Royal Decree 2257 / 1994, Of 25Th November, Which Approves Official Methods Of Analysis Of Feed Or Pet Foods And Their Raw.

Original Language Title: Real Decreto 2257/1994, de 25 de noviembre, por el que se aprueba los métodos oficiales de análisis de piensos o alimentos para animales y sus primeras materias.

Read the untranslated law here: https://www.global-regulation.com/law/spain/1462834/real-decreto-2257-1994%252c-de-25-de-noviembre%252c-por-el-que-se-aprueba-los-mtodos-oficiales-de-anlisis-de-piensos-o-alimentos-para-animales-y-sus-prime.html

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As result of the full integration of Spain into the European Community, and the consequent need to harmonise legislation Spanish with Community rules, the order of 23 May 1989, which approves official methods of analysis of feed or pet foods and their raw was published.


The subsequent enactment of Directive 92/89/EEC, of the Committee, on 3 November, amending Annex I of the Fourth Directive 73/46/EEC, which determines Community methods of analysis for the official control of foodstuffs for animals; Directives of the Commission 92/95/EEC of 9 November, and 94/14/EC, of 29 March, that amending the annex to the seventh Directive 76/372/EEC, laying down Community methods of analysis for the official control of feedingstuffs; and directives 93/70/EEC of the Commission of July 28, 93/117/EEC, the Commission of 17 December, that laying down Community methods of analysis for control official of animal food, and 93/28/EEC, Commission of 4 June, amending Annex I to the third Directive 72/199/EEC by which determines Community methods of analysis for the official control of feedingstuffs for animals, make necessary approval in the internal area of the corresponding rule of transposition in order to incorporate the Community methods of analysis in those envisaged, by modifying what is precise analytical methods initially collected in the order of 23 May 1989 already quoted, or add others that are not referred to therein.


This Royal Decree is issued under cover of the provisions in article 40.2 of the Act 14/1986 of 25 April, General health, and article 149.1.16. ª of the Constitution.


In the processing of this Royal Decree have been consulted entities and organizations in the sector affected by the same.


In his virtue, on the proposal of the Ministers of agriculture, fisheries and food, of health and Consumer Affairs, finance and industry and energy, prior report of the inter-ministerial Commission for food management, according to the Council of State and after deliberation by the Council of Ministers at its meeting of November 25, 1994, I have : Article 1. Object.


Methods of analysis of feedingstuffs or feedingstuffs and their prime materials listed in the annex are approved as official.


Article 2. The so-called official methods.


When there are no official methods for certain analysis of food or feed for animals and their raw, and until they are approved, may be used as laid down in national regulations or those international recognized methods.


Sole additional provision. Basic character.


Provisions of this Royal Decree has the character of basic State legislation in the field of bases and coordination of the general planning of economic activity and health, contained in article 149.1.16. ª of the Constitution.


Sole repeal provision. Repeal legislation.


The provisions of equal or lower rank who are opposed to the present Royal Decree and, in particular, the order of 23 May 1989, which approves official methods of analysis of feed or pet foods and their raw are hereby repealed.


First final provision. Schools of development.


It allows the Ministers of agriculture, fisheries and food, health and Consumer Affairs, finance and economy and industry and energy, to conduct, in the field of their respective competencies, the provisions necessary for the implementation and application of the provisions of this Royal Decree.


Second final provision. Entry into force.


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


Given in Madrid on November 25, 1994.


JUAN CARLOS R.


The Minister of the Presidency, ALFREDO PEREZ RUBALCABA ANEXO official methods of analysis of feedingstuffs and their raw 1. Preparation of the sample for analysis and general provisions 1.1 principle. The preparation of samples for analysis must get that they are as homogeneous and representative possible.


In general, the methods of analysis are applicable to all kinds of feed and its raw materials. Certain feedingstuffs require analytical methods individuals, that are provided for in the description of the corresponding methods.


When two or more methods for the determination of a component of a feedingstuff are quoted, the choice of applicable method, unless otherwise indicated, will correspond to the control laboratory; However, should be given in the analysis report.


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


Moreover, when reference is made to water for the dilutions or washes, means whenever it is distilled water. Similarly, when reference is made to a solution, without further indication, means that you it's a solution in distilled water.


1.3 reagents. All reagents must be of quality compared to the method of analysis (p.a.). For the analysis of trace elements the purity of the reagents must be checked by a blank test. An additional purification may be required depending on the results.


1.4. preparation of the muestra-procedimiento. The chemical analysis must necessarily be on a homogeneous sample. On the contrary, certain macroscopic determinations, as well as the determination of moisture, should be on the sample in the State in which is arriving at the laboratory. To take into account this double requirement, the sample shall be divided into two parts. One of them will be kept separate in the State in which it is located; the other will be prepared for the chemical analysis of mode which is indicated next: divide the sample with the help of a mechanical device or manually, after having carefully mixed the whole in a surface clean and dry. In the latter case, it is convenient to apply the method of the quarters, which consists on taking samples in two opposite sectors. Finally, take for analysis a portion of 100 g around and crush, if need be, so all pass through a 1 mm mesh sieve


Immediately place this sample in a dry container fitted with airtight and cover.


If sample is wet, we will proceed to a predesecacion, so that the degree of humidity set to a value between 8 and 12 per 100. To this end, dry the sample at a temperature right at the right time.


1.5 results. The result that will be referred to in the analysis report shall be the mean value obtained from two determinations at least. Unless otherwise specified, it be expressed as a percentage of the original sample, as you have come to the laboratory. The result should not contain more significant figures than as permitted by the precision of the analytical method.


1.6 references. First Community directive of 15 June 1971. 17/250/EEC. «Official Journal of the European communities» number L 155, 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-tungstico acid, incinerated, and heavy.


2.2 materials and appliances.


2.2.1 mechanical stirrer.


2.2.2 capsule of incineration of Platinum, quartz or porcelain.


2.2.3. electric muffle furnace.


2.3 reagents.


2.3.1. diethyl ether.


2.3.2 chloroform.


2.3.3. solution of sodium hydroxide 4N.


2.3.4 hydrochloric acid 0, 3N.


2.3.5. sodium chloride.


2.3.6 10 per 100 solution (w/v) of silico-tungstico acid (SiO12WO. 26 HO).


2.4 procedure. Weigh, with approximation of 5 mg, 15 g of the sample and place it in a container of some 200 ml stoppered. Add 100 ml of ether diethyl and 50 ml of chloroform exactly 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 lumps, continuing the turmoil at intervals; Let stand until the next day. If the supernatant fluid is not completely clean, add a few drops of water; filter the eter-cloroformo layer. Collect 50 ml of the filtrate into a 50 ml volumetric flask and transfer them quantitatively with the help of 50 ml of diethyl ether to a blister of decanting of 150 ml. remove three successive times with 20 ml of hydrochloric acid, leaving decant and collect the acid extract after each extraction. Meet the acid extracts in a 250 ml flask and remove the last traces of ether and chloroform warmed slightly. Add approximately 1 g of sodium chloride, leave to cool and precipitate the alkaloids by the solution of the silico-tungstico acid (2.6.1). Mechanically stirred for 30 minutes, let stand overnight, filter on known ash filter and wash the precipitate successively twice with 10 ml and twice with 5 ml of hydrochloric acid.


Place the filter containing the precipitate in a capsule of incineration and ash at 900 ° C. Allow to cool and weigh.


2.5 calculations. Percentage of alkaloids = 0, 2 p.


Where: P = weight of the ashes.



Express the results as a percentage of the sample.


2.6 observations. Add solution silico-tungstica until you see that milky white alkaloid more precipitate is formed.


2.7 references. First Community directive of 15 June 1971 (71/250/EEC). Official Journal of the European communities number L 155, 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 alkalizes through a solution of sodium hydroxide. The ammonia is distilled and collected in a measured quantity of sulphuric acid, the excess is entitled by a standard solution of sodium hydroxide.


This method enables you to determine the content of crude protein of animal feed from the content of nitrogen, determined according to the Kjeldahl method.


3.2 materials and appliances. Suitable to mineralize, distilling and holder according to the Kjeldahl procedure.


3.3 reagents.


3.3.1. potassium sulphate.


3.3.2 catalyst:, cupric oxide CuO, or copper sulphate pentahydrate crystallized, CuSO.5HO.


3.3.3. granulated zinc.


3.3.4 sulfuric acid q = 1,84 g/l.


3.3.5 sulfuric acid (HSO ½) C = 0,5 mol/l.


3.3.6 sulfuric acid (HSO ½) C = 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 solution of sodium hydroxide, c = 0.25 mol/l.


3.3.10. sodium hydroxide solution, c = 0,1 mol/l.


3.3.11 pumice granules, washed in hydrochloric acid and calcined.


