the sample into suspension in the mixture (49.1.2.5) and centrifuge. Collect the supernatant liquid to use as it is or dilute it, if necessary, by mixing (49.1.2.5) to obtain antibiotic concentrations of 100 micrograms/ml (49.1.4.2.1) and 5 micrograms/ml (49.1.4.2.2). approximately.

49.1.4.3 Detection and identification.

49.1.4.3.1 Cromatography. Immerse the paper in the buffer solution, pH 3.5 (49.1.2.1). Remove excess liquid by compressing the paper between sheets of dry filter paper. A volume of 0,01 ml of the reference solutions (49.1.4.1.2 and 49.1.4.1.3) and the extract (49.1.4.2 and 48.1.4.2.2) should be deposited on the paper. In order to obtain a good sepration, the appropriate moisture content of the paper is decisive; if appropriate, allow to dry slightly.

Develop by ascending chromatography. Use eluent I (49.1.2.3) for bioautography detection, eluent II (49.1.2.4) for UV light detection. When the solvent front reaches 15 to 20 cm high (approximately one hour thirty minutes) interrupt the chromatography and dry the paper.

49.1.4.3.2 ultraviolet light detection. When the antibiotic content is greater than 1 microgram/cm, yellow fluorescent spots or irradiation under the UV lamp (49.1.3.4) can be observed after treatment of the chromatogram by ammoniacal vapours (49.1.2.7).

49.1.4.3.3 Bioautography Detection. Pour the culture medium (49.1.2.10) previously sown with B. cereus (49.1.2.9) on glass plates (49.1.3.5) and place the paper on the culture medium. After five minutes of contact, remove the paper and place it on a different place from the culture medium where it will be maintained during the incubation period, incubating for one night in a stove at 30 ° C. The presence of an antibiotic from the tetracycline group is marked by clear inhibition zones in the turbid culture medium.

To fix the chromatogram, vaporize the solution (49.1.2.11) on the paper, after incubation.

49.1.4.3.4 Identification. The RF values relative to the 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.

Oxitetracycline (OTC): 0.20.

4-epi-CTC: 0.15.

4-epi-TC: 0.13.

4-epi-OTC: 0.10.

The "epi" compounds have a lower antibiotic activity than normal compounds.

49.1.5 Remarks.

49.1.5.1 Any commercial culture medium of a similar composition may be used and of the same results.

49.2 Chlortetracycline, oxytetracycline and tetracycline.

49.2. A By diffusion on agar.

49.2. A. 1 Principle. The method enables the determination of chlortetracycline, (CTC), oxytetracycline (OTC) and tetracycline (TC) in feed, concentrates and premixtures. The lower limit of the determination is 5 mg/kg. The contents of less than 5 mg/kg can be estimated by graphical interpolation.

For contents equal to or less than 50 mg/kg, the sample shall be extracted by diluted formanide. For the contents of more than 50 mg/kg, it is subjected to extraction by means of a mixture of acetone, water and hydrochloric acid for the determination of the CTC, and a mixture of methanol and hydrochloric acid for the determination of the TBT and the CT.

The extracts shall then be diluted and their antibiotic activity shall be determined by the measurement of the diffusion of the CTC or of the OTC or of the TC in the middle of the agar sown with B. cereus. The diffusion is indicated by the formation of inhibition zones in the presence of the microorganism. The diameter of these zones is directly proportional to the logarithm of the antibiotic concentration.

49.2. A. 2 Reassets.

49.2. A. 2.1 Microorganisms: B. cereus ATCC number 11,778.

49.2. A. 2.1.1 Maintenance of the strain. Seed B. cereus in inclined agar tube formed by the culture medium (49.2.A. 3.1) free of methylene blue and boric acid. Incubate for one night at approximately 30 ° C. Preserve the culture in refrigerator and resembrate every fourteen days on inclined agar.

49.2. A. 2.1.2 Preparation of spores suspension. Collect the germs from an inclined agar tube (49.2.A. 2.1.1) with 2 to 3 ml aid of physiological serum (49.2.A. 2.2.5). Sow with said suspension a vial of Roux containing 300 ml of the culture medium (49.2.A. 2.2.1), free of methylene blue and boric acid, whose concentration in agar is 3 to 4 per 100. Incubate three to five days at 28-30 ° C, then collect the spores in 15 ml of ethanol (49.2.A. 2.2.6), after having checked the sporulation under the microscope, and homogenise. Said suspension may be stored in a refrigerator for at least 5 months.

For preliminary tests on plates with the basis of determination (49.2.A. 2.2.1), determine the quantity of inoculum to be obtained for the different concentrations of antibiotic used, some areas of Inhibition as extensive as possible, and be clear. This amount is usually 0.2 to 0.3 ml/1,000 ml. The sowing of the culture medium is between 50 and 60 ° C.

49.2. A. 2.2 Means of cultivation and reagents.

49.2. A. 2.2.1. Basis of determination (see 49.2.A. 5.1):

Glucose: 1 g.

Peptone triptica: 10 g.

Meat extract: 1.5 g.

yeast extract: 3 g.

Agar, according to quality: 10 to 20 g.

Tween 80: 1 ml.

phosphate buffer, pH 5.5 (49.2.A. 2.2.2): 10 ml.

Solution to 5 per 100 (w/v) of boric acid: 15 ml.

ethanolic solution at 0.5 per 100 methylene blue: 4 ml.

Distilled Water: Up to 1,000 ml.

Adjust to pH 5.8 before your job.

49.2. A. 2.2.2 Tampon phosphate pH 5,5. Potassium dihydrogen phosphate (KHPO) p.a.: 130.86 g.

Monohydrogen Phosphate sodium dihydrate (NaHPO.2HO) p.a.: 6.947 g.

Distilled Water: Up to 1,000 ml.

49.2. A. 2.2.3 Tampon phosphate, pH 5.5, diluted to 1/10.

49.2. A. 2.2.4 Tampon phosphate, pH 8. Potassium dihydrogen phosphate (KHPO) p.a.: 1,407 g.

Monohydrogen Phosphate sodium dihydrate (NaHPO.2HO) p.a.: 57.539 g.

Distilled Water: Up to 1,000 ml.

49.2. A. 2.2.5 Sterile physiological suero.

49.2. A. 2.2.6 Ethanol at 20 per 100 (v/v).

49.2. A. 2.2.7 Hydrochloric acid 0.1N.

49.2. A. 2.2.8 Formamide at 70 per 100 (v/v). Prepare fresh before use and adjust pH 4.5 with the help of approximately 2N sulphuric acid.

49.2. A. 2.2.9 Mix acetone p.a ./agua/hydrochloric acid (d = 1.19) (65/33/2 in volume).

49.2. A. 2.2.10 Mix methanol p.a ./hydrochloric acid (d = 1.19) (98/2 v/v).

49.2. A. 2.2.11 Substance patterns: CTC, OTC, TC whose activity is expressed in hydrochloride.

49.2. A. 3 Procedure.

49.2. A. 3.1 Patterns solutions.

49.2. A. 3.1.1 Chlortetracycline. Prepare from the standard substance (49.2.A. 2.2.11) and with the help of hydrochloric acid (49.2.A. 2.2.7), a standard solution whose concentration corresponds to 500 micrograms/ml of chlortetracine-HCl. This solution is kept for a week in the refrigerator.

From this standard solution, prepare a S8 calibration solution whose concentration corresponds to 0.2 micrograms/ml of chlortetracine-HCl. Dilution is done with the aid of phosphate buffer, pH 5.5 diluted to 1/10 (49.2.A. 2.2.3), with an addiction of 0.01 per 100 black amido (see 49.2.A. 5.2).

Prepare below for successive dilutions (1 + 1) with buffer help (49.2.A. 2.2.3), the following concentrations:

S: 0.1 micrograms/ml.

S: 0.05 micrograms/ml.

S: 0.025 micrograms/ml.

49.2. A. 3.1.2 Oxitetracycline. Proceeding as indicated in (49.2.A. 3. 1.1) prepare, from a standard solution whose concentration corresponds to 400 micrograms/ml of oxytetracine-HCl, a working standard solution S8 of 1.6 micrograms/ml of oxytetracine-HCl and the following concentrations:

S: 0.8 micrograms/ml.

S: 0.4 micrograms/ml.

S: 0.2 micrograms/ml.

49.2. A. 3.1.3 Tetracycline. Proceeding as indicated in (49.2.A. 3.1.1), prepare, from a standard solution of which the concentration is 500 micrograms/ml of tetracine-HCl, a working standard solution S8 of 1.0 micrograms/ml of tetracina-HCl and the following concentrations:

S: 0.5 micrograms/ml.

S: 0.25 micrograms/ml.

S: 0.125 micrograms/ml.

49.2. A. 3.2 Extraction.

49.2. A. 3.2.1 Contents equal to or less than 50 mg/kg. Treat the sample by formamide (49.2.A. 2.2.8), according to the indications given below in the table. Shake for 30 minutes in agitator. Dilute immediately thereafter with the aid of phosphate buffer (49.2.A. 2.3), as shown in the table below, to obtain the concentration U. The concentration of formamide in such a solution should not exceed 40 per 100. Centrifuge or let decant so that a clear solution is obtained.

Prepare the U, U and U concentrations by successive dilutions (1 + 1) with the aid of phosphate buffer (49.2.A. 2.3).

Antibiotic/CTC/DTC/TC

Suspected content in mg/kg. /10/50/10/50/10/50

Shows in g. /10/10/24/9.6/20/10

Ml of formamide (49.2.A. 2.2.8). /100/100/80/100/80/100

dil/dil///dil

Ml phosphate buffer (49.2.A. 2.2.3). /1/5/1/25/70/200/120/1/5

(a)/(b)///(a)

Concentration U in micrograms/ml. /0.2/0.2/1.6/1.6/1.0/1.0

(a) Take 20 ml of extract and complete 100 ml with the tampon in a graduated flask.

(b) Take 4 ml of extract and complete 100 ml with the tampon in a graduated flask.

49.2. A. 3.2.2 Contents above 50 mg/kg.

49.2. A. 3.2.2.1 Chlortetracycline. Treat, according to the presumed antibiotic content of the sample or its manufacturing guarantee, a sample of 1 to 10 grams, for 20 times its volume with the mixture (49.2.A. 2.2). Shake for thirty minutes with agitator.

Check that the pH remains below 3 throughout the extraction; if it is necessary to adjust to pH 3 (for the mineral compounds, with the aid of acetic acid at 10 per 100). Take an aliquot part of the extract and adjust the pH to 5.5 with the aid of phosphate buffer, pH 8 (49.2.A. 2.2.4), in the presence of green bromoresol (turn from yellow to blue). Dilute with the aid of phosphate buffer, pH 5.5, diluted to 1/10 (49.2.A. 2.2.3) to obtain concentration U (49.2.A. 3.2.1).

Then prepare U, U and U concentrations by successive dilutions (1 + 1) with the help of phosphate buffer (49.2.A. 2.2.3).

49.2. A. 3.2.2.2 Oxitetracycline and tetracycline. Proceed as indicated in (49.2.A. 3.2.2.2) by replacing the mixture (49.2.A. 2.2) with the mixture (49.2.A. 2.2.10).

49.2. A. 3.3 Modes of determination.

49.2. A. 3.3.1 Siembre of the culture medium. Sow at 50-60 ° C the base means for determination (49.2.A. 2.2.1) with the suspension of spores (49.2.A. 2.1.2).

49.2. A. 3.3.2 Preparation of the boxes. The spread of agar is carried out in boxes, with the four concentrations of the pattern (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each box must necessarily receive the four concentrations of the pattern and the extract.

To this end, choose the dimensions of the boxes in such a way that at least 8 cavities of 10 to 13 mm in diameter can be practiced in the gelled medium. Calculate the quantity of inoculated culture medium (49.2.A. 3.3.1), which shall be used in such a way as to obtain a uniform coating of approximately 2 mm thick. It is preferable to use as boxes flat glass plates with an aluminium ring or a perfectly flat plastic material, 200 mm in diameter and 20 mm in height.

Introduce by pipette into the cavities a quantity of antibiotic solution, exactly measured, between 0.10 and 0.15 ml, according to the diameter.

For each sample, make at least four diffusion repetitions with each concentration, so that each determination is the subject of an evaluation of 32 inhibition zones.

49.2. A. 3.3.3 Incubation. Incubate the boxes for eighteen hours at approximately 28-30 ° C.

49.2. A. 4 Calculations. Measure the diameter of the inset zones, preferably by projection. Record the measurements on semi-logarithmic paper, representing the logarithm of the concentrations against the diameters of the inhibition zons. Draw the straight lines of the calibration solution and the extract. In the absence of interference, the two straight lines shall be parallel.

The logarithm of the relative activity is calculated as follows:

(U + U + U + U-S-S-S-S) x 0.602

U + U + U + U-S-S-S-S) x 0.602

U + U + S + S-U-U-S-S

Actual activity = assumed activity x relative activity.

The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 per 100 in relative value.

49.2. A. 5 Remarks.

49.2. A. 5.1 Any commercial culture of a similar composition may be used and of the same results.

49.2. A. 5.2 The amido black serves to characterize the inhibition zones of the calibration solutions (blue rings).

49.2. B By turbidimetry.

49.2. B. 1 Principle. The method enables the determination of chlortetracycline (CTC), oxytetracycline (OTC) and tetracycline (TC) in concentrations greater than 1 g/kg, to the extent that no other substance interferes, resulting in cloudy extracts. Said method is faster than the method by diffusion on agar.

The sample is subjected to extraction by mixture of acetone, water and hydrochloric acid for the determination of the CTC, and by a mixture of methanol and hydrochloric acid for the determination of the OTC and the TC.

The extracts are then diluted and their antibiotic effect is determined by the measure of the light transmission of a culture medium sown with Staphylococcus aureus and with added antibiotic. Light transmission is a function of the concentration of the antibiotic.