3.3.12 Acetanilide (melting point = 114 ° C, nitrogen = 10.36 per 100) 3.3.13 sucrose (free of nitrogen compounds).


3.4 procedure.


3.4.1. mineralization. Weigh 1 g of the sample to an accuracy of 0,001 g and transfer such amount to the flask of the apparatus of carbon. Add 15 g of potassium sulphate (3.3.1), an appropriate quantity of catalyst (3.3.2) (0.3 to 0.4 g of cupric oxide) or 0.9 to 1.2 g of cupric sulfate pentahydrate, 25 ml of sulphuric acid (3.3.4) and a few granules of pumice (3.3.1.1). Homogenize. Heat the flask first with moderation and shaking from time to time, if necessary, until the carbonization of the mass and the disappearance of the foam. later, more intensely to the boil adjust fluid. Warming is suitable when the boiling acid condenses on the walls of the flask. Avoid overheating of the walls and the adhesion on them from organic particles. When the solution becomes clear, pale green, keep boiling for another two hours. Then leave to cool.


3.4.2 distillation. Add carefully enough water to dissolve the sulphates completely. Leave to cool and then add a few granules of zinc (3.3.3).


Place in the flask of the appliance manifold Distiller 25 ml, measured with accuracy, sulphuric acid (3.3.5 or 3.3.6), depending on the presumed nitrogen content and a few drops of the methyl (3.3.7) red.


Connect the mineralization flask to the condenser of the distillation apparatus and immerse the end of refrigerant in the liquid in the flask collector to a minimum depth of 1 cm (see observation 3.7.3). Enter slowly into the flask of the digestion 100 ml of solution of sodium hydroxide (3.3.8) without loss of ammonia (3.7.1).


Heat the flask up to complete distillation of the ammonia.


3.4.3. degree. Holder excess sulfuric acid of the collecting flask with the solution of sodium hydroxide (3.3.9 or 3.3.10), depending on the concentration of the sulphuric acid used, until the end point.


3.4.4. blank test. To confirm that the reagents are free from nitrogen, carry out a test blank (mineralization, distillation and titration) using 1 g of sucrose (3.3.13) instead of the sample.


3.5 calculation of results. The content of crude protein 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 titration of the sample.


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


m = mass (g) of the sample.


3.6 checking the method.


3.6.1. repeatability. The difference between the results of two parallel determinations carried out with the same sample must not exceed:-0.2 per 100 in absolute value, for contents of less than 20 per 100 crude protein.


-1.0 per 100 in relative value on the highest value for contents in protein between 20-40 per 100.


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


3.6.2 accuracy. Carry out the analysis (mineralization, distillation and titration) on 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, minimum.


3.7 observations.


3.7.1. the device can be manual, semi-automatic or automatic. If the device requires a transfer between mineralization and distillation, this transfer must be lossless. If the flask of the distillation apparatus is not fitted with a dropping funnel, add the solution of hydroxide immediately before connecting the flask to the condenser, leaving you to slide the liquid slowly down the walls.


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


3.7.3. for products with low content in nitrogen, the volume of sulfuric acid (3.3.6) that has to be introduced in the collecting flask may be reduced, if necessary, to 10 or 15 ml and make up to 25 ml with water.


3.8 references. Directive 93/28/EEC. «Official Journal of the European Communities» L 179, on June 4, 1993.


4 (a) crude fat (without prior hydrolysis) 4 (a).1 principle. The sample is extracted with petroleum ether. The solvent is distilled and the residue dried and weighed.


The method enables you to determine the crude fat content in feed. Does not apply to the analysis of seeds and oleaginous fruit defined in Regulation number 136/66/EEC of the Council of 22 September 1966.


Applicable to the raw materials of vegetable origin, with the exception of those in which its fats are not completely removable with light petroleum without prior hydrolysis. Such exceptions include Glutens, yeasts, soya protein and potatoes among others. This procedure also applies to compound, except feed those containing milk in protective or whose fat are not fully extractable with light petroleum without prior hydrolysis.


4 (a).2 Material and devices.


4 (a). 2.1 Extractor. If the appliance has a siphon (Soxhlet type) you can adjust the volume of reflux so obtained at least 10 cycles per hour. If the device had no siphon, the volume of liquid having reflux must be about 10 ml per minute.


4 (a). 2.2 extraction thimbles, free of matter soluble in petroleum ether, and whose porosity consistent with the requirements of paragraph 4 (a). 2.1.


4 (a). 2.3 range of dewatering well with vacuum at 75 ° C ± 3 ° C or at atmospheric pressure at 100 ° C ± 3 ° C.


4 (a).3 reagents.


4 (a). 3.1 ether petroleum, boiling range: 40 to 60 ° C. Bromine index must be less than 1 and the residue of evaporation less than 2 mg / 100 ml.


4 (a). 3.2 sodium sulfate, anhydrous.


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


4 (a).4 procedure.


4 (a). 4.1 weigh to the nearest 1 mg, 5 g of the sample and insert plug of cotton WAD.


Put the cartridge in the exhaust (4 (a). 2.1) extracting for six hours with light petroleum (4 (a). 3.1). Collect the extract in a dry flask previously weighed, where there are some fragments of pumice stone.


Evaporate the solvent by distillation. Dry residue, by inserting the flask for an hour and a half in drying stove (4 (a). 2.3). Leave to cool in a desiccator and weigh. Dry again for 30 minutes to ensure that the fat content remains constant (the difference in weight between two successive weighings is less than 1 mg).


4 (a). 4.2 for products high in fats, difficult to crush or unsuitable for the taking of a homogeneous test sample, proceed as follows: weigh to the nearest 1 mg, 20 g of the sample and mix with 10 g or more of anhydrous sodium sulfate.


Extract with light petroleum (4 (a). 3.1) as shown in 4 (a). 4.1.


Complete the extract obtained to 500 ml with light petroleum (4 (a). 3.1) and mix thoroughly. Place 50 ml of the solution in a small dry and set, flask containing fragments of pumice stone (4 (a). 3.3). Remove solvent by distillation, drying, and continue with the operative method indicated in the last paragraph of point 4 (a). 4.1. Value (a).


Eliminate the solvent from the residue of extraction that is found in the cartridge. Crush the residue to a particle size of 1 mm. Put it back in to the extraction thimble (do not add sodium sulphate) and continue with the operating method, as shown in the second and third subparagraphs of point (4 (a). 4.1.). (B) value.


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 crude 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 = the mass, in grams, of the residue of the second extraction.


The difference between the results of two parallel determinations carried out on the same sample by the same analyst should not exceed:-0.2 per 100 in absolute value, for contents in crude fats lower than 5 per 100.


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


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


4 (a).6 references. Amendment to Directive 71/393/EEC, directive of the Commission of 20 December 1983. «Official Journal of the European communities» number L 15/28 of 18 January 1984. Method number 4.


4 (b) crude fat (with prior hydrolysis) 4 (b).1 principle. The sample is treated in hot with hydrochloric acid. Cool the mixture and filter. After having it washed and dried, extracted with petroleum ether. The solvent is distilled and the residue dried and weighed.


The method enables you to determine the crude fat content of feedingstuffs. Does not apply to the analysis of seeds and oleaginous fruit defined in Regulation number 136/66/EEC of the Council of 22 September 1966.


Applicable to feedingstuffs of animal origin, as well as those mentioned in method 4 (a).1, as exceptions to this procedure.


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


4 (b).3 reagents.


4 (b). 3.1 ether petroleum, boiling range: 40 to 60 ° C. Bromine index must be less than 1 and the residue of evaporation less than 2 mg / 100 ml.


4 (b). 3.2 3N hydrochloric acid.


4 (b). 3.3 adjuvant of filtration, diatomaceous earth for example.


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


4 (b).4 procedure. Although, with approximation of 1 mg, 2.5 g of sample (to poor fat samples can be increased to 5 g), insert it into a beaker 400 ml, or in a 300 ml conical flask, add 100 ml of hydrochloric acid 3N (4 (b). 3.2) and a few fragments of pumice stone. Cover the beaker with a watch glass or connect a reflux condenser to the Erlenmeyer flask. Bring the mixture to a gentle boil, using a small flame or a hot plate; keep boiling for one hour. Avoid the product from adhering to the walls of the container.


Cool and add sufficient filtration adjuvant (4 (b). 3.3), to prevent the loss of fat during filtration. Filter with double damp, fat-free filter paper. Wash the residue with cold water up to the neutrality of the filtrate. Check that it does not contain fats. Its presence would indicate the need for a sample extraction with petroleum ether, prior to hydrolysis, according to method 4 (a).


Place the double filter paper containing the residue on a watch glass and dry for one and a half hours in the oven at 100 ° C ± 3 ° C.


Insert the double filter containing the dry residue extraction cartridge (4 (a). 2.2) and cover with a cotton WAD plug. Put the cartridge in the exhaust (4 (a). 2.1) and continue operating as indicated in the second and third subparagraphs of paragraph 4 (a). 4.1.


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


4 (b).6 references. The same as in method 4 (a).


5 5.1 chlorides principle. Chlorides solubilize in water, defecating is the solution if it contains organic materials, subsequent acidification with nitric acid and precipitation of chlorides with silver nitrate. The excess nitrate is titrated with a solution of ammonium thiocyanate. Applicable to all feedingstuffs.


5.2 materials and appliances.


5.2.1 agitator of 35 to 40 r.p.m.


5.3 reagents.


5.3.1 0 ammonium thiocyanate solution, 1N.


5.3.2. solution of nitrate of silver 0, 1N.


5.3.3. amonico-ferrico sulphate saturated solution.


5.3.4 nitric acid, d = 1.38.


5.3.5. diethyl ether.


5.3.6. acetone.


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


5.3.9 carbon active, free from Chlorides.


5.4. procedure.


5.4.1. preparation of the solution.


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


5.4.1.2. samples with organic matter (less those cited in 5.4.1.3). Weigh with 1 mg, about 5 g of the sample and place it with 1 g of active carbon in a volumetric flask of 500 ml. add 400 ml of water at 20 ° C approximately and 5 ml of Carrez I solution, shake, and then add 5 ml of the Carrez II. Shake for thirty minutes, make up to volume, mix and filter.


5.4.1.3 cake and flour of linen, products rich in flax flour and other products rich in mucilage or in colloidal substances (for example, hydrolyzed starch).


Prepare the solution as indicated in 5.4.1.2, but without filtering. Decant (if necessary centrifuge), separate 100 ml of the supernatant fluid and place them in a flask of 200 ml. mix with acetone and make up the volume with this solvent, homogenize and filter.


5.4.2. evaluation. Take from 25 to 100 ml of filtrate (with less than 150 mg chlorine content) obtained in 5.4.1.1, 5.4.1.2 or 5.4.1.3, and insert it into a conical flask, dilute if necessary, up to 50 ml with water. Add 5 ml of nitric acid, 20 ml of saturated solution of ferric ammonium sulphate and two drops of ammonium thiocyanate solution, added by means of a burette filled up to the stroke of zero. Then add by means of a burette solution of silver nitrate to an excess of 5 ml. Add 5 ml of diethyl ether and shake strongly to collect the precipitate. Titrate the excess of nitrate of silver by ammonium thiocyanate solution until the shift to dark red persists for a minute.


5.5 calculations. The amount of chlorine (p) expressed in sodium chloride present in the volume of separate filtering for valuation is given by the formula: P = 5,845 (V1 - V) mg is: V = the volume, in ml, of added silver nitrate solution.


V = the volume, in ml, solution of ammonium thiocyanate 0, 1N used in the assessment.


Carry out a blank no sign to analyze and if you use a solution of nitrate of silver 0, 1N this detract from the volume (V - V).


Express the result as a percentage of the sample.


5.6 observations. For fat products, previously degreased by diethyl ether according to 4 (a).


5.7 references. First the Commission directive of 15 June 1971. (71/250/EEC). «Official Journal of the European communities» number L 155, 12 July 1971.


6 humidity 6.1 principle. The sample is dried out under defined conditions, varying according to the nature of the product. The loss of mass is determined by weighing. It is necessary to proceed to a predesecacion when it comes to solid substances, with a high moisture content. It does not apply to products derived from milk considered as feedingstuffs, compound feed consisting essentially of minerals and seeds and oleaginous fruits, mineral substances. For cereals and their products, except products of hydrolyzed cereals and rootlet of barley (6.3.2.2), applies the official method for cereals and derivatives.


6.2 material and equipment.


6.2.1 mill crusher, easy to clean, allowing a rapid crushing and uniform, without causing sensitive overheating or condensation, avoiding the most contact with the air.


6.2.2. analytical balance accuracy 0.5 mg.


6.2.3 containers dry stainless metal or glass, fitted with a lid that ensures a watertight closing; useful surface that allows to obtain a breakdown of the sample of the order of 0.3 g per cm.


6.2.4 isothermal oven (± 1 ° C) electrical heating, which ensure a suitably ventilated and rapid temperature regulation.


6.2.5 stove vacuum, electric heating, equipped with a pump oil or air introduction device hot dried or a drying agent.


6.2.6. desiccator with plate of metal or porcelain, containing an efficient drying agent.


6.3 procedure.


6.3.1. preparation of the sample.


6.3.1.1. all samples, with the exception of those referred to in 6.3.1.2. Separate previously at least 50 g of the sample, crushing it or treating it previously properly, if necessary, to avoid any variation in moisture content.


6.3.1.2 food liquid or Pasty, consisting essentially of fats. Previously separate and weigh 25 g of sample to 10 mg around approach. Add an appropriate quantity of anhydrous, heavy sand with approximation of 10 mg and mix until you obtain a homogeneous product.


6.3.2 desiccation.



6.3.2.1. all foods, with the exception of those mentioned in 6.3.2.2. Tare, approximation of 0.5 mg, with a provided container with lid. Although, with 1 mg, in the container weighed about 5 g of the sample and spread evenly. Place the container, without cover, in a previously heated oven at 103 ° C. To prevent the stove temperature does not drop too, place the container quickly. Leave to dry for four hours from the time that the stove again reaches the temperature of 103 ° C. Place the lid on the bowl, remove it from the oven, let cool for thirty to forty-five minutes in a desiccator and weigh to approximation of 1 mg.


Specimens consisting essentially of fats, make an additional drying of thirty minutes in the oven at 103 ° C. The difference between the two weighings must not exceed 0.1 per 100 of moisture.


6.3.2.2 feed compounds containing more than the 4 by 100 of sucrose or lactose, hydrolyzed cereal products, rootlet of barley, carob, head of beet, sugar and soluble fish and compound feed containing more than 25 per 100 of mineral salts with water of crystallization. Tare with an approximation of 0.5 mg, a container with a lid. Although, with an approximation of 1 mg in the container weighed approximately 5 g of the sample and spread evenly. Place the container in the vacuum oven previously heated at a temperature of 80 ° c to 85 ° C, without lid. To prevent that the stove temperature drops too, place the container quickly. Bring the pressure to 100 torrs, and let dry to this pressure for four hours, under a stream of hot, dry air or with the help of a drying agent (300 g approximately for 20 samples). In the latter case, cut the connection with the vacuum pump when the prescribed pressure is reached. Counting drying time from the moment that the stove again reaches the temperature of 80 to 85 ° C. Take with caution the stove at atmospheric pressure.


Open range, immediately cover the Pan and remove it. Let cool for thirty to forty-five minutes in the desiccator and weigh to within 1 mg. proceed to an additional dewatering of 30 minutes in the vacuum oven at 80 ° c to 85 ° C temperature and weigh again. The difference between the two weighings must not exceed 0.1 per 100 of moisture.


6.3.3. predesecacion. Solid foods, whose moisture content is high and make the difficult grinding must be predesecados as shown below: weigh approximately 50 g of sample not ground to an approximation of 10 mg (if necessary can become a previous division in the case of pellets or agglomerates) in a suitable container.


Allow to dry in an oven at the temperature of 60 ° c to 70 ° C, until the moisture content is reduced to a value between 8 and 12 per 100.


Remove from the stove and let cool air in the laboratory for one hour and weigh to within 10 mg. then crush immediately as indicated in 6.3.1 make dewatering as in 6.3.2.1.


6.4 calculations. The moisture content, as per cent of sample, is obtained by the following formulas: 6.4.1 desiccation without predesecacion: 100 / M (M - m) is: M = the initial mass, in g of the sample.


m = mass, in g of the dry sample.


(6.4.2 desiccation with predesecacion: (M'-m) M / M' + and - M) 100 / E = 100 (1 - mm / E M') being: E = initial mass, in g of the sample.


M = mass, in g, of the predesecada 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 the 0.2 per 100 of moisture.


6.5 references. Second Commission directive of 18 of November 1971. (71/393/EEC. «Official Journal of the European communities», number L 279, 20 December 1971.


7. determination of crude fiber (cellulose gross) 7.1 principle. Determination in feed, organic substances free from fat and insoluble in acid and alkaline medium, conventionally called crude fiber.


Sample, defatted where, is successively treated with solutions in boiling sulfuric acid and potassium hydroxide, of certain concentrations. Separates the residue by filtration using porous glass filter, washed, dried, weighed and is calcined at a temperature of between 475 and 500 ° C. Weight loss due to the calcination is crude fiber of the test sample.


7.2 material and devices.


7.2.1. apparatus for warming to digestion with sulfuric acid or solution of potassium hydroxide, equipped with a holder for the filter Crucible (7.2.2) and an outlet tube fitted with a tap to make vacuum and evacuate the liquid and, where appropriate, with compressed air to apply back pressure. Every day, before use, Preheat it with water boiling for five minutes.