49.2. B. 2 Reassets.

49.2. B. 2.1 Microorganism: Staphylococcus aureus K 141. See (49.2.B. 5.1).

49.2. B. 2.1.1 Maintenance of the strain. Sow S. aureus on the inclined agar tube formed by the culture medium (49.2.B. 2.2.1), added from 1.5 to 3 per 100 (according to the quality). Incubate for one night at 37 ° C. Preserve the culture in refrigerator and resembrate four weeks over inclined agar. To prepare sub-crops for laboratory use at the same time.

49.2. B. 2.1.2 Preparation of the inoculum. Twenty-four hours before you are employed, you will resembre a subculture on inclined agar and incubate for one night at 37 ° C. Put in suspension the entire culture of an agar tube in 2 ml approximately of the base medium (49.2.B. 2.2.1) and then transfer the suspension in sterile conditions to approximately 100 ml of the same base medium. (49.2.B. 2.2.1). Incubate in water bath at 37 ° C until the growth of the strain enters its logarithmic phase (one hour thirty minutes to two hours).

49.2. B. 2.2 Means of cultivation and reagents.

49.2. B. 2.2.1 The basis of the determination. (See 49.2.B. 5.2.)

Peptone: 5 g.

yeast extract: 1.5 g.

Meat extract: 1.5 g.

Sodium Chloride: 3.5 g.

Glucose: 1 g.

potassium dihydrogen phosphate (KHPO) p.a.: 1.32 g.

Potassium Monohydrogen Phosphate (KHPO) p.a.: 3.68 g.

Distilled Water: Up to 1,000 ml.

pH after sterilisation: 6.8 to 7.

49.2. B. 2.2.2 Tampon phosphate pH 4.5. Potassium dihydrogen phosphate (KHPO) p.a.: 13,6 g.

Distilled Water: Up to 1,000 ml.

49.2. B. 2.2.3 Hydrochloric acid 0.1N.

49.2. B. 2.2.4 Mix acetone p.a ./agua/hydrochloric acid (d = 1.19) (65/33/2 in volume).

49.2. B. 2.2.5 Mezcla methanol p.a ./hydrochloric acid (d = 1.19) (98/2 in volume).

49.2. B. 2.2.6 Solution to 10 per 100 approximately (w/v) formaldehyde.

49.2. B. 2.2.7 Pattern substances: OTC, CTC, CT, the activity of which is expressed as hydrochloride.

49.2. B. 2.3 Pattern solution. Prepare, from the standard substance (49.2.B. 2.2.7) and with the aid of hydrochloric acid (49.2.B. 2.2.3) a standard solution whose concentration is 400 to 500 micrograms/ml of CTC-HCI, OTC-HCI or TC-HCI. This solution is kept for a week in the refrigerator.

49.2. B. 3 Procedure.

49.2. B. 3.1 Extraction.

49.2. B. 3.1.1 Chlortetracycline. Insert in a graduated flask of 200 or 250 ml, a sample of 1 to 2 grams. Add approximately 100 ml of the mixture (49.2.B. 2.2.4) and shake for thirty minutes with agitator. Complete the volume with the phosphate buffer, pH 4.5 (49.2.B. 2.2.2). Homogenize and let it be deposited.

49.2. B. 3.1.2 Oxitetracycline and tetracycline. Insert a sample of 1 to 2 grams in a graduated flask of 200 to 250 ml. Add approximately 100 ml of the mixture (49.2.B. 2.2.5) and shake for thirty minutes with agitator. Complete the volume by the phosphate buffer, pH 4.5 (49.2.B. 2.2.2). Homogenize and let it be deposited.

49.2. B. 3.2 Modes of determination.

49.2. B. 3.2.1 Preparation of standard series and extract. Dilute appropriately with the aid of phosphate buffer, pH 4.5 (49.2.B. 2.2.2), the standard solution (49.2.B. 2.3) and the extract (49.2.B. 3.1), so that a series of concentrations can be obtained to establish a curve for each determination. of calibration, and interpolation on said curve of at least two values relative to the extract. Dilutions should be chosen according to the growth conditions of the strain, which may vary from one laboratory to another. It is derived from the following form: 49.2.B. 3.2.1.1 Chlortetracycline. Dilute the standard solution (49.2.B. 2.3.) with the aid of phosphate buffer (49.2.B. 2.2.2) to obtain a standard solution for which concentration corresponds to 0.2 micrograms/ml CTC-HCl. Prepare then with the aid of a phosphate buffer (49.2.B. 2.2.2), as follows, and in tubes intended for determination, 6 dilutions with a repetition of each dilution.

Ml working pattern solution/Ml of phosphate buffer (49.2.B. 2.2.2)/Concentration in CTC-HCl (x/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 (49.2.B. 3.1.1) with the aid of phosphate buffer (49.2.B. 2.2.2) to obtain an assumed concentration in CTC-HCl of 0.12 micrograms/ml. Insert 1 ml of such solution into two tubes and 0.75 ml (= 0.09 micrograms) in two other tubes. Complete the volume of the last two tubes to 1 ml with the phosphate buffer (49.2.B. 2.2.2).

49.2. B. 3.2.1.2 Oxitetracycline and tetracycline. Dilute the calibration solution (49.2.B. 2.3) with the aid of phosphate buffer (49.2.B. 2.2.2) to obtain a working standard solution for which the concentration corresponds to 0,6 micrograms/ml of OTC-HCl or TC-HCl. Prepare then with the aid of the phosphate buffer (49.2.B. 2.2.2) as follows and in the tubes for determination, 7 dilutions with a repetition of each dilution.

Ml working pattern solution/Ml of phosphate buffer (49.2.B. 2.2.2)/Concentration in CTC-HCl (x/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 (49.2.B. 3.1.2) with the phosphate buffer (49.2.B. 2.2.2) to obtain an assumed concentration in OTC-HCl or TC-HCl of 0.48 micrograms/ml. Insert 1 ml of such solution into two tubes and 0.5 ml (= 0.24 micrograms) in two other tubes. Complete the volume of the last two tubes to 1 ml by the phosphate buffer (49.2.B. 2.2.2).

49.2. B. 3.2.2 Inoculation of the culture medium. Seed the base of the determination (49.B. 2.2.1) with the inoculum (49.2.B. 2.1.2) so that it can be obtained by the photometer at 590 nm a luminous transmission of 85 per 100 in a 5 cm or 92 per 100 bucket in a 2 cm bucket, The apparatus is regulated at 100 per 100 transmission on the non-inoculated base medium (49.2.B. 2.2.1).

49.2. B. 3.2.3 Siembra. Insert into each tube (49.2.B. 3.2.1.1) or (49.2.B. 3.2.1.2) 9 ml of the inoculated culture medium (49.2.B. 3.2.2). The filling operation of the tubes must be done in an appropriate manner, but not necessarily in sterile conditions.

49.2. B. 3.2.4 Incubation. The incubation must be done in a water bath whose temperature is kept homogeneous at 37 ° C ± 0.1 ° C by agitation. The incubation time should be chosen in such a way that it is possible to draw a transmission curve whose inclination is appropriate to precise measurements (usually two hours thirty minutes to three hours). Then block the growth by rapidly introducing 1 ml of formaldehyde solution (49.2.B. 2.2.6) into each tube.

49.2. B. 3.2.5 Measure of growth. Measure the transmissions using the photometer at 590 nm, regulating the apparatus at 100 per 100 transmission over the most clear calibration solution (corresponding to the highest antibiotic content). Due to the small differences in turbidity presented by the different tubes, it is recommended to use buckets of 2 cm at least, and preferably 5 cm.

49.2. B. 4 Calculations. Graphically plot the calibration curve on millimeter paper, putting the photometric transmissions in relation to the antibiotic concentrations. Interpolate on the curve the transmissions relative to the extract. Calculate the antibiotic content of the sample.

The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 per 100, in relative value.

49.2. B. 5 Remarks.

49.2. B. 5.1 This strain, isolated by the LUFA in Kiel, charges faster growth than S. aureus ATCC 6538 P.

49.2. B. 5.2 Any means of commercial cultivation of a similar composition can be used and of the same results.

49.2. B. 6 References. Third Commission Directive of 27 April 1972. 72 /199/EEC. 'Official Journal of the European Communities' number L, 123/6 of 29 May 1972.

50. Oleandomycin

(By Broadcast on agar)

50.1 Principle. The method allows for the determination of oleandomycin in feed, concentrates and premixtures, even in the presence of tetracyclines. The lower limit of the determination is 0.5 mg/kg.

The sample is subjected to extraction by a diluted methanolic solution of tris (hydroxymethyl) aminomethane. After centrifugation, the extract is diluted and its antibiotic activity is determined by the measurement of the diffusion of the oleandomycin in an agar medium sown with B. cereus. The diffusion shall be indicated by the formation of inhibition zones in the presence of the micro-organism. The diameter of these areas is directly proportional to the logarithm of the antibiotic concentration.

50.2 Reassets.

50.2.1 Microorganism: B. cereus K 205 TR (see 50.5.1) (resistant to tetracyclines).

50.2.1.1 Maintenance of the strain. Seed B. cereus on tube agar inclined with the culture medium (50.3.2.2.1) with the addition of 100 micrograms of oxytetracycline by 5 ml. Incubate for one night at approximately 30 ° C. Preserve the culture in the refrigerator and resembrate every four weeks over inclined agar.

50.2.1.2. Preparation of the suspension of spores. Collect the germs from an inclined agar tube (50.2.1.1) with approximately 3 ml of physiological serum (50.2.2.3). Sow with said suspension a vial of Roux containing 300 ml of the culture medium (50.2.2.1) whose concentration is 3 to 4 per 100. Incubate three to five days at 28-30 ° C, then collect the spores in 15 ml of ethanol (50.2.2.4), after having checked the sporulation under the microscope, and homogenize. Such suspension may be kept in the refrigerator for at least five months.

By pre-testing on plates with the base medium for determination (50.2.2.2) to determine the amount of inoculum to be obtained for the different concentrations of oleandomycin used, some inhibition zones as extensive as possible and clear. This amount is usually 0.1 to 0.2 ml/1,000 ml. The sowing of the culture medium shall be 60 ° C.

50.2.2 Crop media and reagents.

50.2.2.1 Strain maintenance medium (see 50.5.2.)

Glucose: 1 g.

Peptone trypsica: 10 g.

Meat extract: 1.5 g.

yeast extract: 3 g.

Agar according to quality: 10 to 20 g.

Distilled Water: Up to 1,000 ml.

Adjust the pH to 6.5 at the time of your job.

50.2.2.2 Base of determination (see 50.5.2). Medium (50.2.2.1) adjusted at pH 8.8.

50.2.2.3 Sterile physiological suero.

50.2.2.4 Ethanol at 20 per 100 (v/v).

50.2.2.5 Methanol p.a.

50.2.2.6 Solution to 0.5 per 100 (w/v) tris (hydroxymethyl) amino methane p.a.

50.2.2.7 Extraction solution.

Pure Methanol: 50 ml.

distilled water: 50 ml.

Tris (hydroxymethyl) amino methane p.a.: 0.5 g.

50.2.2.8 Pattern substance: oleandomycin of known activity.

50.3 Procedure.

50.3.1 Pattern solution. Dissolve standard substance (50.2.2.8) in 5 ml of methanol (50.2.2.5) and dilute with the solution (50.2.2.6) to obtain a concentration in oleandomycin of 100 micrograms/ml.

From such a standard solution, prepare diluting by solution (50.2.2.6) a working standard solution S containing 0.1 micrograms/ml of oleandomycin.

Prepare next for successive dilutions (1 + 1) with the help of the solution (50.2.2.6) the following concentrations:

S: 0.05 micrograms/ml.

S: 0.025 micrograms/ml.

S: 0.0125 micrograms/ml.

50.3.2 Extract. Take according to the supposed content of oleandomycin of the sample, a quantity of 2 to 10 grams, add 100 ml of the solution (50.2.2.7) and shake for thirty minutes with agitator.

Centrifuge, take an aliquot of the extract and dilute by solution (50.2.2.6) to obtain an assumed concentration in oleandomycin of 0.1 micrograms/ml (U). Prepare the U, U and U concentrations by successive dilutions (1 + 1) with the help of the solution (50.2.2.6).

50.3.3. Modalities of the determination.

50.3.3.1. Sowing of the culture medium.

Sow at 60 ° C the base of the determination (50.2.2.2) with the suspension of spores (50.2.1.2).

50.3.3.2 Preparing the boxes. The agar diffusion is verified in boxes with the four concentrations of the calibration solution (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each box must necessarily receive the four calibration and extract concentrations.

To this end, choose the dimensions of the boxes in such a way that at least eight cavities of 10 to 13 mm in diameter can be made in the gelled medium. Calculate the amount of seed culture medium (50.3.3.1) to be used to obtain a uniform coating of approximately 2 mm thickness. It is preferable to use as boxes flat glass plates with an aluminium ring or a perfectly flat plastic material of 200 mm in diameter and 20 mm in height.

Introduce exactly measured amounts of antibiotic solution between 0.10 and 0.15 ml according to the diameter with the pipette in the cavities.

For each sample, make at least four diffusion repetitions with each concentration, so that each determination is the subject of an evaluation of 32 inhibition zones.

50.3.3.3 Incubation. Incubate the boxes for eighteen hours at approximately 28-30 ° C.

50.4 Calculations. Measure the diameter of the inhibition zones, preferably by projection. Record the measurements on semi-logarithmic paper, representing the logarithm of the concentrations against the diameters of the inhibition zones. Draw the straight lines of the calibration solution and the extract. In the absence of interference, the two straight lines must be parallel.

The logarithm of the relative activity will be calculated by the following formula:

U + U + U + U-S-S-S-S) x 0.602

U + U + U + U-S-S-S-S) x0.602

U + U -S-S-U-U-S-S

Actual activity = assumed activity x relative activity.

The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 per 100 in relative value.

50.5 Remarks.

50.5.1 Cepa isolated by the LUFA in Kiel.

50.5.2 Any commercial culture medium of analogous composition can be employed and of the same results.

50.6 References. Third Commission Directive of 27 April 1972. 72 /199/EEC. 'Official Journal of the European Communities' number L, 123/6 of 29 May 1972. Annex II.