7.2.2 Crucible, 50 ml glass, with glass, including porosity filter plate filter between 40 and 90 xm. Before using it for the first time, heated to 500 ° C for a few minutes and cool (7.7.6).


7.2.3 column resistant to boiling, 270 least with reflux condenser.


7.2.4 stove drying with thermostat.


7.2.5 oven muffle, with thermostat.


7.2.6. appliance of cold extraction, consisting of a support for the filter Crucible (7.2.2) and a discharge tube with a tap to make vacuum and evacuate the liquid.


7.2.7 connection joints to join the heating (7.2.1), the Crucible (7.2.2), & (7.2.3) column and connect the extractor in the Crucible and cold (7.2.6).


7.3 reagents.


7.3.1 sulfuric acid, c = 0.13 mol/l.


7.3.2. defoaming agent (e.g., n-octanol).


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


7.3.4. acetone.


7.3.5 ether oil ligero-intervalo of boiling 40-60 ° C.


7.3.6 hydrochloric acid, c = 0,5 mol/l.


7.3.7 solution of potassium hydroxide, c = 0.23 mol/l.


7.4 procedure. Weigh 1 g of sample with an approximation of 1 mg, if necessary, previous preparation (see 7.7.1 and 7.7.2, 7.7.3), and put it in a crucible (7.2.2) and add one gram of adjuvant filtration (7.3.3).


Connecting the device of heating (7.2.1) and filter Crucible (7.2.2) and then join the spine (7.2.3) and the crucible. Put in jar 150 ml of sulphuric acid (7.3.1) heated previously until the boiling point and add a few drops of anti-foaming agent (7.3.2) as necessary. Bring liquid to a boil in 5 ± 2 minutes and let boil briskly for 30 minutes flat.


Open the tap of the spout (7.2.1) and filter vacuum sulfuric acid through the filter crucible. Wash three times the residue to the filter Crucible using 30 ml of boiling water each time. After every wash, dry residue of the suction filter.


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


After the last wash, dry residue by extraction, disconnect the Crucible and its content and connect it to the pump cold (7.2.6). Apply vacuum and wash the residue in the Crucible, three times using 25 ml of acetone each time, drying it by aspiration after every wash.


Dry the filter Crucible at 130 ° C in an oven until constant weight. After each drying, cool in the desiccator and weigh rapidly. Place the crucible in the muffle furnace and burnt the contents at a temperature of between 475 and 500 ° C during 30 minutes as minimum.


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


Test blank no sign. The loss of weight due to the ignition should not exceed 4 mg.


7.5 calculations.


The content of crude fibre as a percentage of the sample is expressed by the formula: (b - c) x 100 / to being: a = mass of the sample in g.


b = mass loss 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. repeatability. The difference between two parallel determinations carried out within the same sample must not exceed: - 0.3 in absolute value, for crude fibre contents lower than 10 per 100.


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


7.7 observations.


7.7.1. feedingstuffs containing fat exceeding 10 per 100 gross should be degreased with light petroleum (7.3.5) before carrying out the analysis. Connect the Crucible filter (7.2.2), with the pre-weighed sample, to exhaust wash three times the vacuum and cold (7.2.6) 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 pursuant to section 7.4.


7.7.2. feedingstuffs containing fats that may not be directly extracted with petroleum ether (7.3.5) should be degreased pursuant to point 7.7.1 and undergo a new degreasing after having been treated with acid at boiling point.



After treatment with acid at boiling and washing, join the Crucible and its contents to cold (7.2.6) exhaust, wash three times using 30 ml of acetone each time and then three times using 30 ml of petroleum ether every time. Dry the filter by suction and continue the analysis pursuant to section 7.4, beginning with the treatment with potassium hydroxide.


7.7.3 If the feedingstuffs contain more than 5 per 100 of carbonates, expressed as calcium carbonate, the Crucible (7.2.2), with heavy sample, connect to the appliance of warming (7.2.1). Wash the sample three times with 30 ml of hydrochloric acid (7.3.6). After each addition, let stand the sample for one minute approximately before filtering. Wash once with 30 ml of water and then follow in accordance with point 7.4.


7.7.4 If using a battery of equipment (various crucibles attached to the heating appliance itself), two determinations from the same sample must not be made in the same series.


7.7.5 If, after boiling, it is difficult to filter out the acid and alkaline solutions, introduce compressed air through heating and follow then filtering apparatus discharge tube.


7.7.6 in order to lengthen the duration of glass filter crucibles, agrees that the calcination temperature does not exceed 500 ° C. Also, avoid sudden thermal changes in the cycles of warming and cooling.


7.8 references. Directive 92/89/EEC. «Official Journal of the European communities», L 344, 26 November 1992.


8 8.1 sugars principle. The method enables you to determine the reducing sugars and total sugars prior investment, expressed in glucose or, where appropriate, sucrose, by conversion with the help of the factor 0.95. This method is applicable to compound feedingstuffs. Is foreseen for other food types. In your case, proceeds to determine separately the lactose and take it into account when calculating the results.


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


8.2 additional materials and appliances. Mechanical stirrer.


8.3. reagents: 8.3.1 40 per 100 ethanol (v/v) (d = 0,948 at 20 ºc).


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


8.3.3. Carrez II solution. Dissolve in water 10.6 g hexaciano ferrato (II) potassium (K (FeCN) 3HO ·) and add distilled water to 100 ml.


8.3.4 the 0.1 per 100 methyl Orange solution (w/v).


8.3.5 4N hydrochloric acid.


8.3.6 acid corhidrico 0, 1N.


8.3.7. 0 sodium hydroxide solution, 1N.


8.3.8. copper sulphate solution II. Dissolve 25 g of copper sulphate (CuSO · 5HO) p.a., free from 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. solution of sodium carbonate. Dissolve 143.8 g of anhydrous soda ash (NaCO) p.a. in 300 ml of hot water, allow to cool.


8.3.11. 0 Sodium Thiosulphate solution, 1N.


8.3.12. starch solution. Add a mixture of 5 g of starch, soluble in 30 ml of water, a liter of boiling water. Boil for three minutes. Leave to cool. Add 10 mg of iodide of mercury (II) as a preservative.


8.3.13 acid sulfuric 6N.


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


8.3.15 pumice boiled with hydrochloric acid and rinsed with water.


8.3.16 Isopentanol.


8.3.17. Luff - Schoorl reagent. Pour carefully stir the citric acid solution (8.3.9) on the solution of soda ash (8.3.10). Immediately add the copper sulphate solution (8.3.8) and make up to 1 litre with water. Let stand overnight and filter. Check the normality of the reagent retrieved (Cu 0, 1N; NaCO2N). The solution's pH should be approximately 9.4.


8.4 procedure.


8.4.1. preparation of the sample. Weigh to approximation of 1 mg to 2.5 g of the sample, and insert it into a volumetric flask of 250 ml. add 200 ml of ethanol to 40 by 100 (v/v) and mix for one hour in the mixer. Add 5 ml of the Carrez I solution and shake for one minute, add and stir during the same time with 5 ml of the Carrez II solution.


Make up to 250 ml with ethanol 8.3.1 solution, mix and filter. Take 200 ml of the filtrate and evaporate approximately half of the volume, in order to eliminate most of the ethanol. Transfer all the residue of evaporation with help of hot water to a 200 ml graduated flask and cool, then make up the volume with water and filter if necessary. This solution will be used for the determination of reducing sugars and after the investment for the determination of total sugars.


8.4.2. determination of reducing sugars. As Max 25 ml of the solution prepared according to 8.4.1 and containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and determine the amount of reducing sugars according to Luff-Schoorl. The result will be expressed as per cent of glucose.


8.4.3. determination of total sugars prior investment. Take 50 ml of the solution 8.4.1 and bring a flask of 100 ml. Add a few drops of methyl Orange solution and add slowly waving 4N hydrochloric acid solution to turn to red. Add 15 ml of hydrochloric acid 0, 1N and immerse it in boiling hot water bath for 30 minutes. Cooling up to 20 ° C and then add 15 ml of 0, 1N (8.3.7) sodium hydroxide solution. Make up to 100 ml with water and mix thoroughly.


Take an amount that does not exceed 25 ml and contain less than 60 mg of reducing sugars expressed as glucose. If it is necessary to make 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 per cent of glucose. Express it in sucrose it should 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 lead to a 300 ml Erlenmeyer flask add 25 ml accurately measured of the defecada solution of sugars, add a bit of pumice and heat waving over the flame of the burner. Immediately place the Erlenmeyer flask on a wire mesh, perforated by a 6 cm diameter opening and regulating the flame in a way that only the bottom of the erlenmeyer is heated. Adapt immediately a refrigerant of reflux on the Erlenmeyer flask, from this moment do boil the solution and keep at boiling point for 10 minutes exactly. Immediately cool to cold water for five minutes and proceed to their assessment as follows: add 10 ml of potassium iodide solution (8.3.14) immediately after and carefully 25 ml of sulphuric acid 6N (8.3.13). Rating below with the solution of Sodium Thiosulphate 0, 1N (8.3.9) until the appearance of yellow color, add the starch solution at this time and finish rating.