51. Amprolium

51.1 Principle. The method allows for the determination of amprolium in feed, concentrates and premixtures. The lower limit of detection is 40 mg/kg.

The sample is subjected to extraction by diluted methanol. The extract is purified on aluminium oxide column and is treated by a methanolic solution of 2,7-dihydroxynaphthalene, potassium hexacane (III), potassium cyanide and sodium hydroxide. A purple coloring develops. The amprolium is determined by spectrophotometry at 530 nm.

51.2 Material and apparatus.

51.2.1 Erlenmeyer 50, 250 and 500 ml screw cap.

51.2.2 Agitator.

51.2.3 Filter crucible, porosity G 3, diameter: 60 mm.

51.2.4 Glass column for chromatogafia (inside diameter: 9 mm, length: 400 to 500 mm).

51.2.5 Centrileakage, with 25 ml screw cap tubes.

51.2.6 Spectrophotometer, with 10 mm thick buckets.

51.3 Reassets.

51.3.1 Methanol p.a.

51.3.2 Diluted Methanol.

Mix two volumes of methanol (51.3.1) and a volume of water.

51.3.3 Solution to 0.2 per 100 (w/v) potassium hexacianoferde (III) (K Fe (CN)) p.a.

This solution remains stable for two weeks.

51.3.4 Solution to 1 per 100 (w/v) potassium cyanide p.a. This solution remains stable for two weeks.

51.3.5 Solution to 1,125 per 100 (w/v) sodium hydroxide p.a.

51.3.6 Sodium hydroxide methanolic solution.

Take 15 ml of the solution (51.3.5) and complete 200 ml by methanol (51.3.1).

51.3.7 Solution to 0.0025 per 100 (w/v) 2,7-dihydroxynaphthalene. Dissolve 25 mg of 2,7-dihydroxynaphthalene p.a. in methanol (51.3.1) and complete 1,000 ml by methanol (51.3.1).

51.3.8 Reactive coloring. Introduce 90 ml of 2,7-dihydroxynaphthalene solution (51.3.7) into a 250 ml erlenmeyer (51.2.1), add 5 ml of potassium hexanocyaniderde (III) solution (51.3.3) and homogenise. Add 5 ml of potassium cyanide solution (51.3.4), cover and homogenise. Leave to rest for thirty-thirty-five minutes, add 100 ml of sodium hydroxide methanolic solution (51.3.6), homogenise and filter on a filter crucible (51.2.3). Use that reagent in the seventy-five minutes that follow the leak.

51.3.9 Aluminium oxide for column chromatography. Before use, shake for thirty minutes, 100 g of aluminium oxide with 500 ml of water, filter, wash three times the sediment over the filter with 50 ml of methanol (51.3.1) each time, dry by aspiration, leave to rest one night and dry for two hours at 100 ° C in a vacuum stove. Allow to cool in desiccator. Control the activity by analyzing, from the point (51.4.2), a certain amount of standard solution (51.3.11). The recovery rate of the amprolium should be 100 per 100 ± 4 per 100.

51.3.10 Pattern Substance. Pure amprolium that responds to the following characteristics:

-Merge point (decomposition): 248 ºC.

-Molecular extinction coefficient at 265 and 235 nm in distilled water: 11.0 x 103.

51.3.11 Pattern solution. Weigh, with accuracy of 0.1 mg, 50 mg of standard substance (51.3.10). Dissolve with diluted methanol (51.3.2) in a 500 ml graduated flask, complete by volume by the same solvent and homogenise. Take 10.0 ml to complete 50 ml by diluted methanol (51.3.2) in a graduated flask and homogenise 1 ml of the solution contains 20 micrograms of amprolium.

51.4 Procedure.

51.4.1 Extraction and Purification.

51.4.1.1 Feed and premixtures. Weigh, with precision of 1 mg, 10 g of finely divided and homogenized sample. For premixtures, weigh 3 to 6 g, with precision of 1 mg. Insert the sample into a 250 ml erlenmeyer (51.2.1) and add 100 ml of exactly diluted methanol (51.3.2). Shake for sixty minutes and filter. Dilute if necessary by diluted methanol (51.3.2) to obtain a solution containing 5 to 15 micrograms of amprolium per ml. Insert in a column for chromatography (51.2.4) previously provided in its lower extremity of a cotton stopper, 5 g of aluminium oxide (51.3.9) and then 25.0 ml of the extract. Let the liquid pass, remove the first 5 ml and collect the next 12 ml in a graduated test piece.

51.4.1.2 Concentric. Weigh, with precision of 1 mg, 0.5 g finely divided and homogenized sample, introduce them into a 500 ml erlenmeyer (51.2.1), add 250 ml of diluted methanol (51.3.2), shake for sixty minutes and filter. Take 5.0 ml of the filtrate and complete 200 ml by diluted methanol (51.3.2) in a graduated flask.

51.4.2 Development of the coloration and measurement of the absorbance. Take 5.0 ml of the solution obtained in (51.4.1.1) or (51.4.1.2) and introduce them into a centrifuge tube A (51.2.5). Introduce 5.0 ml of diluted methanol (51.3.2) into a centrifuge tube B (51.2.5). Add in each tube 10,0 ml of the colouring reagent (51.3.8), cover the tubes, homogenise and leave to rest for eighteen minutes. Centrifuge then for three minutes to obtain clear solutions and decant the solutions A and B in the 50 ml erlenmeyer (51.2.1).

Measure immediately in the spectrophotometer at 530 nm the absorbance of solution A, using as white the solution B. Determine the amount of amprolium referring to the calibration curve (51.4.3).

51.4.3 Calibration curve. Enter in the centrifugal tubes (51.2.5) respective volumes of 1.0-2.0-3.0-4.0 and 5.0 ml of standard solution (51.3.11). Complete the volume of the first four tubes up to 5.0 ml with diluted methanol (51.3.2). Add in the five tubes 10.0 ml of colouring reagent (51.3.8), cover the tubes, homogenise and leave to rest for eighteen minutes. Centrifuge then for three minutes and decant the solutions in 50 ml erlenmeyer (51.2.1).

Measure immediately in the spectrophotometer at 530 nm the absorbance of the solutions, using as white a mixture of 5 ml of diluted methanol (51.3.2) and 10 ml of the colouring reagent (51.3.8). Draw the calibration curve by taking the values of the absorbances and the abscissa into the corresponding amounts of amprolium in mg.

51.5 Calculations.

51.5.1 Feed and premixtures. The mg content of amprolium per kg of sample is given by the formula:

A x F x 20,000

A x F x 20,000

P xFx 20,000

Being:

A = Quantity, in mg, of amprolium determined by spectrophotometric measurement.

P = Weight, in g, of the sample.

F = dilution coefficient (performed if the case was given in 51.4.1.1.).

51.5.2 Concentric. The amprolium content in sample percentage is given by the formula:

A x 200

A x 200

P x 200

Being:

A = Quantity, in mg, of amprolium determined by spectrophotometric measurement.

P = Weight, in g, of the sampling.

The difference between the results of two parallel determinations performed on the same sample must not exceed:

-10 mg/kg in absolute value for the amprolium content of less than 100 mg/kg.

-10 per 100, in relative value, for contents between 100 and 5,000 mg/kg.

-500 mg/kg, in absolute value, for contents between 5,000 and 10,000 mg/kg.

-5 per 100, in relative value, for contents greater than 10,000 mg/kg.

51.6 References. Fifth Commission Directive of 25 March 1974. (74/203/EEC). 'Official Journal of the European Communities' number L, 108/7 of 22 April 1974.

52 Etopabato

(Methyl-4-acetamido-2-ethoxybenzoate)

52.1 Principle. The method allows for the determination of etopabate in feed, concentrates and premixtures. The lower limit of detection is 2 mg/kg.

The sample is subjected to extraction by diluted methanol. The solution is acidified and extracted with chloroform. The chloroformic extract is first washed with an alkaline solution and then with water. The purified extract is concentrated, the etopabate is hydrolyzed by the diluted hydrochloric acid. The aminated derivative thus formed is diazota and is copulates with the N-(1-naphthyl) ethylenediamine. The coloured complex is extracted by butanol and the absorbance of the solution is measured at 555 nm.

52.2 Material and apparatus.

52.2.1 Erlenmeyer 250 ml, with screw cap.

52.2.2 100 ml decantation, with a grinding plug.

52.2.3 Agitator.

52.2.4 Empty rotary evaporator with 250 ml flasks.

52.2.5 Bath of water.

52.2.6 Centrileakage, with tubes of 15 and 50 ml, of standard grinding.

52.2.7 Air Refrigerant, with grinding mouth.

52.2.8 Spectrophotometer, with 10 mm thick buckets.

52.3 Reassets.

52.3.1 Methanol p.a.

52.3.2 Methanol at 50 per 100 (v/v). Mix equal volumes of methanol (52.3.1) and water.

52.3.3 Hydrochloric acid p.a. (d = 1.19).

52.3.4 Diluted hydrochloric acid to 1/10. Take 10.0 ml of hydrochloric acid (52.3.3), complete 100 ml with water.

52.3.5 Approximately 0.3N hydrochloric acid. Take 25.0 ml of hydrochloric acid (52.3.3), complete 1,000 ml with water.

52.3.6 Chloroform p.a.

52.3.7 Solution to 4 per 100 (w/v) sodium carbonate. Dissolve 40 g of sodium carbonate anhydrous p.a. in water and complete 1,000 with water.

52.3.8 Solution to 0.2 per 100 (w/v) sodium nitrite.

Dissolve in water 100 mg sodium nitrite p.a. and complete 50 ml with water in a graduated flask. Prepare immediately before your employment.

52.3.9 Solution to 1.0 per 100 (w/v) ammonium sulfamate. Dissolve in water 500 mg of ammonium sulfamate p.a. and complete 50 ml with water in a graduated flask. Prepare immediately before your employment.

52.3.10 Solution at 0.2 per (w/v) N-(1-naphthyl) ethylenediamine. Dissolve in water 100 mg N-(1-naphthyl) ethylenediamine p.a. and complete 50 ml with water in a graduated flask. Prepare immediately before your employment.

52.3.11 Sodium chloride anhydrous, p.a.

52.3.12 N-butanol p.a.

52.3.13 Pattern Substance. Pure etopabate.

52.3.14 Pattern solution.

52.3.14.1 Solution of 0.040 mg of etopabate per ml. Weigh to the nearest 0,1 mg, 40 g of standard substance (52.3.13). Dissolve with diluted methanol (52.3.2) in 100 ml graduated flask. Complete by volume using the same solvent and homogenize. Take 10.0 ml, complete 100 ml with methanol (52.3.2) in a graduated flask and homogenise. This solution remains stable for one month.

52.3.14.2 Solution of 0.016 mg etopabate in 20 ml. Take 5.0 ml of the solution (52.3.14.1) complete to 250 ml with diluted methanol (52.3.2) in a graduated flask and homogenise. Prepare before your employment.

52.4 Procedure.

52.4.1 Extraction. Weigh, with precision of 1 mg, a finely divided and homogenized sample quantity containing 80 micrograms approximately etopabate. Introduce the sample into a 250 ml erlenmeyer (52.2.1) and add 100.0 ml of diluted methanol (52.3.2). Mix, close the erlenmeyer and shake for an hour using the agitator (52.3.4). Let decant, filter and remove the first ml of the filtrate.

52.4.2 Purification. All operations described at this point must be carried out quickly.

Introduce 20.0 ml of the limpid extract into a 100 ml decantation funnel (52.2.2), add 5.0 ml of dilute hydrochloric acid to 1/10 (52.3.4) and 20.0 ml of chloroform (52.3.6). Shake first with prudence and then vigorously for three minutes. Leave to rest until the separation of the phases and collect the chloroformic phase in a second decantation funnel of 100 ml (52.2.2).

Extract the acid phase twice successive by 30.0 ml of chloroform (52.3.5) each time. Gather the chloroformic extracts in the second decantation funnel and remove the acid phase. Add 10 ml of sodium carbonate solution (52.3.7) to the chloroformic solution, shake for three minutes and leave to rest until the phases are separated. Collect the chloroformic phase in a third 100 ml decanting funnel (52.2.2) and remove the aqueous phase. Add 10 ml of sodium carbonate solution (52.3.7) to the chloroformic solution, shake for three minutes and leave to rest until the phases are separated.

Collect the chloroformic phase in a fourth decantation funnel of 100 ml (52.2.2), wash twice successively with 25 ml of water each time, separate the aqueous phases and quantitatively collect the chloroformic extract in a flask 250 ml (52.2.4). Assemble the aqueous phases, wash the empty decantation funnels by some ml of chloroform (52.3.6) then wash the aqueous phase using said chloroform. Separate the chloroformic phase and add it to the extract collected in a flask.

52.4.3 Hydrolysis. Evaporate the chloroformic extract up to 2 ml approximately on a water bath at 50 ° C in the rotary vacuum evaporator (52.2.4). Dissolve the residue with 2 or 3 ml of methanol (52.3.1). Quantitatively transfer the solution to a 50 ml centrifuge tube (52.2.6) with two 10 ml portions and a 5 ml portion of 0.3N hydrochloric acid (52.3.5). Add some fragments of pumice stone, homogenize and attach to the tube an air coolant (52.2.7). Dip the tube into a boiling water bath and keep it for forty-five minutes. Leave to cool below under a cold water stream.

52.4.4 Development of colouring and measurement of absorbance. Add 1.0 ml of sodium nitrite solution (52.3.8), shake and leave to rest for two minutes. Add 1.0 ml of ammonium sulfamate solution (52.3.9), shake and leave to rest for two minutes. Add 1.0 ml of N-(1-naphthyl) ethylenediamine solution (52.3.10), shake and leave to rest for ten minutes. Add 5.0 g of sodium chloride (52.3.11) and 10.0 ml of n-butanol (52.3.12), shake vigorously until the complete dissolution of sodium chloride.

Transfer the supernatant butanolic layer with the aid of a pipette, to a 15 ml centrifuge tube (52.2.6) and centrifuge. Then measure the AA absorbance in spectrophotometer at 555 nm by comparison with n-butanol (52.3.12).

52.4.5 Test blank. Carry out a blank test using the same operating method, starting from point 52.4.2 with 20.0 ml of diluted methanol (52.3.2). Measure the absorbance AB at 555 nm by comparison with n-butanol (52.3.12).

52.4.6 Pattern Test. Carry out a test using the same operating method, starting from point 52.4.2, with 20.0 ml of standard solution (52.3.14.2). Measure the absorbance AC at 555 nm by comparison with n-butanol (52.3.12).

52.5 Calculations. The content in mg of etopabate per kg of sample is given by the formula:

A-A x 80

A-A x 80

A-A x P

Being:

A = Absorbance of the solution from the sample.

A = Absorbance of the solution resulting from the blank test.

A = Absorbance of the solution resulting from the pattern test.

P = Weight in grams of the sample.

The difference between the results of two parallel determinations performed on the same sample must not exceed:

-20 per 100, in relative value, for the contents in etopabate less than 7.5 mg/kg.

-Of 1,5 mg/kg, in absolute value, for contents between 7,5 and 10 mg/kg.

-15 per 100, in relative value, for contents greater than 10 mg/kg.

52.6 References. Fifth Commission Directive of 25 March 1974. (74/203/EEC). 'Official Journal of the European Communities' number L, 108/7 of 22 April 1974.

53. Dinitolmida (DOT 6 Zoalene)

(3,5-dinitro-o -toluamide)

53.1 Principle. The method enables the determination of dinitolmides (DOT) in feed, concentrates and premixtures. Nitrofuran derivatives interfere. The lower limit of detection is 40 mg/kg.

The sample is subjected to extraction with acetonitrile. The extract is purified on aluminium oxide and filtered. An aliquot portion of the filtrate evaporates to dryness. The residue is collected by dimethylformamide and is treated with ethylenediamine. A purple coloring develops. The dinitolmide is determined by spectrophotometry at 560 nm.

53.2 Material and apparatus.

53.2.1 Erlenmeyer 250 ml, standard grinding.

53,2.2 Normalized, low-ebb, reflux refrigerant.

53.2.3 Filter crucible, porosity G 3, diameter 60 mm.

53.2.4 Empty filtration apparatus (e.g. Witt apparatus).

53.2.5 Bath of water, regulated at 50 ° C.

53.2.6 Spectrophotometer, with a 10 mm thick cubed.

53.3 Reassets.

53.3.1 Acetonitrile at 85 per 100 (v/v). Mix 850 ml of pure acetonitrile and 150 ml of water, distil the mixture before use; collect the fraction that distills between 75 and 77 ° C.

53.3.2 Aluminium oxide for column chromatography. Calcined for two hours at least 750 °C, cool in desiccator and consign in glass bottle topaz of a frosted stopper. Before use, wetting as indicated: insert in a glass bottle topaz 10 g of aluminium oxide and 0,7 ml of water, cover hermetically and heat for five minutes in a boiling water bath, stirring throughout the time with energy. Let cool stirring it. Control the activity by subjecting the analysis, from the point (53.4.1), to a given quantity of standard solution (53.3.6). The recovery of the dinitolmide should be 100 per 100 ± 2 per 100.

53.3.3 N, N-dimethylformamide 95 per 100 (v/v): mix 95.0 ml of N, N-dimethylformamide p.a. and 5.0 ml of water.

53.3.4 Ethylenediamine p.a., maximum water content 2.0 per 100.

53.3.5 Pattern substance: 3,5 dinitro-o-pure toluamide that responds to the following characteristics:

Merge point: 177 ° C.

Molecular Extinction Coefficient at 248 nm in acetonitrile: 13.1/103.

Molecular Extinction Coefficient at 266 nm in N, N-dimethylformamide: 10.1/103.

53.3.6. Standard solution. Weigh, with accuracy of 0.1 mg, 40 mg of standard substance (53.3.5). Dissolve with acetonitrile (53.3.1) in a 200 ml graduated flask. Complete volume with the same solvent and homogenize. Take 20.0 ml, complete 100 ml with acetonitrile (53.3.1) in a graduated flask and homogenise. 1 ml of this solution contains 40 micrograms of dinitolmida.

53.4 Procedure.

53.4.1 Extraction and Purification. Weigh, with precision of 1 mg, 10 g of finely divided and homogenized sample. For concentrates and premixtures, weigh with precision of 1 mg, 1 g. Insert the sample into a 250 ml erlenmeyer (53.2.1) and add 65 ml of acetonitrile (53.3.1). Mix, attach to the erlenmeyer the reflux coolant (53.2.2) and heat in the water bath (53.2.5) for thirty minutes continuously stirring it. Allow to cool with cold water current. Add 20 g of aluminium oxide (53.3.2), shake for three minutes and allow decanting.

Place a 100 ml graduated flask into the filtration apparatus (53.2.4), adjust the filter crucible (53.2.3) and filter the liquid by vacuuming. Then transfer the sediment to the crucible with a few ml of acetonitrile (53.3.1) and aspire. Cut the vacuum, put the sediment back into suspension with a few ml of acetonitrile (53.3.1) and aspire again. Repeat these last operations until the volume of the filtrate reaches approximately 95 ml. Complete the volume to 100 ml by the acetonitrile (53.3.1) and homogenise.

If necessary, take an aliquot part and dilute with acetonitrile (53.3.1) to obtain a solution containing 5 to 15 micrograms of dinitolmide per ml.

53.4.2 Development of colouring and measurement of absorbance. Introduce respectively in three vessels of 50 ml, A, B and C, 4.0 ml of the solution obtained in (53.4.1). Add only to the glass C, 1.0 ml of standard solution (53.3.6). Bring the three glasses over the water bath (53.2.5) located under a well-ventilated gas campaign and evaporate to dry under current air. Allow to cool down to room temperature.

Add 10.0 ml of N, N-demethylformamide (53.3.3) to vessel A and 2.0 ml respectively to vessels B and C. Let in contact for a few minutes agitating slightly until complete dissolution of the residue. Add 8.0 ml of ethylenediamine (53.3.4) in the B and C vessels and homogenise. Measure, five minutes exactly after the addition of ethylenediamine, the absorbance of the three solutions by the spectrophotometer (53.2.6) at 560 nm, using as white the N, N-dimethylformamide (53.3.3).

53.5 Calculations. The mg content of dinitolmide per kg of sample is given by the formula:

A-A x F

A-A x F

A-B 3 P

Being:

A = Absorbance of solution A (witness).

A = Absorbance of solution B (sample).

A = Absorbance of solution C (internal pattern).

P = Weight in grams of the sample.

F = Dilution coefficient (performed if the case was given (53.4.1)).

The difference between the results of two parallel determinations performed on the same sample must not exceed:

-10 mg/kg, in absolute value, for the contents of dinitolmidm less than 100 mg/kg.

-10 per 100, in relative value, for contents between 100 and 5,000 mg/kg.

-500 mg/kg, in absolute value, for contents between 5,000 and 10,000 mg/kg.

-5 per 100, in relative value, for contents greater than 10,000 mg/kg.

53.6 References. Fifth Commission Directive of 25 March 1974 (74/203/EEC). 'Official Journal of the European Communities' number L, 108/7 of 22 April 1974.

54. Nicarbazine

(4,4-dinitrocarbanilide Echimolecular Mix

and 2-hydroxy-4,6-dimethyl pyrimidine)

54.1 Principle. The method allows for the determination of nicarbazine in feed, concentrates and premixtures that do not contain more than 5 per 100 of forregrate flours. Nitrofuran derivatives, acetylheptin and carbadox interfere. The limit of detection is 20 mg/kg.

The sample is subjected to extraction with N, N-dimethylformamide. The extract is purified in aluminium oxide chromatographic column: the nicarbazine is eluted with ethanol. Eluate is treated with an ethanolic solution of sodium hydroxide: a yellow colouring is developed. The nicarbazine is determined spectrophotometrically at 430 nm.

54.2 Material and apparatus.

54.2.1 Erlenmeyer 250 ml with normalized mouth.

54.2.2 Reflux coolant, with normalized mouth.

54.2.3 Bath of boiling water.

54.2.4 Centrileakage with 120 ml tubes

54.2.5 Glass chromatographic column (inside diameter: 25 mm, length 300 mm).

54.2.6 Spectrophotometer with 10 mm thick buckets.

54.2.7 Buretas graduated to 1/10 ml.

54.3 Reassets.

54.3.1 N, N-dimethylformamide p.a.

54.3.2 Aluminium oxide for column chromatography. Calcined for two hours at least 750 °C, cool in desiccator and store in glass bottle topaz with stopper. Before using it, control its activity by subjecting to analysis from the point (54.4.2), a determined amount of pattern (54.3.8.3). The recovery of nicerbazine should be 100 per 100 ± 2 per 100.

54.3.3 Ethyl alcohol of 95 per 100 (v/v).

54.3.4 ethyl alcohol of 80 per 100 (v/v).

54.3.5 Solution to 50 per 100 (w/v) sodium hydroxide p.a.

54.3.6 A 1 per 100 (w/v) ethanolic solution of sodium hydroxide. Take 1 ml of the solution (54.3.5) in a 50 ml graduated flask and make up to 80 per 100 ethanol (54.3.4). Prepare at the time of employment.

54.3.7 Pattern Substance. Pure nicarbazine, molecular extinction coefficient at 362 nm in N, N-dimethylformamide is 37.8 x 10 3.

54.3.8 Pattern solution.

54.3.8.1 1.25-mg solution of nicarbazine per ml. Weigh with a precision of 0.1 mg, 125 mg of standard substance (54.3.7). Dissolve in 75 ml N, N-dimethylformamide (54.3.4), in a 100 ml graduated flask, heating slightly. Leave to cool, complete with volume with the same solvent and homogenize. Keep the coat of light.

54.3.8.2 Solution of 0.125 mg of nicarbazine per ml. Take 10 ml of the solution (54.3.8.1) and complete 100 ml with N, N-dimethylformamide (54.3.1) in a graduated flask and homogenise.

54.3.8.3 Solution of 0.025 mg of nicarbazine per ml. Take 20 ml of the solution (54.3.8.2), complete 100 ml with N, N-dimethylformamide (54.3.1) in a graduated flask and homogenise.

54.4 Procedure.

54.4.1. Extraction. Weigh, with precision of 1 mg, 10 g of finely divided and homogeneous sample. For concentrates and premixtures weigh 1 g with the accuracy of 1 mg, enter the sample into a 250 ml erlenmeyer (54.2.1) and add 100 ml of exactly N, N-dimethylformamide (54.3.1). Mix, attach to a reflux coolant (54.2.2) and heat in water bath (54.2.3) for 15 minutes stirring occasionally. Allow to cool under cold water current.

Then transfer the supernatant liquid to a centrifuge tube (54.2.4) and centrifuge for approximately three minutes.

If pipette 25 ml of the supernatant and dilute with N, N-dimethylformamide (54.3.1) need to be pipette to obtain a solution containing 2 to 10 micrograms of nicarbazine per ml.

54.4.2. Chromatography. Insert in a chromatographic column (54.2.5) 30 g of aluminium oxide (54.3.2) in suspension in N, N-dimethylformamide (54.3.1). Leave the liquid down to 1 cm above the aluminium oxide column and then insert into the column 25 ml of the extract obtained at (53.4.1). Let the liquid pass by avoiding leaving the column dry and wash the column three times the column with 10 ml of N, N-dimethylformamide (54.3.1) each time. Elude immediately with 70 ml of ethanol at 95 per 100 (54.3.3). Remove the first 10 ml of eluate and collect the remainder in fractions as follows:

-A fraction (a) of 5 ml.

-A fraction b) of 50 ml in a graduated flask.

-A fraction c) of 5 ml.

Control that fractions a) and c) do not show yellow coloration for the addition of ethanolic sodium hydroxide solution (54.3.6). Continue operations on fraction b) as indicated in (54.4.3).

54.4.3. Development of the colouring and measurement of the absorbance. Insert 20 ml of the fraction (b) respectively into two 25 ml graduated flasks A and B. Add to the flask A 5.0 ml of ethanolic sodium hydroxide solution (54.3.6) and the flask B 5.0 ml of ethanol of 95 per 100 (54.3.3). Homogenize.

Measure in the first five minutes the absorbance of the solutions at 430 nm using as white a mixture of 20.0 ml of ethanol of 95 per 100 (54.3.3) and 5 ml of ethanolic solution of sodium hydroxide (54.3.6).

The difference of the absorbents between solution B and A is interpolated in the calibration curve (54.4.4) to obtain the amount of nicarbazine.

54.4.4. Calibration curve. Treat 25 ml of the standard solution (54.3.8.3) by chromatography as indicated in (54.4.2). Transfer to a graduated burette (54.2.7) the fraction (b) of the eluate and pipette into dry flasks of 25 ml, respectively, the following volumes: 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 nicarbazine). Add to each flask 5.0 ml of ethanolic sodium hydroxide solution (54.3.6) to make up to 95 per 100 ethanol (54.3.3) and to homogenise.

Measure in the first five minutes the absorbance of the solutions at 430 nm, using as white a mixture of 20 ml of ethanol of 95 per 100 (54.3.3) and 5.0 ml of ethanolic solution of sodium hydroxide. Plot the pattern curve representing the value of the absorbance and the corresponding amounts of nicarbazine in mg in order.

54.5 Calculations. The amount of nicarbazine in mg per kg of sample is given by the formula:

A x F x 10,000

A x F x 10,000

P x F x 10,000

Being:

A = Quantity, in mg, of determined nicarbazine per measure spectrophotometric.

P = Weight, in g, of the sample.

F = Dilution coefficient (eventually made in 54.4.1).

The difference between the results of two parallel determinations performed on the same sample must not exceed the following values:

-10 mg/kg, in absolute value, for contents in nicarbazine less than 100 mg/kg.

-10 per 100, in relative value, for contents between 100 and 5,000 mg/kg.

-500 mg/kg, in absolute value, for contents ranging from 5,000 to 10,000 mg/kg.

-5 per 100, in relative value, for contents greater than 10,000 mg/kg.