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


8.5 calculations. By means of table I establish the amount of glucose in mg corresponding to the difference between the two valuations, according to the ml of Sodium Thiosulphate 0, 1N spent on each of the evaluations.


Express the results as per cent of the sample.


8.6. observations.


8.6.1. in the case of feed very rich, molasses or little homogeneous feed weigh 20 g and place in a volumetric flask of 1 litre with 500 ml of water. Mix for one hour in the mixer. Defecate through reagents Carrez I and II (8.3.2 and 8.3.3) as described in 8.4.1, using all the reagents doses four times over. Lead to 1000 ml with ethanol 80 per 100 (v/v) (8.3.1). Homogenize and filter, then remove the ethanol according to 8.4.1.


In the absence of starch products free of hydrolyzed, made up to 100 ml with distilled water.


8.6.2. in the case of molasses and feed simple, rich in sugars and practically free starch, weigh 5 g and place in a 250 ml volumetric flask, add 200 ml of distilled water and mix for one hour or more in the agitator. Bowel movement immediately by means of the Carrez I and II (8.3.2 and 8.3.3) reagents according to 8.4.1.


Make up to 250 ml with water, mix and filter, to determine the 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 avoid the formation of foam.


8.6.4. the difference between the amount of total sugars after inversion, expressed as glucose, and the amount of reducing sugars also expressed as glucose, multiplied by 0,95 gives the amount as per cent of sucrose.


8.6.5. in order to calculate the amount of reducing sugars, excluding lactose, can be determined in the following ways: 8.6.5.1 for a rough estimate, multiply the amount of lactose obtained, by separate determination by 0,675, and subtract the result of quantity in sugars.



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


8.6.5.1 and 8.6.5.2 cases determines the amount of sugars present according to Luff-Schoorl, expressed in mg of glucose.


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


8.7 references. First the 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 of Luff-Schoorl reagent, Na2S2O30, 1N: ml / glucose fructose C6H12O6 inverted sugars: 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 fat this determination shall apply to those fats which are used as raw material.


The procedure shall be as set out in the method number 10 of the official methods of analysis of oils and greases approved by order of 31 January 1977 («Official Gazette» of 14 July 1977).


In the event that it is necessary to prepare the sample will follow procedures collected in the method number 1 of the aforementioned order.


10 calcium 10.1 principle. The method enables you to determine the total feed calcium content.


Sample ash, ashes are treated with hydrochloric acid and the calcium precipitates in the form of calcium oxalate. After dissolving the precipitate in sulphuric acid, oxalic acid formed is valued through a solution of potassium permanganate.


10.2 material and devices.


10.2.1. volumetric flask of 250 ml capacity.


10.2.2 Erlenmeyer of 250 ml capacity.


10.2.3. Platinum, quartz or porcelain crucible.


10.2.4. filter crucibles of G4 porosity, glass.


10.2.5 electric oven with air circulation and automatic controller, to regulate at 550 ° C.


10.2.6. water bath.


10.3 reagents.


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 ammonium oxalate in cold, p.a.


10.3.6 30 per 100 solution (w/v) of citric acid, p.a.


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


10.3.8 the 0.04 per 100 solution (w/v) of bromocresol green.


10.3.9. potassium permanganate solution; 0, 1N, p.a.


10.4 procedure. Weigh to approximation of 1 mg, 5 g of the sample to be analyzed (or more) if necessary, burnt it at 550 ºc and transfer the ash to a conical flask of 250 ml. Add 40 ml of hydrochloric acid (10.3.1), 60 ml of water and a few drops of nitric acid (10.3.2). Bring to the boil and keep it that way for 30 minutes. Cool, quantitatively transfer the solution into a 250 ml volumetric flask, rinse the Erlenmeyer flask and make up to volume with water, homogenize and filter.


A pipette according to presumed quantity of calcium, an aliquot containing 10 to 40 mg of calcium, and insert it into an Erlenmeyer flask of 250 ml. Add 1 ml of the citric acid solution (10.3.6) and 5 ml of ammonium (10.3.7) chloride solution. Approximately complete the volume to 100 ml with water. Bring to a boil, adding 8 to 10 drops of solution of bromocresol green (10.3.8) and 30 ml of hot ammonium (10.3.5) oxalate solution. If you see a precipitate, dissolve it by adding some drops of hydrochloric acid (10.3.1).


Then neutralize very slowly with ammonia (10.3.4), stirring constantly, until a pH 4, 4-4, 6 (turning of indicator). Place the Erlenmeyer flask in a boiling water bath for thirty minutes, steeping the precipitate formed. Remove from the bath, leave it to stand for one hour and filter in a G4 filter crucible. Rinse the Erlenmeyer flask the crucible with water until the total elimination of excess ammonium oxalate (the absence of chloride in the washing water indicates that the wash is sufficient).


Dissolve the precipitate on the filter with 50 ml of sulfuric acid hot (10.3.3), rinse the crucible with water hot until the filtrate to 100 ml approximately. Heated to 70-80 ° C and rating by potassium permanganate solution (10.3.9) until a persistent pink colour is obtained for a minute.


10.5 calculations. 1 ml of 0 potassium permanganate, 1N corresponds to 2,004 mg of calcium.


Express the result as per cent of the sample.


10.6 observations.


10.6.1. for small amounts of calcium, proceed as in 10.5. Filtration the precipitate calcium oxalate on an ashless filter paper and calcined in a crucible at 550 ° C. Retrieve the residue with a few drops of acid sulphuric (10.3.3), evaporate to dryness, calcine again at 550 ºc and weigh.


If P is the weight of the calcium sulphate obtained, the calcium of the aliquot taken amount will be equal to P x 0,2944.


10.6.2. If the sample consists solely of mineral materials, proceed to dissolution by hydrochloric acid without previous incineration. For products such as difficult to dissolve in acids aluminico-calcicos phosphates, proceed to an alkaline fusion before the dissolution. Intimately mixing the part taken with five times the weight of a mixture of equal parts of sodium carbonate and potassium carbonate in a crucible. Heat carefully until the complete fusion of the mixture. Refrigerate and dissolve in hydrochloric acid.


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


10.7 references. First the Commission directive of 15 June 1971. (71/250/EEC). «Official Journal of the European communities» number L 155, 12 July 1971.


11 fat 11.1 principle. Determination of fat by the Gerber method.


11.2 material and devices.


11.2.1 butyrometers contrasted, with graduations 0 to 6 per 100 of fat and divisions of 0.1 per 100. Butyrometers can use with graduations from 0 to 5 per 100 and the 0.1 per 100 divisions, when the sample has a content less than 5 per 100 grease.


11.2.2. tapered rubber or other appropriate Stoppers.


11.2.3 contrasted 1-10 ml pipettes.


11.2.4 clock glasses.


11.2.5 water bath adjustable at 65 ± 1 ° C.


11.2.6 centrifuge capable of reaching 1,200 RPM.


11.2.7 funnel devoid of neck and stem, for the introduction of the sample in the butirometro.


11.3 reagents.


11.3.1 sulfuric acid density d = 1.82.


11.3.2 alcohol isoamyl of density d = 0,815 and 128 ° C 132 ° c distillation range


11.4 procedure.


11.4.1 weigh to the nearest 0,1 mg, 1,100 grams of product and insert it into the butirometro through the funnel. Through the funnel, add 10 ml of warm water (40 ° C), dragging what has failed to adhere to it. Close and shake strongly to dissolve the milk.


Then add 10 ml of sulphuric acid, making it gently sliding down the walls of the butirometro. Add 1 ml of isoamyl alcohol. Close the butirometro with rubber stopper, gently swirl and spin at 1,200 rpm for three to five minutes. Remove the butirometro centrifuge and place in the water bath at 65 ° C. Allow some minutes and read.


11.5. expression of the results. Read in the butirometro, in the split level, the height reached by the fat column, having been adjusted to zero liquid containing no fat. The degrees of the scale indicate the tens, and tenths units per cent. You can see perfectly 0.5 per 100.


11.6 observations.


11.6.1. in the case of Sera devoid of casein, can produce carbonisation which hinder the reading (because the sulfuric acid of density d = 1.82 is too focused). In this case can be diluted to 10 per 100 (v/v) with distilled water.


11.6.2 fat very micronized products, should repeat for three times the centrifugation, heating at 65 ° C and readings, to get consistent results.