54.6 References. Fifth Commission Directive of 25 March 1974 (74/203/EEC). 'Official Journal of the European Communities' number L, 108/7 of 22 April 1974.

55. Buquinolate

(ethyl-4-hydroxy-6.7 Carboxylate

diiso-butoxy-3-quinoline)

55.1 Principle. The method allows the determination of buquinolate in feed, concentrates and premixtures. The lower limit of detection is 10 mg/kg. Decoquinate interferes in dosing.

The buquinolate is extracted from the sample with chloroform. The extract evaporates to dryness, the residue is collected with chloroform and the solution is chromatography in thin layer. The buquinolate is eluted by ethanol and determined spectrophotomically by comparison with standard solutions.

55.2 Material and apparatus.

55.2.1 Erlenmeyer 50 and 250 ml with screw cap.

55.2.2 Agitator.

55.2.3 Centrileakage with 15 ml tubes with screw cap.

55.2.4 Bath of thermoregulated water at 50 ° C.

55.2.5 Equipment for thin layer chromatography.

55.2.6 Glass plates for 200 x 200 mm thin-layer chromatography, prepared as follows: uniformly extend over the plates a layer of 0.5 mm thick silica gel G (55.3.5) (55.6.1). They are allowed to dry for fifteen minutes in the air. Then they are introduced for two hours at 110 ° C in the stove (55.2.11). They are cooled in desiccator containing silica gel as dehydrating.

Equivalent plates can be used already prepared.

55.2.7 Micropipettes of 0.50 ml.

55.2.8 Zone Colector for thin layer chromatography.

55.2.9 Short-wave ultraviolet Lamp.

55.2.10 Espectroluorimeter equipped with a xenon lamp and two monochrome lamps.

55.2.11 Forced air and temperature adjustable from 0 ° C to 150 ° C.

55.2.12 Rotary Vacuum Evaporation Apparatus with 250 ml flask

55.3 Reassets.

55.3.1 Chloroform p.a.

55.3.2 Ethanol 96 per 100 (v/v) p.a.

55.3.3 Chloroform-ethanol Mix. Mix 10 volumes of chloroform (55.3.1) and volume of ethanol (55.3.2).

55.3.4 Ethanol 80 per 100 (v/v) p.a.

55.3.5 Gel of silica G for thin layer chromatography.

55.3.6 Buquinolate pattern quality.

55.3.7 Pattern solutions:

55.3.7.1 Standard solutions 0.1 mg/ml buquinolate. Weigh 50 mg of standard substance (55.3.6) with the accuracy of 0.1 mg. Dissolve in chloroform (55.3.1) and hot, in a water bath at 50 ° C. Transfer quantitatively to a 250 ml graduated flask. Allow to cool to room temperature. Complete with chloroform (55.3.1) up to the aphoro and homogenize.

55.3.7.2 Job Pattern Solutions. Pipette into 25 ml volumetric flasks; 5,0, 10,0, 15,0, 20,0 and 25.0 ml of the standard solution (55.3.7.1). Complete up to the aphoro with chloroform (55.3.1) and homogenise. Prepare at the time of use. These solutions contain 0.04, 0.08, 0.12, 0.16 and 0.20 mg/ml, respectively.

55.4 Procedure.

55.4.1 Sample Preparation. Grind the sample in such a way that it passes through a 1 mm sieve (according to the ISO R 565 recommendations).

55.4.2 Extract. Weigh to the nearest 1 mg an amount of prepared sample according to (55.4.1) containing approximately 1.25 mg of buquinolate and to be quantitatively introduced into a 250 ml erlenmeyer (55.2.1). Add 100.0 ml of chloroform (55.3.1). Mix, cover and shake for an hour in the agitator (55.2.2). Let the first ml of the filtrate decant, filter and despise.

Introduce 80 ml of the limpid filtrate into the flask of the rotavaby (55.2.12). Evaporate almost until dryness over a water bath (55.2.4). Quantitatively transfer the oily residue to a 10 ml flask with the aid of chloroform and through a narrow stem funnel. Complete the volume with chloroform (55.3.1) and if the solution is not clear, centrifuge for three minutes at 3,000 rpm in closed tube.

55.4.3 Fine-Layer Cromatography. Deposit punctually on a chromatography plate (55.2.6) with the help of a micropipette (55.2.7) and at a respective distance of 2 cm, volumes of 0.25 ml of the extract obtained in (55.4.2) and of the five standard solutions (55.3.7.2).

Elude the chromatogram in the development chamber using chloroform (55.3.1) as eluent until the solvent front reaches the top edge of the plate. Dry with the aid of an air stream. Reposition the plate in the development chamber, using as eluent the mixture of chloroform/ethanol (55.3.3) until the solvent front has advanced about 12 cm. Take out the camera plate. Let the solvent evaporate. Examine the plaque under the short-wave ultraviolet light (55.2.9) and delimit the buquinolate spots (Rf value 0.4 to 0.6) with the help of a needle.

55.4.4 Elution. Collect the silica from each delimited area with the help of a zone collector (55.2.8) in a centrifuge tube (55.2.3). Add to each tube 10 ml of ethanol (55.3.4), shake for twenty minutes and then centrifuge for five minutes at 3,000 rpm. Decant the limpid solutions in the 50 ml erlenmeyer (55.2.1).

55.4.5 Measurement of fluorescence. Adjust to 100 the scale of the spectrophotometer (55.2.10) with the aid of eluate (55.4.4) obtained from the most concentrated pattern solution. Use for excitation the wavelength between 300 and 289 nm which of the most intense fluorescence and for emission the wavelength of 375.

Measure in these conditions the fluorescence intensity of the other eluates (55.4.4). Determine from the values found the amount (C) of buquinolate in mg contained in the 10 ml of the sample alluate.

55.5 Calculation. The mg content of buquinolate per kg of sample is given by the formula:

C x 50,000

C x 50,000

P x 50,000

Being:

C = Quantity in mg of buquinolate determined by the spectrophotometric measure.

P = Weight, in grams, of the sample taken for analysis.

The difference between the results of two parallel determinations performed on the same sample should not differ from:

-50 per 100 of the highest result for the buquinolate values between 10 and 20 mg/kg.

-10 mg/kg in absolute value for the values between 20 and 100 mg/kg.

-10 per 100 of the highest result for values between 100 and 5,000 mg/kg.

-500 mg/kg in absolute value for the values between 5,000 and 10,000 mg/kg.

-5 per 100 of the highest result for values greater than 10,000 mg/kg.

55.6 Remarks.

55.6.1 The preparation of the plates is as follows: Mix 60 g of silica gel G (55.3.5) with 120 ml of distilled water, in a 250 ml erlenmeyer with stopper, stirring vigorously for thirty to forty-five seconds. The once well-homogenized suspension is quickly dumped in the applicator and spreads evenly over the plates, a layer of 0.5 mm thick. They are allowed to dry for fifteen minutes in the air; then they are inserted into the stove (55.2.11) for two hours at 110 ° C. They are cooled in desiccator containing silica gel as dehydrating.

55.7 References. Sixth Commission Directive of 20 December 1974 (75/84/EEC). 'Official Journal of the European Communities' number L, 32/26 of 5 February 1975.

56. Sulfaquinoxaline

(2 sulfanilamidoquinoxaline)

56.1 Principle. The method enables the determination of sulfaquinoxaline in compound feed, concentrator and premixtures. The lower limit of the determination is 20 mg/kg. Interfere with other sulfonamides and arsanilic acid.

Sulfaquinoxaline is extracted from the sample with dimethylformamide and chloroform. It is hydrolyzed in alkaline medium. After neutralization of the aminated derivative is diazota and copula with the N-(1-naphthyl) ethylenediamine. The absorbance of the solution is measured at 545 nm.

56.2 Material and apparatus.

56.2.1 250 ml Erlenmeyers with normalized mouth and glass stopper.

56.2.2 Agitator.

56.2.3 Filterings of porosity 3, diameter 80 mm, with kitasates.

56.2.4 250 ml decantation.

56.2.5 Graduated 50, 100, 250, and 500 ml.

56.2.6 150 mm x 25 mm test tubes

56.2.7 Bath of boiling water.

56.2.8 Spectrophotometer with 20 mm optical step buckets.

56.3 Reassets.

56.3.1 N, N-Dimethylformamide p.a.

56.3.2 Chloroform p.a.

56.3.3 Absolute Ethanol.

56.3.4 alkaline solution. Dissolve in distilled water 10 g of sodium hydroxide p.a. and 25 g of sodium chloride p.a. Complete up to 500 ml with distilled water and homogenise.

56.3.5. Concentrated hydrochloric acid p.a. (d = 1.18).

56.3.6 Solution 0.1 per 100 (w/v) sodium nitrate. Dissolve in distilled water 100 mg sodium nitrite, complete with distilled water up to 100 ml and homogenise. Prepare immediately before use.

56.3.7 Solution 0.5 per 100 (w/v) of ammonium sulfamate. Dissolve in distilled water 500 mg of ammonium sulfamate p.a. to complete up to 100 ml with distilled water and homogenise. Prepare immediately before use.

56.3.8 Solution 0.1 per 100 (w/v) of N-(1-naphthyl) ethylenediamine dihydrochloride. Dissolve in hydrochloric acid p.a. 0.1 per 100 (v/v) 100 mg dihydrochloride of N-(1-naphthyl) ethylenediamine p.a. to complete up to 100 ml with the same acid and homogenise. Prepare immediately before use.

56.3.9 Pattern Substance. Pure sulfaquinoxaline.

56.3.10 Pattern solution. Weigh to the nearest 0.1 mg, 250 mg standard substance (56.3.9). Dissolve in about 50 ml of a sodium hydroxide solution (25 ml of a sodium hydroxide solution 0.1 N ° 25 ml of water), complete up to 500 ml with distilled water. Take 5 ml of this solution and dilute to 100 ml with distilled water. 1 ml of this solution contains 25 micrograms of sulfaquinoxaline.

56.4 Procedure.

56.4.1 Sample Preparation. Grind the sample in such a way that it passes in its entirety through a mesh of 1 mm (according to the recommendation ISO R. 565).

56.4.2 Extract. Weigh with the accuracy of mg a sample amount containing 0.25 to 1.25 mg of sulfaquinoxaline. Enter quantitatively into a 250 ml erlenmeyer (56.2.1) and add 20 ml of N, N, dimethylformamide (56.3.1). Mix and heat for thirty minutes on water bath (56.2.7). Allow to cool under cold water current. Add 60 ml of chloroform (56.3.2) plug the erlenmeyer and shake for thirty minutes with the help of the agitator. (56.2.2).

Filter the liquid on the filter crucible (56.2.3) placed in kitasates and aspire slightly. Wash the erlenmeyer with four portions of 5 ml of chloroform (56.3.2) and successively transfer the liquids to the crucible. Transfer the filtrate to a decantation funnel (56.2.4), wash the kitasate with the aid of approximately 15 ml of chloroform (56.3.2) and transfer the liquid to the decantation funnel.

56.4.3 Hydrolysis. Add 50 ml of alkaline solution (56.3.4) and 5 ml of ethanol (56.3.3) to the decanting funnel. Mix well by avoiding emulsion formation, either by reversing the funnel about 20 times or by stirring horizontally. Leave to rest until the separation of the two phases (separation is usually completed around fifteen minutes).

Transfer the upper phase (aqueous phase) to a 250 ml graduated flask (56.2.5). Remove again from the chloroformic phase with three portions of the alkaline solution (56.3.4) and transfer the aqueous extract after each extraction to the graduated flask (56.2.5). Complete up to the force with distilled water and homogenise.

Enter 25.0 ml of the solution into a 50 ml volumetric flask (56.2.5), add 5.0 ml of hydrochloric acid (56.3.5). Complete up to the force with distilled water and homogenise. Filter if necessary and remove the first 15 ml of the filtrate. Insert 10 ml of the solution into two test tubes (56.2.6) A and B respectively.

56.4.4 Development of color and measure of absorbance. Add to each tube 2.0 ml of sodium nitrite solution (56.3.6) shake and leave to rest for three minutes. Add 2.0 ml of ammonium sulfamate solution (56.3.7), shake and leave to rest for two minutes. Add 1.0 ml of N (1-naphthyl) ethylenediamine dihydrochloride solution (56.3.8) in tube A and 1.0 ml of distilled water in the B tube. Mix the contents of each tube well. Connect the tubes to a water tube with the help of rubber gaskets and apply a small vacuum to remove the dissolved nitrogen.

Measure after ten minutes the absorbance Aa and Ab, of the solutions in the spectrophotometer (56.2.8) at 545 nm taking the distilled water as white. Determine from the Aa-Ab value the amount (C) of sulfaquinoxaline present in the sample solution referring to the pattern curve (56.4.5) previously made.

56.4.5 Curva pattern. Enter the 100 ml graduated flasks (56.2.5), 2.0 volumes; 4.0; 6.0; 8.0; and 10.0 ml of the standard solution (56.3.10) corresponding to 50, 100, 150, 200 and 250 micrograms of sulfaquinoxaline. Add 8 ml of hydrochloric acid (56.3.5) in each flask by completing the volume with distilled water and homogenising.

Take 10 ml of each solution, corresponding to 5, 10, 15, 20, and 25 micrograms of sulfaquinoxaline, respectively, and introduce them into test tubes (56.2.6). Develop the color as indicated in 56.4.4, first paragraph. Then measure the absorbance at 545 nm using distilled water as white. Draw the pattern curve by placing the corresponding amount of sulfaquinoxaline expressed in microgram in order of the absorbance value and the abscissa.

56.5 Calculations. The amount in mg of sulfaquinoxaline per kg of sample is given by the following formula:

Sulfaquinoxaline mg/kg = C x 50

Sulfaquinoxaline mg/kg = C x 50

Sulfaquinoxaline mg/kg = P x 50

Being:

C = Amount of sulfaquinoxaline in microgram determined by spectrophotometric measurement.

P = Weight, in grams, of the sample taken for the test.

The difference between the results of two parallel determinations performed on the same sample should not be greater than:

-10 mg/kg in absolute value for sulfaquinoxaline contents between 20 and 100 mg/kg.

-10 per 100 of the highest result for contents between 100 and 5,000 mg/kg.

-500 mg/kg in absolute value for the contents between 5,000 and 10,000 mg/kg.

-5 per 100 of the highest result for contents greater than 10,000 mg/kg.

56.6 References. Sixth Commission Directive of 20 December 1974 (75/84/EEC). 'Official Journal of the European Communities' number L 32/26 of 5 February 1975.

57. Furazolidone

(n-(5-nitro-2-furfurylidene) -3-amino-2-oxazolidone)

57.1 Principle. The method enables the determination of furazolidone in feed, concentrates and premixtures. The lower limit of the determination is 10 mg/kg.

The furazolidone is extracted with acetone after the sample has been previously degreased with petroleum ether. The extract is purified in an aluminium oxide chromatographic column and the furazolidone is eluted with the acetone. The eluate evaporates to dryness, and the residue is collected in amyl alcohol. The furazolidone is extracted with a urea solution and the absorbance of the extract is measured at 375 nm.

57.2 Material and apparatus.

57.2.1 100-and 250-ml topaz-colored glass-filled matrices.

57.2.2 100 Ml Topaz Color Glass Decantation

57.2.3 Soxhlet or Twisselman extraction apparatus.

57.2.4 Extraction Cartridges 25 x 80 or 28 x 100 mm.

57.2.5 Glass column for chromatography, internal diameter 10 mm and length 300 mm.

57.2.6 Bath of thermoregulated water up to 150 ° C.

57.2.7 Spectrophotometer with 10 mm thick buckets.

57.3 Reassets.

57.3.1 Acetona p.a.

57.3.2 Neutral aluminium oxide for chromatography. Granulometry 100 to 240 meshes prepared as follows. Mix 500 g of aluminium oxide with 1000 ml of hot distilled water and then decant the supernatant liquid. Repeat this operation twice and then filter through a Buchner filter. Dry the aluminium oxide at 105 ° C to constant weight.

57.3.3 Amyl Acetate p.a.

57.3.4 Amyl alcohol. Mixture of isomers should be appropriate.

57.3.5 Ether oil, p. b 40-60 ° C.

57.3.6 Urea solution. Mix 90 g urea p.a. with 100 ml of distilled water, heat slightly to facilitate dissolution.

57.3.7 Pattern Substance. Pure furazolidone.

57.3.8 Pattern solutions. Weigh to the nearest 0,1 mg about 25 mg of standard substance (57.3.7). Enter it quantitatively into a 250 ml graduated flask (57.2.1) complete up to the aphorum with acetone (57.3.1) and homogenise (1 ml of this solution contains 100 micrograms of furazolidone).

57.4 Procedure. All manipulations must be done by avoiding direct light:

57.4.1 Sample Preparation. Grind the sample in such a way that it passes through a 1 mm mesh sieve, in accordance with the ISO R 565 recommendation.

57.4.2 Extract. Weigh exactly 1 mg, 5 to 20 g of well-homogenized sample (containing at most 1 mg of furazolidone) in an extraction cartridge (57.2.4). Place it in the extraction apparatus (57.2.3) and extract with petroleum ether (57.3.5). For the Soxhlet apparatus, 13 to 17 cycles of solvent are required; for other devices the duration of the extraction must not be less than thirty minutes. Then remove the cartridge from the apparatus, remove the residual solvent by drying the cartridge and its contents with the aid of a hot air stream.

For the extraction, proper, place the cartridge back into the extraction apparatus and extract with the acetone (57.3.1). For the Soxhlet apparatus, at least 25 solvent cycles are required; for other devices, the conditions necessary for obtaining a complete extraction must be determined in advance. Evaporate the acetonic extract on the water bath (57.2.6) to a volume of 5 to 10 ml and allow to cool down to room temperature.

57.4.3 Cromatography. Insert a small glass wool stopper at the lower end of the chromatographic column (57.2.5) and tighten the stopper with the aid of a rod, to obtain a thickness of 2 to 3 mm. Prepare an aluminium oxide suspension (57.3.2) in acetone (57.3.1). Insert the suspension into the chromatographic column (57.2.5) and leave to rest. The column thus obtained must be approximately 200 mm in height. Leave the acetone (57.3.1) down to the upper surface of the column.

Transfer the extract obtained in (57.4.2) onto the column, wash the flask several times with acetone (57.3.1) and transfer the liquids onto the column. Place an appropriate flask under the column and elude the furazolidone with the acetone (57.3.1).

The total volume of the used acetone, including the used for washing, should not be greater than 150 ml.

57.4.4 Extraction and measurement of absorbance. Evaporate the eluid obtained in 57.4.3 until dry water bath (57.2.6) (see 57.5.1). Dissolve the residue in 10 ml of amyl alcohol (57.3.4) and transfer the solution to a decantation funnel (57.2.2). Wash the flask with successive portions of 10 ml of amyl acetate (57.3.3) and 10 ml of the urea solution (57.3.6). Transfer these solutions to a decantation funnel (57.2.2) and shake vigorously for two minutes. Leave to rest for three to four minutes and collect the aqueous phase in a 100 ml graduated flask (57.2.1). Repeat the washing and extraction with the aid of four portions of 10 ml of urea solution (57.3.6) and collect each time the aqueous phase in the graduated flask. Complete the contents of the flask up to 100 ml with the urea solution (57.3.6) and homogenise. Measure the absorbance of the solution in the spectrophotometer (57.2.7) at 375 nm by using the urea solution (57.3.6). Determine the amount of furazolidone by referring to the pattern curve (57.4.5).

57.4.5 Curva pattern. Prepare four chromatographic columns (57.2.5) according to the indicated operative model (57.4.3 first paragraph). Enter volumes of 2.5, 5.0, 7.5, and 10 ml of standard solution (57.3.8) in the column.

Elude each column with 150 ml of acetone (57.3.1) and continue the operative mode as indicated in 57.4.4. Draw the pattern curve by placing the corresponding amounts of furazolidone in microgram in order of the absorbance and the abscesses.

57.5 Calculations. The value in milligrams of furazolidone per kg of sample is given by the following formula:

C

--

P

Being:

C = Quantity in micrograms of furazolidone determined by the photometric measurement.

P = Weight in grams of the sample taken for analysis.

The difference between the results of two determinations made in parallel and performed on the same sample should not differ from:

-50 per 100 of the highest result for the values of furazolidone between 10 and 20 mg/kg.

-10 mg/kg in absolute value for the values between 20 and 100 mg/kg.

-10 per 100 of the highest result for values between 100 and 5,000 mg/kg.

-500 mg/kg in absolute value for the values between 5,000 and 10,000 mg/kg.

-5 per 100 of the highest result for values greater than 10,000 mg/kg.

57.6 Observations.

57.6.1 Occasionally you get a small amount of diacetone-alcohol, produced by condensation of the acetone on aluminum oxide, that does not hinder subsequent extractions.

57.7 References. Sixth Commission Directive of 20 December 1974 (75/84/EEC). 'Official Journal of the European Communities' number L 32/26 of 5 February 1975.

58. Determination of halofuginone

DL-trans-7-bromo-6-chloro-3 (3-(3-hydroxy-2 -piperidyl) acetonyl)-quinazolin-4-(3H) -one

58.1 Principle. Determination of halofuginone in animal feed. The limit of determination is 1 mg/kg.

After treating it with hot water, halofuginone is extracted as a free base with ethyl acetate, and subsequently passed as hydrochloride to an aqueous acidic solution. The extract is purified by an ion exchange chromatography. The halofuginone content is determined by high reverse phase (CLAR) liquid chromatography using an ultraviolet detector.

58.2 Material and apparatus.

58.2.1 Ultrasonic Bath.

58.2.2 Rotating Evaporator.

58.2.3 Centrileak.

58.2.4 CLAR equipment with variable wavelength ultraviolet detector or photodiode array detector.

58.2.4.1 Liquid chromatography column (300 mm x 4 mm) with C, 10 lm, or equivalent column fill.

58.2.5 Glass column (300 nm x 10 mm) equipped with a porous glass filter and a key.

58.2.6 Glass fiber filters, of a diameter of 150 mm.

58.2.7 Membrane Filters, of 0.45 lm.

58.2.8 Membrane Filters, of 0.22 lm.

58.3 Reassets.

58.3.1 Acetonitrile, CLAR grade.

58.3.2 Resina amberlita XAD-2.

58.3.3 Ammonium acetate.

58.3.4 Ethyl Acetate.

58.3.5 Glacial acetic acid.

58.3.6 Halofuginone normalized pattern, DL-trans-7-bromo-6-chloro-3-(3-(3-hydroxy-2-piperidyl) acetonyl)-quinazolin-4-(3H)-one hydrobromide, E 7.64.

58.3.6.1 Solution standard solution of halofuginone (100 lg/ml). Weigh 50 mg halofuginone (58.3.6) with a precision of 0.1 mg in a 500 ml volumetric flask; dissolve the product in ammonium acetate buffer solution (58.3.18), complete the container to the mark with the buffer solution and mix. This solution, stored in the dark and at 5 ° C, remains stable for three weeks.

58.3.6.2 Calibration solutions. Pour 1,0, 2,0, 3,0, 4,0 and 6,0 ml of the standard reserve solution (58.3.6.1) into a series of 100 ml graduated flasks. Fill up to the mark with mobile phase (58.3.21) and mix the liquids. These solutions have halofuginone concentrations of 1.0, 2.0, 3.0, 4.0 and 6.0 lg/ml respectively. These solutions must be prepared whenever they are to be used.

58.3.7 Hydrochloric acid (density at 20 ° C approx. 1,16 g/ml)

58.3.8 Methanol.

58.3.9 Silver Nitrate.

58.3.10 Sodium Ascorbate.

58.3.11 Sodium carbonate.

58.3.12 Sodium chloride.

58.3.13 EDTA (ethylenediaminotetacetic acid, disodium salt).

58.3.14 Water, CLAR grade.

58.3.15 Sodium carbonate solution c = 10 g/100 ml.

58.3.16 Sodium chloride saturated sodium carbonate solution, c = 5 g/100 ml. Dissolve 50 g of sodium carbonate (58.3.11) in water, dissolve up to one litre and add sodium chloride (58.3.12) until the solution is saturated.

58.3.17 Hydrochloric acid, approx. 0.1 mol/l. Dilute 10 ml of hydrochloric acid (58.3.7) in water, up to one litre.

58.3.18 Ammonium Acetate Buffer Solution, approx. 0.25 mol/l. Dissolve 19,3 g of ammonium acetate (58.3.3) and 30 ml of acetic acid (58.3.5) in water (58.3.14), and dilute up to one litre.

58.3.19 XAD-2 amberlite resin preparation. Wash the appropriate quantity of resin (58.3.2) with water until all chloride ions have been removed, which will be checked by the silver nitrate test (58.3.20) in the discarded aqueous phase. Then wash the resin with 50 ml of methanol (58.3.8), discard the methanol and keep the resin under new methanol.

58.3.20 Silver nitrate solution, approx. 0.1 mol/l. Dissolve 0.17 g of silver nitrate (58.3.9) in 10 ml of water.

58.3.21 CLAR Mobile Phase. Mix 500 ml of acetonitrile (58.3.1) with 300 ml of ammonium acetate buffer (58.3.18) and 1,200 ml of water (58.3.14). Adjust the pH to 4.3 using acetic acid (58.3.5). Filter the mixture by a filter of 0.22 um (58.2.8) and degasify the solution (e.g. by means of ultrasounds for ten minutes). This solution, stored in the dark and in a closed container, is stable for a month.

58.4 Procedure. Observation: halofuginone as a free base is unstable in alkaline and ethyl acetate solutions. It must not remain in ethyl acetate for more than 30 minutes.

58.4.1 General.

58.4.1.1 A blank feed must be analysed to ensure that it does not contain halofuginone or interfering substances.

58.4.1.2 A recovery test should be performed, analyzing the blank feed after enriching it by addition of a quantity of halofuginone, similar to the present in the test sample. To enrich at a level of 3 mg/kg, add 300 ll of the standard reserve solution (58.3.6.1) to 10 g of the blank feed, mix and wait ten minutes before proceeding to the extraction (58.4.2).

Note: For the purposes of this method, the blank feed should be similar to that of the test sample and halofuginone should not be detected in its analysis.

58.4.2 Extract. Weigh 10 g of the prepared sample with a precision of 0.01 g in a 200 ml centrifuge tube, add 0.5 g of sodium ascorbate (58.3.10), 0.5 g of EDTA (58.3.13) and 20 ml of water, and mix everything. Immerse the tube in a water bath at 80 ° C for five minutes. After cooling to room temperature, add 20 ml of sodium carbonate solution (58.3.15) and mix. Immediately add 100 ml of ethyl acetate (58.3.4) and shake vigorously by hand for 15 seconds. Place the tube in the ultrasound bath (58.4.1) for three minutes and loosen the plug. Centrifuge for two minutes and decant the ethyl acetate phase into a 500 ml separator funnel through a glass fibre filter (58.2.6). Repeat the extraction of the sample with a second portion of 100 ml of ethyl acetate. Wash the combined extracts for one minute with 50 ml of sodium chloride saturated sodium carbonate solution (58.3.16) and discard the aqueous layer.

Extract the organic layer for one minute with 50 ml of hydrochloric acid (58.3.17). Pass the lower acid layer to a 250 ml separator funnel. Remove the organic layer again for one and a half minutes with another 50 ml of hydrochloric acid and combine with the first extract. Wash the two acid extracts combined by stirring them for approximately 10 seconds with 10 ml of ethyl acetate (58.3.4).

Quantitatively transfer the aqueous layer to a 250 ml round-bottom flask and discard the organic phase. Evaporate all ethyl acetate remaining in the acidic solution using a rotaavapor (58.4.2). The temperature of the water bath must not exceed 40 ° C. At a vacuum of approximately 25 mbar all residual ethyl acetate is removed in five minutes at 38 ° C.