11.7 references. Institute of rationalization and standardization of work. A standard Spanish, 64.029.


12 ashes 12.1 gross principle. Incineration of the sample at 550 ºc and weighing of the residue until constant weight.


12.2 material and devices.


12.2.1. heating plate.


12.2.2. electric oven with temperature regulation.



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


12.2.4 ammonium nitrate: 20 per 100 solution (w/v).


12.3 procedure. Weigh approximately 5 g of sample, with an approximation of 1 mg (for products that have tendency to fluff it weigh 2.5 g in a previously calcined and Tared Crucible). Place the Crucible on the hotplate until carbonization of the sample. Insert the crucible in the furnace set at 550 ± 5. º C. Keep at this temperature until ash white, light grey or reddish, seemingly devoid of carbonaceous particles. Place the crucible in a desiccator, allow to cool and weigh immediately.


12.4 calculations. The percentage of ash on natural material is obtained by the following formula: % Ash (natural material) = (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 observations.


12.5.1 difficult to incinerate materials undergo a first three-hour incineration, cool and some drops of a solution to 20 is added per 100 of ammonium nitrate. Continuing the incineration after drying in an oven.


Eventually repeat until complete incineration.


12.5.2. for earlier treatment-resistant materials, operate as follows: after a three-hour incineration, drag the ashes with hot water and filter through a small filter of known ashes. Ash filter and its contents in the original crucible.


Lead filtering to the cold Crucible, evaporate to dryness, incinerate and weigh.


12.5.3. in the case of oils and fats, weigh 25 g into a melting pot of suitable capacity. Char inflaming the sample through an ashless filter paper wick. After combustion, moisten with the minimum amount of water possible. Dewatering and continue as shown at 12.5.


12.6 references. First the Commission directive of 15 June 1971. (71/250/EEC). «Official Journal of the European communities» number L 155, 12 July 1971.


13 (a) volatile nitrogenous Bases (ammonia nitrogen) (by microdiffusion) 13 (a).1 principle. Method enables you to determine the content of volatile nitrogen bases, expressed as ammonia, animal feed.


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


13 (a).2 Material and devices.


13 (a). 2.1 mixer: approximately 35 to 40 oscillations per minute.


13 (a). 2.2 cells (see diagram) Conway, glass or plastic material.


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


(Omitted figure) 13 (a).3 reagents.


13 (a). 3.1 20 per 100 solution (w/v) of trichloroacetic acid.


13 (a). 3.2 Indicator: dissolve 33 mg of bromocresol green and 65 mg of methyl red in 100 ml of ethanol to 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 to 95-96 per 100 and 700 ml of water. Add 10 ml of indicator (13 (a). 3.2.). Mix and adjust if necessary, the colour of the solution to light red by adding a solution of sodium hydroxide. 1 ml of this solution enables you to set as maximum 300 g of NH3.


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


13 (a). 3.5. Acid sulfuric 0, 02N.


13 (a).4 procedure. Weigh to the nearest 1 mg, 10 g of the sample and insert it into a volumetric flask of 200 ml 100 ml of water. Mix for 30 minutes in the mixer. Add 50 ml of trichloroacetic acid solution (13 (a). 3.2), make up to volume with water, shake vigorously and filter through a pleated filter.


Insert the pipette in the central part of the Conway cell 1 ml of boric acid solution (13 (a). 3.3) and the Crown of 1 ml of the filtrate of the sample cell. Cover partially with the help of the greased lid. Dropping quickly on Crown 1 ml of saturated potassium carbonate solution (13 (a). 3.4) and close the lid tightly. Remove with caution the cell giving you a movement of rotation to the horizontal plane, to ensure the mixture of the two reagents. Leave well incubate for four hours at least at room temperature or for one hour at 40 ° C.


Rating the volatile bases in the solution of boric acid with sulphuric acid 0, 02N (13 (a). 3.5) using a Microburette (13 (a). 2.3).


Carry out a blank test applying the same operating method.


13 (a).5 calculations of the 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 relative value, for ammonia contents of less than 1.0 per 100; 0.1 in absolute value, for ammonia contents equal to or greater than 1.0 per 100.


13 (a).6 observations. If the ammonia from the sample content was greater than 0.6 per 100 to dilute the initial filtrate.


13 (a).7 references. Second Commission directive of 18 of November 1971. (71/393/EEC). «Official Journal of the European communities» number L 279, 20 December 1971.


13 (b) 13 volatile nitrogenous Bases (b).1 principle. Method enables you to determine the content of volatile nitrogen bases, expressed as ammonia, fish flours not containing practically urea. It is only applicable for ammonia contents of less than 0.25 per 100.


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


13 (b).2 Material and devices.


13 (b). 2.1 35 to 40 strokes-per-minute mixer approximately.


13 (b). 2.2 type Kjeldahl distillation apparatus.


13 (b). 3. Reagents.


13 (b). 3.1 20 per 100 solution (w/v) of trichloroacetic acid.


13 (b). 3.2 oxide of magnesium, p.a.


13 (b). 3.3 anti-foaming 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 the 0.3 per 100 solution (w/v) of methyl red in 95-96 per 100 ethanol.


13 (b).4 procedure. Weigh to an accuracy of 1 mg, 10 g of the sample and place with 100 ml of water in a graduated flask of 200 ml. mix for 30 minutes in the mixer. Add 50 ml of trichloroacetic acid solution (13 (b). 3.1), make up to volume with water, shake vigorously and filter through a pleated filter.


Take a clear amount of filtering based on the content in volatile nitrogenous bases (100 ml are sufficient, in general). Dilute to 200 ml and add 2 g of magnesium oxide (13 (b). 3.2) and a few drops of anti-foaming emulsion (13 (b). 3.3). The solution should be alkaline in litmus paper; If it is not, add magnesium oxide (13 (b). 3.2). Distil about 150 ml solution in an apparatus of the type Kejldahl and collect the distillate in an Erlenmeyer flask containing an accurately measured volume (25 to 50 ml) of sulphuric acid 0, 1N (13 (b). 3.4). During distillation, avoid an overheating of the walls. Lead sulfate solution to boil for two minutes, cool and titrate the excess sulphuric acid with sodium 0 hydroxide solution, 1N (13 (b). 3.5) in the presence of the methyl red indicator (13 (b). 3.6).


Carry out a blank test applying the same operating method.


13 (b).5 calculations. 1 ml of sulphuric acid 0, 1N corresponds to 1.7 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 should not exceed in relative value, from 10 per 100 of ammonia.


13 (b).6 references. Second Commission directive of 18 of November 1971. (71/393/EEC). «Official Journal of the European communities» number L 279, 20 December 1971.


14 14.1 carbonates principle. Decomposition of carbonates by the action of hydrochloric acid and comparison of the volume of carbon dioxide evolved against a known amount of calcium carbonate that are measured in the same conditions.


14.2 material and devices.


14.2.1. appliance of Scheibler - Dietrich, as shown in Figure 14.1 in.


14.3 reagents.


14.3.1 hydrochloric acid, d = 1.10.


14.3.2. calcium carbonate.


14.3.3. solution of sulphuric acid 0, 1N or approximate, coloured with methyl red.


14.4 procedure. Depending on the concentration in carbonates of the sample weigh: 0.5 g when wealth is between 50 and 100 per 100 expressed as calcium carbonate.


1 g when wealth is between 10 and 50 per 100 expressed as calcium carbonate.


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


Once heavy the ideal amount of sample to analyse to insert it into the flask (4) of the apparatus of Scheibler-Dietrich, which must be equipped with a small tube of unbreakable material containing 10 ml of the solution 14.3.1. Then connect the flask with the appliance. Turn the three-way stopcock (5) in such a way that the tube (1) communicate with the outside.



By the mobile tube (2), which is full of acid sulphuric colored (14.3.3) and attached to the graduated tube (1), bring the fluid level zero graduation. Turn the key (5) so that it will communicate the tubes (1) and (2) and check the level to zero.


Previous inclination of the flask (4), slowly let all HCL (14.3.3) on the sample. Equalize pressure by lowering the tube (2). Shake the flask (4) until it stops completely the release of carbon dioxide.


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


Carried out under the same conditions a comparative test with 0.5 g of carbonate of calcium (14.3.2).


14.5 calculations.


% CO3Ca = V. 100:T. 2 p is: V = the volume, in ml, of CO emitted by the sample.


T = the volume, in ml, of CO emitted by 0.5 grams of COCa (14.3.2).


P = weight, in grams, of the sample.


14.6 observations.


14.6.1. when the sample is greater than 2 grams previously introduce 15 ml of distilled water in the flask (4) and mix before starting the analysis. Use the same volume of water for comparative analysis.


14.6.2 If you use a volume other than the Scheibler-Dietrich apparatus, it is necessary to adapt the amount of sample taken of boiling and the substance to compare, as well as the calculation of results.