58.4.3 Cleaning.

58.4.3.1 Preparation of the amberlite resin column.

Prepare an XAD-2 column for each sample extract. Pour 10 g of prepared resin (58.3.19) into a glass column (58.2.5) using methanol (58.3.8). Place a small piece of glass wool on top of the resin bed. Let the methanol in the column elude and wash the resin with 100 ml of water, cutting the flow when the liquid reaches the top of the resin bed. Before you use it, leave the column at rest for ten minutes to balance it. The column should never be allowed to dry.

58.4.3.2 Sample cleanup. Transfer the extract (58.4.2) quantitatively to the top of the prepared resin column (58.4.3.1.) and elude, discarding the eluid. The elution speed shall not exceed 20 ml/minute. Rinse the round-bottom flask with 20 ml of hydrochloric acid (58.3.17) and use this liquid to wash the resin column. Remove all other acidic solution with a jet of air. Discard the washing water. Add 100 ml of methanol (58.3.8) to the column and elude 5-10 ml, collecting the eluid in a 250 ml round-bottom flask. Leave the remaining methanol for ten minutes to be equated with the resin and continue the elution at a rate not exceeding 20 ml/minute, collecting the eluate in the same round-bottom flask. Evaporate the methanol in the rotavaby (58.2.2); the water bath temperature must not exceed 40º C. Transfer quantitatively the residue to a 10 ml graduated flask using the mobile phase (58.3.21). Fill up to the root with mobile phase and mix. Filter an aliquot by a membrane filter (58.2.7). Reserve this solution for determination by CLAR (58.4.4).

58.4.4 Determination by CLAR.

58.4.4.1 Parameters. The following conditions are offered as a guide; other conditions may be used as long as they give the same results.

Liquid chromatography column (58.2.4.1).

CLAR mobile phase (58.3.21).

Flow: 1.5 to 2 ml per minute.

Detection wavelength: 243 nm.

Injection volume: 40 to 100 ll.

Check the stability of the chromatographic system by injecting multiple times the calibration solution (58.3.6.2) with a content of 3.0 lg/ml, until peak heights (areas) and constant retention times have been reached.

58.4.4.2 Calibration curve. Inject each calibration solution several times (58.3.6.2) and measure the peak heights (areas) for each concentration. Draw a calibration curve using the heights or mean areas of the peaks of the calibration solutions as ordered and the corresponding concentrations in lg/ml as abscesses.

58.4.4.3 Sample Solution. Inject the sample extract (58.4.3.2) several times, using the same volume used for the calibration solutions and determine the mean height (area) of the halofuginone peaks.

58.5 Calculations. From the mean height (area) of the halofuginone peaks of the sample solution, determine the concentration of the sample solution in lg/ml by referring to the calibration curve (58.4.4.2)

The content of halofuginone w (in mg/kg) of the sample is obtained by the following formula:

c x 10

w =

m

Being:

c: Concentration of halofuginone of the sample solution in lg/ml.

m: Mass of the sample used for the test in grams.

58.6 Validation of results.

58.6.1 Identity. The identity of the analysed product can be confirmed by a co-chromatography, or by using a diode network detector (diode array), by which the spectra of the sample extract and the calibration solution are compared (58.3.6.2). with a content of 6,0 lg/ml.

58.6.1.1 Co-chromatography. Reinforce a sample extract by adding an appropriate amount of calibration solution (58.3.6.2). The amount of halofuginone added should be similar to the calculated amount of halofuginone found in the sample extract.

Only the height (area) of the halofuginone peak should be increased by the amount added, taking into account the dilution of the extract. The peak width, at half its height, shall be equal to the original width of + 10 per 100.

58.6.1.2 Diode network detection (diode array). The results are evaluated according to the following criteria:

a) The wavelength of the maximum absorption of the spectra of the sample and the pattern, recorded at the apex of the chromatographic peak, must be the same, within a margin determined by the resolution of the detector. In the case of the diodes network detector, the margin is generally located at + 2 nm.

(b) Between 225 and 300 nm, the spectra of the sample and the pattern recorded at the apex of the chromatographic peak shall not be different for those parts of the spectrum between 10 and 100 per 100 relative absorption. This criterion is met when the same maximums are present and at no point the deviation observed between the two spectra exceeds 15 per 100 of the absorption of the product analyzed pattern;

c) Between 225 and 300 nm, the spectra of the climb slope, the apex and the slope of the peak of the chromatogram of the sample extract, must not be different from each other as regards the parts of the spectrum. included in the range of 10-100 per 100 relative absorption. This criterion is met when the same maximums are present and at no point the deviation observed between the spectra exceeds 15 per 100 of the absorption of the spectrum at the apex of the peak.

If you do not meet any of these criteria, the presence of the analyzed product is not confirmed.

58.6.2 Repetibility. The difference between the results of two parallel determinations carried out on the same sample shall not exceed 0,5 mg/kg for a halofuginone content of up to 3 mg/kg.

58.6.3 Recovery. The recovery of the reinforced white sample shall be at least 80 per 100.

58.7 References. Commission Directive 93 /70/EEC. 'Official Journal of the European Communities', L 234 of 28 July 1993.

58.8 Results of a collaborative study. A collaborative study was organized * in which three samples were analyzed in eight laboratories.

* The Analyst 1983, 108: 1252-1256.

Results

Sample A (white) after receipt/Sample B (flour): After receipt/After two months/Sample C (granules): After receipt/After two months

Media (1)/n.d. /2.80/2.42/2.89/2.45

DR/-0.45/0.43/0.40/0.42

CV/-/16/18/14/17

rec//86/74/88/75

(1) Units in mg/kg.

n.d.: was not detected.

DR: standard deviation of reproducibility.

CVR: coefficient of variation (percent).

rec: recovery (percent).

59. Determination of methyl benzoate

7-benzyloxy-6-butyl-3-methoxycarbonyl-4-quinolone

59.1 Principle. The methyl benzoate is extracted from the sample with methanolic methanesulfonic acid solution. The extract is purified by partition with dichloromethane and ion exchange chromatography and then by new extraction with dichloromethane. The content of methyl benzoate is determined by high-resolution liquid chromatography (CLAR) using an ultraviolet detector.

This method is intended for the determination of methyl benzocuate in animal feed. The lower limit of determination is 1 mg/kg.

59.2 Material and appliances.

59.2.1 Lab Agitator.

59.2.2 Rotating Evaporator.

59.2.3 Glass column (250 mm x 15 mm) provided with a key and a reservoir of approximately 200 ml capacity.

59.2.4 CLAR equipment with variable wavelength ultraviolet detector or diode network detector.

59.2.4.1 Column for liquid chromatography of 300 mm x 4 mm, with C fill of 10 lm, or equivalent.

59.2.5 Membrane filters of 0.22 lm.

59.2.6 Membrane Filters of 0.45 lm.

59.3 Reassets.

59.3.1 Dichloromethane.

59.3.2 Methanol, quality for CLAR.

59.3.3 Mobile Phase of CLAR. Mixture of methanol (59.3.2) and water (CLAR quality) 75 + 25 (V + V).

Filter through a filter of 0.22 lm (59.2.5) and degasify the solution, (for example, by means of ultrasound treatment for ten minutes).

59.3.4 Methanesulfonic acid solution, c = 2 per 100. Dilute 20,0 ml of methanesulfonic acid to 1,000 ml with methanol (59.3.2).

59.3.5 Hydrochloric acid solution, c = 10 per 100. Dilute 100 ml of hydrochloric acid (q c.a. 1,18 g/ml) to 1,000 ml with water.

59.3.6 Cationic exchange resin Amberlite CG-120 (Na), 100-200 meshes. The resin must be treated before use: prepare a slurry with 100 g of resin and 500 ml of hydrochloric acid solution (59.3.5) and bring to the boil on a hot plate, stirring continuously. Allow to cool and decant the acid. Filter in vacuum through a filter paper. Rinse the resin twice with 500 ml portions of water and then with 250 ml of methanol (59.3.2). Rinse the resin with another 250 ml portion of methanol and dry by passing air through the filter cake. Save the dried resin in the capped bottle.

59.3.7 Pattern substance: pure methyl benzocuate (7-benzyloxy-6-butyl-3-methoxycarbonyl-4-quinolone).

59.3.7.1 Methyl benzocuate stem pattern solution, 500 lg/ml. Weigh 50 mg of the standard substance (59.3.7) with accuracy of 0.1 mg, dissolve in methanesulfonic acid solution (59.3.4) in a 100 ml volumetric flask, make up and mix.

59.3.7.2 Methyl benzocuate intermediate standard solution, 50 lg/ml. Pass 5.0 ml of the parent solution of methyl benzocuate (59.3.7.1) to a 50 ml graduated flask, make up to the mark with methanol (59.3.2) and mix.

59.3.7.3 Calibration solutions. Transfer 1,0, 2,0, 3,0, 4,0 and 5,0 ml of the intermediate standard solution of methyl benzoate (59.3.7.2) to a series of 25 ml graduated flasks. Make up with mobile phase (59.3.3) and mix. These solutions have methyl benzocuate concentrations of 2.0, 4.0, 6.0, 8.0 and 10.0 lg/ml, respectively. These solutions must be prepared whenever they are to be used.

59.4 Procedure.

59.4.1 General.

59.4.1.1 A blank feed must be analysed to ensure that it does not contain methyl benzocuate or interfering substances.

59.4.1.2 A recovery test should be performed, analyzing the blank feed after enriching it by addition of a quantity of methyl benzocuate similar to the one in the sample. To enrich at a level of 15 mg/kg, add 600 ll of the stock standard solution (59.3.7.1) to 20 g of the blank feed, mix and wait ten minutes before proceeding to extraction 59.4.2.

Note: For the purposes of this method, the blank feed should be of a similar type to the sample and in its analysis methyl benzocate should not be detected.

59.4.2 Extract. Weigh with a precision of 0,01 g approximately 20 g of the prepared sample and transfer to a 250 ml flask erlenmeyer flask. Add 100.0 ml of the methanesulfonic acid solution (59.3.4) and shake mechanically (59.2.1) for thirty minutes. Filter the solution through filter paper and preserve filtering for the liquid-liquid partition phase (59.4.3).

59.4.3 Liquid-liquid partition. Transfer 25.0 ml of the filtrate obtained at point 59.4.2 to a 500 ml decantation ampoule containing 100 ml of hydrochloric acid solution (59.3.5). Add 100 ml of dichloromethane (59.3.1) to the ampoule and shake for one minute. Wait for the separation of the layers to occur and pour the lower layer (dichloromethane) into a 500 ml round flask. Repeat the extraction of the aqueous phase with two other portions of 40 ml of dichloromethane and combine them with the first extract contained in the round flask. Evaporate the dichloromethane extract until dryness in the rotary evaporator (59.2.2) at 40 ° C at reduced pressure. Dissolve the residue in 20-25 ml of methanol (59.3.2), cover the flask and store the whole extract for ion exchange chromatography (59.4.4).

59.4.4 ion exchange chromatography.

59.4.4.1 Preparation of the cationic exchange column. Cover with glass wool the lower end of the glass column (59.2.3). Prepare a slurry with 5.0 g of the treated cationic exchange resin (59.3.6) and 50 ml of hydrochloric acid (59.3.5), pour in the glass column and leave to rest. Remove the excess acid, until its level is just above the resin surface and wash the column with water until the eluid from neutral to the tornasol. Transfer 50 ml of methanol (59.3.2) to the column and allow to elude to the surface of the resin.

59.4.4.2 Column chromatography. By pipette, carefully transfer the extract obtained at point 59.4.3 to the column. Rinse the round flask with two portions of 5 to 10 ml of methanol (59.3.2) and transfer these washing liquids to the column. Let the extract pass to the resin surface and wash the column with 50 ml of methanol, ensuring that the flow does not exceed 5 ml per minute. Discard the eluid. Elude the methyl benzocuate of the column using 150 ml of methanesulfonic acid solution (59.3.4) and collect the eluid from the column in a 250 ml erlenmeyer flask.

59.4.5 Liquid-liquid partition. Transfer the eluid obtained in point 59.4.4.2 to a one-litre decantation ampoule. Rinse the flask erlenmeyer with 5 to 10 ml of methanol (59.3.2) and mix the washing liquids with the contents of the decantation ampoule. Add 300 ml of a hydrochloric acid solution (59.3.5) and 130 ml of dichloromethane (59.3.1). Shake for a minute and let the phases separate. Transfer the lower layer (dichloromethane) to a 500 ml round flask. Repeat the extraction of the aqueous phase with two new portions of 70 ml of dichloromethane and mix in the round flask these two extracts with the first one. Evaporate the dichloromethane extract in the rotary evaporator (59.2.2) at 40 ° C and reduced pressure until dryness. Dissolve the residues in the flask with about 5 ml of methanol (59.3.2) and transfer this solution quantitatively to a 10 ml graduated flask. Rinse the round flask with two new portions of 1 to 2 ml of methanol to the root and mix. Filter an aliquot portion by a membrane filter (59.2.6). Reserve this solution for determination by CLAR (59.4.6).

59.4.6 Determination by CLAR.

59.4.6.1 Parameters. The following conditions are offered as a guide: other conditions may be used whenever the same results are produced.

Liquid chromatography column: (59.2.4.1).

CLAR mobile phase: mixture of methanol and water (59.3.3).

Flow: 1 to 1.5 ml/minute.

Detection wavelength: 265 nm.

Injection volume: 20 to 25 ll.

Check the stability of the chromatographic system by injecting multiple times the calibration solution (59.3.7.3) with a content of 4 lg/ml, until peak heights (or areas) and constant retention times have been reached.

59.4.6.2 Calibration curve. Inject each calibration solution several times (59.3.7.3) and measure the heights of the peaks (areas) for each concentration. Draw a calibration curve using the means of the heights or areas of the peaks of the calibrated solutions as ordered and the corresponding concentrations in lg/ml as abscesses.

59.4.6.3 Sample Solution. Inject the sample extract (59.4.5) several times, using the same volume used for calibration solutions. Determine the mean height (area) of the methyl benzocate peaks.

59.5 Calculations. From the mean height (area) of the methyl benzocuate peaks of the sample solution, determine the concentration of the sample solution in lg/ml by reference to the calibration curve (59.4.6.2)

The content of methyl benzoate w (mg/kg) is obtained by the following formula:

w =/c x 40

m

Being:

c = Methyl benzoquate concentration of the sample solution in lg/ml.

m = Mass of the sample used for the test, in grams.

59.6 Check the results.

59.6.1 Identity. The identity of the analyte can be confirmed by a co-chromatography or by using a diode network detector (diode array), which compares the spectra of the sample extract and the calibration solution (59.3.7.3) with a content 10 lg/ml.

59.6.1.1 Co-chromatography. Reinforce a sample extract by adding an appropriate amount of the intermediate standard solution (59.3.7.2). The amount of methyl benzoate added shall be similar to the calculated amount of methyl benzocuate found in the sample extract. Only the height of the methyl benzoate peak shall be increased by taking into account both the amount added and the dilution of the extract. The width of the peak, half of its maximum height, shall not vary by ± 10 per 100 from the original width.

59.6.1.2 Diode network detection. The results are evaluated according to the following criteria:

a) The wavelength of the maximum absorption of the spectra of the sample and the pattern, recorded at the apex of the chromatographic peak, must be the same, within a margin determined by the resolution of the detector. In the case of the diodes network detector, the margin is generally located within ± 2 nm.

(b) Between 220 and 350 nm, the spectra of the sample and the pattern, recorded at the apex of the chromatographic peak, shall not be different for those parts of the spectrum between 10 and 100 per 100 relative absorption. This criterion is met when the same maximums are present, and at no point observed the deviation between two spectra exceeds 15 per 100 of the absorption of the standard analyte.

(c) Between 220 and 350 nm, the spectra of the climb slope, the apex and the slope of the decrease of the chromatographic peak of the sample extract, must not be different from each other as far as the parts of the spectrum are concerned. included in the range of 10-100 per 100 relative absorption. This criterion is met when the same maximums are present and at no point observed the deviation between the spectra exceeds 15 per 100 of the absorption of the spectrum at the apex of the peak.

If any of these criteria are not met, the presence of the analyte is not confirmed.

59.6.2 Repetibility. The difference between the results of two parallel determinations carried out on the same sample shall not exceed 10 per 100 relative to the highest result for a methyl benzocuate content ranging from 4 to 20 mg/kg.

59.6.3 Recovery. The recovery of the reinforced white sample shall be at least 90 per 100.

59.7 Results of a collaborative trial. Five samples were analyzed in ten laboratories. The analyses were carried out in duplicate for each sample.

Results

Blank Sample/Flour 1/Granules/Flour 2/Granules

(mg/kg)/n.d. /4.50/4.50/8.90/8.70

S mg/kg/-/0.30/0.20/0.60/0.50

CV (percent)/-/6.70/4.40/6.70/5.70

S (mg/kg)/-/0.40/0.50/0.90/1.00

CV (percent)/-/8.90/11.10/10.10/11.50

Recovery (percent)/-/92.00/93.00/92.00/89.00

S = Standard deviation of repeatability.

CV = Repeatability variation coefficient.

S = Standard deviation of reproducibility.

CV = Coefficient of reproducibility variation.

59.8 References. Directive 93 /117/EEC. 'Official Journal of the European Communities' L 329 of 30 December 1993.

60. Determination of robenidine

1,3-bis (4-chlorobenzylidene) amino hydrochloride)

guanidine

60.1 Principle. The sample is extracted with acidified methanol. The extract is dried and an aliquot part is purified in an aluminium oxide column. Robenidine is eluted from the column with methanol, concentrated and completed up to a suitable volume with a mobile phase. The content of robenidine is determined by high-performance, reverse-phase (CLAR) liquid chromatography using an ultraviolet detector.

This method is intended for the determination of robenidine in animal feed. The minimum limit for determination is 5 mg/kg.

60.2 Material and apparatus.

60.2.1 Glass column. Amber glass column provided with a key and a deposit of approximately 150 ml capacity, with an inside diameter of 10,15 mm and a length of 250 mm.

60.2.2 Laboratory oscillator Agitator.

60.2.3 Rotating Evaporator.

60.2.4 CLAR equipment with variable wavelength ultraviolet detector or diode network detector operating in the range 250-400 nm.

60.2.4.1 Column for liquid chromatography: 300 mm x 4 mm with fill of 10 lm of C or equivalent.

60.2.5 Glass fiber filter paper (Whatman GF/A or equivalent).

60.2.6 Membrane Filters of 0.22 lm.

60.2.7 Membrane Filters of 0.45 lm.

60.3 Reassets.

60.3.1 Methanol.

60.3.2 Acidified Methanol. Transfer 4.0 ml of hydrochloric acid (P ca, 1,18 g/ml) to a 500 ml graduated flask, make up to the mark with methanol (58.3.1.) and mix. This solution should be prepared before each use.

60.3.3 Quality Acetonitrile for CLAR.

60.3.4 Molecular Tamiz. Type 3A, pearls of 8-12 meshes (pearls of 1,6-2,5 mm, crystalline aluminosilicate, 0,3 mm of pore diameter).

60.3.5 Aluminium oxide: acid, activity grade I for column chromatography. Transfer 100 g of aluminium oxide to an appropriate container and add 2,0 ml of water. Cover and shake for approximately twenty minutes. Store in a closed container.

60.3.6 Potassium solution dihydrogen phosphate, c = = 0.025 mol/l. In a 1,000 ml graduated flask dissolve 3.40 g of potassium dihydrogen phosphate in water (quality for CLAR), make up and mix.

60.3.7 Phosphate disodium solution, c = = 0.025 mol/l. Dissolve 3.55 g of anhydrous sodium phosphate (or 4.45 g) in a 1,000 ml volumetric flask. dihydrate or 8.95 g dodecahydrate), make up and mix.

60.3.8 Mobile Phase of CLAR. Mix the following reagents:

650 ml of acetonitrile (60.3.3).

250 ml of water (quality for CLAR).

50 ml of potassium dihydrogen phosphate solution (60.3.6).

50 ml phosphate disodium solution (60.3.7).

Filter by a filter of 0.22 lm (60.2.6) and degasify the solution (for example, by applying ultrasounds for ten minutes).

60.3.9 Pattern Substance. Pure Robenidine: 1,3-bis ((4-chlorobenzylidene) amino) guanidine hydrochloride (E 758).

60.3.9.1 Robenidine stem pattern solution: 300 lg/ml. Weigh 30 mg of robenidine standard substance (60.3.9) with accuracy of 0.1 mg. In a 100 ml graduated flask dissolve in acidified methanol (60.3.2), make up to the same solvent and mix. Wrap the flask with foil and keep the light coat.

60.3.9.2 Robenidine standard solution: 12 lg/ml. Transfer 10,0 ml of the stock standard solution (60.3.9.1) to a 250 ml graduated flask, make up to the mobile phase (60.3.8) and mix. Wrap the flask with foil and keep the light coat.

60.3.9.3 Calibration solution. Transfer 5,0, 10,0, 15,0, 20,0 and 25.0 ml of the intermediate standard dissolution (60.3.9.2) to a series of 50 ml graduated flasks. Make up with mobile phase (60.3.8) and mix. These solutions correspond, respectively, to 1,2; 2.4; 3.6; 4.8 and 6.0 lg/ml of robenidine. These solutions should be prepared before each use.

60.4 Procedure.

Note: Robenidine is sensitive to light. Amber glass material shall be used in all operations.

60.4.1 General.

60.4.1.1 A blank feed must be analysed to check that it contains neither robenidine nor interfering substances.

60.4.1.2 A recovery test shall be carried out, which shall consist of the analysis of the white feed enriched with the addition of a quantity of robenidine similar to that contained in the sample. To enrich at a level of 60 mg/kg, transfer 3.0 ml of the stock standard solution (60.3.9.1) to a 250 ml erlenmeyer flask. Evaporate the solution in a nitrogen stream until approximately 0.5 ml is subtracted. Add 15 g of blank feed, mix and wait ten minutes before the extraction (60.4.2).

Note: For the purpose of this method, the blank feed must be of a similar type to that of the sample and robenidine should not be detected in its analysis.

60.4.2 Extract. Weigh, with a precision of 0,01 g, approximately 15 g of the prepared sample. Transfer to a 250 ml erlenmeyer flask and add 100.0 ml of acidified methanol (60.3.2), cover and shake for an hour in the agitator (60.2.2). Filter the solution by a fiberglass filter paper (60.2.5) and collect all the filtrate in a 150 ml erlenmeyer flask. Add 7.5 g of molecular sieve, cover and shake for five minutes. Filter immediately by a fiberglass filter paper. Preserve this solution for the purification phase (60.4.3).

60.4.3 Purification.

60.4.3.1 Preparation of the aluminium oxide column. Insert a small glass fibre cap at the lower end of the glass column. Weigh 11.0 g of prepared aluminium oxide (60.3.5) and transfer to the column. During this phase, efforts should be made to minimise exposure to the atmosphere. Gently tap the lower end of the loaded column so that the aluminum oxide is thirsty.

60.4.3.2 Purification of the sample. Transfer with a pipette to the column 5.0 ml of the sample extract prepared at point 60.4.2. Place the tip of the pipette near the wall of the column and allow the solution to be absorbed into the aluminium oxide. Elude the robenidine of the column with 100 ml of methanol (60.3.1), maintaining a flow of 2-3 ml/minute, and collect the eluid in a 250 ml round flask. Evaporate until dry the solution of methanol at reduced pressure and at a temperature of 40 ° C, using a rotary evaporator (60.2.3). Dissolve the residue again in 3-4 ml of mobile phase (60.3.8) and transfer quantitatively to a 10 ml graduated flask. Wash the flask with several portions of 1-2 ml of mobile phase and transfer the washings to the graduated flask. Make up to the same solvent and mix. Filter an aliquot by a filter of 0.45 lm (60.2.7). Preserve this solution for determination by CLAR (60.4.4).

60.4.4 Determination by CLAR.

60.4.4.1 Parameters. The following conditions are offered as a guide; other conditions may be used as long as they give the same results.

Column for liquid chromatography (60.2.4.1).

CLAR Mobile Phase (60.3.8).

Flow: 1.5 to 2 ml per minute.

Detector wavelength: 317 nm.

Injection volume: 20 to 50 ll.

Check the stability of the chromatographic system by injecting multiple times the calibration solution (60.3.9.3) with a content of 3.6 lg/ml, until heights or peak areas and constant retention times have been reached.

60.4.4.2 Calibration curve. Inject each calibration solution several times (60.3.9.3) and measure the peak heights (areas) for each concentration. Draw a calibration curve using the heights or mean areas of the peaks of the calibration solutions as ordered and the corresponding concentrations in lg/ml as abscesses.

60.4.4.3 Sample Solution. Inject the sample extract (60.4.3.2) several times, using the same volume used for the calibration solutions and determine the mean height (or area) of the robenidine peaks.

60.5 Calculations. From the mean height (area) of the robenidine peaks of the sample solution, determine the concentration of the sample solution in lg/ml by referring to the calibration curve (60.4.4.2).

The content of robenidine w (in mg/kg) of the sample is obtained by the following formula:

w = (c x 200): m Being:

c = Robenidine concentration of the sample solution lg/ml.

m = Mass, in grams, of the sample used for the test.

60.6 Check the results.

60.6.1 Identity. The identity of the analyte can be confirmed by a co-chromatography, or by using a diode network detector, by which the spectra of the sample extract and the calibration solution (60.3.9.3) are compared with a content of 6.0. lg/ml.

60.6.1.1 Co-chromatography. Reinforce a sample extract by adding an appropriate amount of calibration solution (60.3.9.3). The amount of robenidine added must be similar to the calculated amount of robenidine found in the sample extract.

You should only increase the height of the robenidine peak, taking into account both the amount added and the dilution of the extract. The width of the peak, at half its maximum height, shall be within ± 10 per 100 of the original width.

60.6.1.2 Diode network detection. The results are evaluated according to the following criteria:

a) The wavelength at which the maximum absorption of the sample and pattern spectra is given, recorded at the apex of the chromatographic peak, must be the same within a margin determined by the resolution of the detector. In the case of the diodes network detector, the margin is generally located within ± 2 nm.

(b) Between 250 and 400 nm, the spectra of the sample and the pattern recorded at the apex of the chromatographic peak shall not be different for those parts of the spectrum between 10 and 100 per 100 of the absorbance on. This criterion is met when the same maximums are present and at no point observed the deviation between the two spectra exceeds 15 per 100 of the absorbance of the standard analyte.

c) Between 250 and 400 nm the spectra of the climb slope, the apex and the slope of the peak of the chromatogram of the sample extract must not be different from each other as far as the parts of the spectrum are concerned. between 10 and 100 per 100 of relative absorbance. This criterion is met when the same maximums are present and at no point observed the deviation between the spectra exceeds 15 per 100 of the absorbance of the apex apex spectrum.

If any of these criteria are not met, the presence of analyte is not confirmed.

60.6.2 Repetibility. The difference between the results of two parallel terminations carried out with the same sample shall not exceed 10 per 100 relative to the highest result obtained for robenidine content exceeding 15 mg/kg.

60.6.3 Recovery. The recovery of the reinforced white sample shall be at least 85 per 100.

60.7 Results of a collaborative trial. The EEC organised a collaborative trial in which four samples of feed for birds and rabbits were analysed in 12 laboratories, both in the form of flours and in granules. Duplicate analyses were performed for each sample and the following results were obtained:

Birds: Flour/Granule/Rabbits: Flour/Grash

Media (mg/kg)/27.00/27.99/43.6/40.1

S (mg/kg)/1.46/1.26/1.44/1.66

CV (percent)/5.4/4.5/3.3/4.1

S (mg/kg)/4.36/3.36/4.61/3.91

CV (percent)/16.1/12.0/10.6/9.7

Recovery (Percent)/90.0/93.3/87.2/80.2

S = Standard deviation of repeatability.

CV = Repeatability variation coefficient.

S = Standard deviation of reproducibility.

CV = Coefficient of reproducibility variation.

60.8 References. Directive 93 /117/EEC. 'Official Journal of the European Communities' L 329 of 30 December 1993.