14.7 references. First the Commission directive of 15 June 1971. 71/250/EEC. «Official Journal of the European communities» number L 155, 12 July 1971.


(FIGURE OMITTED)


FIGURE 14.1. (1/8 scale in mm)


Appliance according to Scheibler-Dietrich to determine CO 15 (to) aflatoxin B (monodimensional thin layer chromatography) 15 (a).1 principle. The method enables you to determine the content of aflatoxin in raw materials and feedingstuffs. This method can not be used in the presence of citrus pulp. The lower limit of determination is 0'01 mg/kg (10 ppb).


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


15 (a).2 Material and devices.


15 (a). 2.1 Triturador-mezclador.


15 (a). 2.2 device to agitate or magnetic stirrer.


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


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


15 (a). 2.5 Rotary apparatus of evaporation in vacuum, with 500 ml flask.


15 (a). 2.6 tapered bottles of 500 ml, with ground glass stopper.


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


15 (a). 2.8 glass for thin layer chromatography plates, 200 x 200 mm, prepared in the following manner (with the amounts indicated can be five plates): add 30 g of silica G-HR gel (15 (a). 3.1. 15) in a conical flask, add 60 ml water, stopper and shake for one minute. Extend the suspension on the plates in order to obtain a uniform layer 0,25 mm thick. Leave to air dry and then store in a desiccator provided with silica gel. At the time of its use, activate the plates keeping them for an hour in the oven at 110 ° C. Plates ready-to-use longer practices in so far as they give results similar to the prepared plates as shown.


15 (a). 2.9 long waves (360 nm) UV lamp. The intensity of irradiation should be allowed to differentiate, even with clarity, a spot of 1.0 ng of aflatoxin B on a plate for thin layer chromatography, at a distance of 10 cm from the lamp.


15 (a). 2.10 tubes of 10 ml, graduated, with polyethylene caps.


15 (a). 2.11 ultraviolet spectrophotometer.


15 (a). 2.12 Fluordensitometro (if any).


15 (a).3 reagents.


15 (a). 3.1 acetone.


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


15 (a). 3.3 n-hexane.


15 (a). 3.4 methanol.


15 (a). 3.5 ether diethyl anhydrous, free from peroxides.


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


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


15 (a). 3.8 Silica Gel, for column particle size 0,05 to 0,20 nm chromatography.


15 (a). 3.9 cotton previously degreased, using 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 solution of sulphuric acid per 100 50 (v/v).


15 (a). 3.14 kieselguhr washed with acid.


15 (a). 3.15 Silica Gel for thin layer chromatography.


15 (a). 3.16 solution pattern of 0,1 micrograms of aflatoxin B per ml in chloroform (15 (a). 3.2) or the benzene/acetonitrile mixture (15 (a). 3.6), prepared and controlled as outlined in 15 (a). 6.


15 (a). 3.17 solution qualitative pattern of 0,1 micrograms of aflatoxin B and B per ml by chloroform (15 (a). 3.2) or the mixed benzene/acetonitrile (15 (a). 3.6). These concentrations are given as an indication and must be adjusted to obtain the same intensity of fluorescence for two aflatoxins.


15 (a). 3.18 developing solvents.


15 (a). 3.18. 1 chloroform (15 (a). 3.2) / acetone (15 (a). 3.1): 9/1 (v/v), unsaturated bucket.


15 (a). 3.18. 2 ether diethyl (15 (a). 3.5) / methanol (15 (a). 3.4) / water: 96/3/1 (v/v/v), unsaturated bucket.


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 tray.)


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 of fats should be degreased with light petroleum (40-60 ° C boiling point) after the preparation indicated in 15 (a). 5.1. In these cases, the results of the analysis are expressed in non-defatted sample weight.


15 (a). 4.2 extraction. Introduce 50.0 g of milled and homogenized sample into a conical flask of 500 ml (15 (a). 2.6). Add 25 g of kieselguhr (15 (a). 3.14), 25 ml of water and 250 ml of chloroform (15 (a). 3.2.). Cover the bottle, shake or stir for 30 minutes with the help of the appliance (15 (a). 2.2) and filter through a pleated filter (15 (a). 2.3). Remove the first 10 ml of the result of the filtration and then collect 50 ml.


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


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


Stir carefully after each addition in order to eliminate air bubbles. Leave that he pose for fifteen minutes and then add with caution, 15 g of sodium sulphate (15 (a). 3.10). Let descend the liquid up to the immediate vicinity of the upper surface of the sodium sulphate 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 descend liquid to the upper surface of the sodium sulphate layer. Remove the filtrate. Then add 100 ml of diethyl ether (15 (a). 3.5) and leave to again descend liquid to the upper surface of the sodium sulphate layer. During these operations, to ensure that the flow is 8-12 ml per minute and that column is not empty. Delete filtrates. Elute later by 150 ml of the mixture chloroform-methanol (15 (a). 3.7) and collect the whole of the eluate.


This evaporate almost to dryness under a stream of inert gas (15 (a). 3.11) and at a temperature not exceeding 50 ° C, by rotary evaporation in vacuum apparatus (15 (a). 2.5). Introduce quantitatively the waste by means of chloroform (15 (a). 3.2) or mix benzene/acetonitrile (15 (a). 3.6) in a tube of 10 ml (15 (a). 2.10). Concentrate the solution under a stream of inert gas (15 (a). 3.11) and then bring volume to 2.0 ml with chloroform (15 (a). 3.2) or mix benzene/acetonitrile (15 (a). 3.6).


15 (a). 4.4-thin-layer chromatography. Deposit promptly in a thin layer chromatography plate (15 (a). 2.8), 2 cm from the lower edge and at intervals of 2 cm, the volumes of the standard solution and the extract below:-10, 15, 20, 30 and 40 µl of the standard solution of aflatoxin B (15 (a). 3.16).


-10 µl of the extract obtained in (15 (a). 4.3) and, superimposed on the same point, 20 µl of standard (15 (a).3) solution.


-10 and 20 µl of the extract obtained in (15 (a). 4.3).


Develop the chromatogram away from light, with help from one of the development solvent (15 (a). 3.16). The choice of the solvent must be determined previously deposited on the plate 25 microliters of the qualitative standard solution (15 (a). 3.17) and ensuring that, during development, aflatoxins B and B are fully separated:-25 per 100 of the higher result for contents in 10 to 20 micrograms/kg aflatoxin B.


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


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



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


15 (a). 5.1 determination of the concentration of aflatoxin b


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


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


Calculate the concentration in micrograms of aflatoxin B per millilitre of solution from the following formulas: 312 · · 1000 to cloroformica solution 312 · · 1000 to cloroformica 206000 solution cloroformica solution 312 · · 1000 312 · · 1000 for the solution in the mix 19800 benzene/acetonitrile without the reach of the light, make suitable dilutions to get a solution whose concentration of aflatoxin B work pattern is 0.1 micrograms per milliliter approximately.


Preserved in the refrigerator, this solution is stable for two weeks.


15 (a). 5.2. Chromatographic purity control.


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


Develop the chromatogram as indicated in (15 (a). 4.4). With ultraviolet light, fluorescence must only give rise to the perception of a single spot and no fluorescence in the area of the original tank should not be seen.


(Figure omitted) 15 (a). 5.3 reproducibility of the results of A method. The reproducibility of the results, i.e. the variation between the results obtained by two or more laboratories on the same sample has been estimated at: ± 50 per 100 of the mean value of the results for mean values of aflatoxin B 10 to 20 xg/kg ± 10 xg/kg average to average between 20 to 50 xg/kg.


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


15 (a). 5.4 references.


Directive 92/95/EEC. «Official Journal of the European Communities» L 327, on 9 November.


Directive 94/14/EC. «Official Journal of the European Communities» L 94 of 13 April.


15 (b) determination of aflatoxin B, using high resolution 15 (b) liquid chromatography.1 principle. The method is based on separation by high resolution liquid chromatography with detection by fluorescence. The extraction of the sample is carried out with chloroform. The extract is filtered and an aliquot of this is purified in a florisil cartridge and then in a cartridge C. Final separation and determination is done by liquid chromatography of high resolution (HPLC) using a reversed-phase column C followed by a reaction of post-columna with aqueous solution of iodine and detection by fluorescence.


The method enables you to determine B aflatoxin in feed, including those containing citrus pulp. The lower limit of determination is 0,001 mg/kg.


Note: Mycotoxins are extremely toxic. Manipulations should be conducted in the cooker hood. They should take special precautions when toxins are in solid form, since due to its electrostatic nature they tend to disperse in the work areas.


15 (b).2 Material and devices. Attention: The use of glassware that have not been washed with acid, aqueous solutions of aflatoxins, can lead to losses of aflatoxin. New or disposable glassware special precautions must be taken. For example, auto-sampler vials and Pasteur pipettes. Therefore, glassware that is going to be in contact with aqueous solutions of aflatoxins should immerse yourself for several hours in a dilute acid (e.g. sulfuric acid, c = 2 mol/l) and then wash thoroughly with distilled water to remove all remaining acid (for example, three rinses, followed by a check with pH paper). In particular, this treatment should be applied to round flasks (15 (b). 2.4), to the volumetric flasks, test tubes, vials or tubes used for calibration solutions and extracts final (in particular, auto-sampler vials) and Pasteur pipettes if used to transfer calibration solutions or extracts.


15 (b). 2.1 grinder/mixer.


15 (b). 2.2 (ISO R 565) 1.0 mm mesh sieve.


15 (b). 2.3 mechanical stirrer.


15 (b). 2.4-evaporator rotary vacuum equipped with a 150 ml to 250 ml round flask.


15 (b). 2.5 liquid chromatograph for high resolution, with a loop of injection that allows to inject 250 xl.


See the manufacturer's instructions for partial or complete loop filling.


15 (b). 2.6 for HPLC analytical column: stuffed C of 3 or 5 xm.


15 (b). 2.7 pump pulses supplied reagent free iodine for example, pump for HPLC, post-column reaction to or designed for post-column reaction.


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


15 (b). 2.9 of reaction, teflon or stainless steel coil. 3,000 x 0.5 mm to 5,000 x 0.5 mm dimensions are appropriate in combination with 5-3 xm HPLC columns. 15 (b). 2.10 water bath termostatizado at 60 ° C with a variation in temperature of less than 0.1 ° C.


15 (b). 2.11 fluorescence Detector that provides approximately and 435 nm emission approximately 365 nm excitation wavelengths. (For the filtering apparatus: issue 400 nm wavelength). It must be possible to carry out the detection of 0.05 ng of B, as a minimum. Apply some pressure (for example, a constrictor or a teflon or stainless steel coil connected to the outlet of the detector port) in order to remove the air bubbles in the flow cell.


15 (b). 2.12 paper Web Registrar.


15 (b). 2.13 electronic Integrator (optional).


15 (b). 2.14 filter folds 24 cm. Macherey-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 conical flask of 500 ml with glass stopper.


15 (b). 2.17 glass column (with an internal diameter of 1 cm approximately and a length of 3 cm approximately) equipped with a Luer tip.


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


15 (b). 2.19 10 ml syringe resistant to chemicals, lockable Luer.


15 (b). 2.20 syringe of 250 xl suitable for HPLC injection (see 15 (b). 2.5).


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


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


15 (b). 2.23 suitable spectrophotometer for measurements in the UV spectrum region.


15 (b). 2.24. Equipment to perform the confirmation test (15 (b).5).


15 (b). 2.24. 1 funnel of 100 ml (with teflon tap), cleaning with acid.


15 (b). 2.24. 2 source of heat to 40-50 ° C.


15 (b).3 reagents.


15 (b). 3.1 chloroform stabilized with 0.5 to 1.0 per 100 of ethanol, mass.


See 15 (b). 9.2.


15 (b). 3.2 methanol, grade HPLC for preparation of 15 (b). 3.6.


15 (b). 3.3 acetone.


15 (b). 3.4 acetonitrile, HPLC grade.


15 (b). 3.5 Elution solvents; prepare one day before use or eliminate air containing ultrasonically.


15 (b). 3.5. 1 acetone mixture (15 (b). 3.3) and water, 98 + 2 (v/v).


15 (b). 3.5. 2 water / methanol mixture (15 (b). 3.2), 80 + 20 (v/v).


15 (b). 3.5. 3 water / acetone mixture (15 (b). 3.3), 85 + 15 (v/v).


15 (b). 3.6. mobile phase for HPLC. 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 in the mobile phase, in accordance with the characteristics of the HPLC column used.


15 (b). 3.7 aqueous saturated iodine. Add 2 g of iodine to 400 ml of water. Mix for 90 minutes as a minimum and filter through a membrane filter (15 (b). 2.15). Protect light saturated solution, in order to prevent photodegradation.


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


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


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


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


15 (b). 3.12 solution pattern of aflatoxin B in chloroform with a concentration of 10 xg/ml. check the concentration of the solution in the following way: determine the absorption spectrum of the solution referred to between 330 and 370 nm by means of the spectrophotometer (15 (b). 2.23). Measure the absorbance (a) at the maximum near 363 nm. Calculate the concentration of aflatoxin B, in micrograms per millilitre of solution, using the following formula: concentration (xg/ml) = 312 · · 1000 = 13,991 · A concentration (xg/ml) = 312 · · 1000 = 13,991 · A concentration (xg/ml) = 22300 = 13,991 · 15 (b). 3.12. 1 solution mother of aflatoxin B in chloroform. Transfer quantitatively 2,5 ml of the standard solution of aflatoxin B (15 (b). 3.12) to a volumetric flask of 50 ml and make up to volume with chloroform. Store this solution in a cool place (4 ° C) away from light, adequately covered and wrapped in a sheet of aluminium (15 (b). 3.13). Aflatoxin B for calibration of HPLC solutions.


Note: The preparation of these solutions should be used for glassware washed with acid (see point 15 (b). 2.) Appliances).



15 (b). 3.13. 1 solution for calibration of 4 ng/ml. (few hours) leave the stock solution (15 (b). 3.12. 1), contained in the flask wrapped in aluminum sheet, until it reaches room temperature. Transfer 400 xl solution (200 ng aflatoxin B) mother to a volumetric flask of 50 ml and evaporate the solution to dryness in current of inert gas (15 (b). 3.11). Dissolve the residue in 20 ml approximately of water / acetone mixture (15 (b). 3.5. 3), make up to volume with the same mixture and mix thoroughly.


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 flask with the water / acetone mixture (15 (b). 3.5. 3) and mix thoroughly.


15 (b). 3.13. 3 solution for calibration of 2 ng/ml. transfer quantitatively 25 ml of the calibration solution (15 (b). 3.13. 1) to a 50 ml graduated flask, make up to volume with the water / acetone mixture (15 (b). 3.5. 3) and mix thoroughly. This solution is also called «Standard» and is used, in particular, to carry out injections again and during the HPLC procedure.


15 (b). 3.13. 4 solution for calibration of 1 ng/ml. transfer quantitatively 2,5 ml of the calibration solution (15 (b). 3.13. 1) into a 10 ml volumetric flask, make up to volume with water / acetone mixture (15 (b). 3.5. 3) and mix thoroughly.


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 test solution. Transfer the mixture (15 (b). 3.14) to a test tube with glass stopper or a bottle with screw cap. Transfer 40 ael of this solution to a plug of glass - washing with acid - test tube (15 (b). 2.22). Evaporate the chloroform in a stream of inert gas (15 (b). 3.11) and dissolve in 10 ml of the water / acetone mixture (15 (b). 3.5. 3).


15 (b). 3.15 reagents for the confirmation test (15 (b).5). 15 (b). 3.15. 1 aqueous saturated sodium chloride solution.


15 (b). 3.15. 2 sodium sulphate anhydrous, granular.


15 (b).4 procedure.


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


15 (b). 4.2 portion of the trial. Although in the Erlenmeyer flask (15 (b). 2.16) 50.0 g of the sample prepared problem.


15 (b). 4.3 extraction. Add a portion of test 25 g of Celite (15 (b). 3.8) 250 ml of chloroform (15 (b). 3.1) and 25 ml of water. Close the flask and shake for 30 minutes on the Shaker mechanical (15 (b). 2.3). Filter through a folded filter (15 (b). 2.14). Collect 50 ml of filtrate. If necessary, take an aliquot of the prepartion and dilute to 50 ml with chloroform to produce aflatoxin B concentration not greater than 4 ng/ml.


15 (b). 4.4. Purification (the procedure should follow without significant interruptions). The following precautions must be taken:-conveniently protect the analysis laboratory of natural light.


This can be used: 1) leaves that absorb UV rays to cover windows and sieved light (avoid direct sun light).


(2) curtains or blinds in combination with artificial light (can be used fluorescent tubes).


-Protect all possible light solutions containing aflatoxin (kept in the dark and using aluminum foil).


15 (b). 4.4. 1 with florisil SEP-PAK purification.


15 (b). 4.4.1.1. Preparation of the columna-cartucho set. Attach a wrench (15 (b). 2.18) to the shorter of a florisil cartridge branch (15 (b). 3.9) (see Figure 15 (b).1).


Wash the cartridge and remove the air taking with a syringe (15 (b). 2.19) 10 ml of chloroform (15 (b). 3.1) and quickly 8 ml of chloroform by pretending the key through the cartridge. Join the branch more long cartridge to the glass column (15 (b). 2.17) and insert into the column through the cartridge the 2 ml of chloroform remaining. Close the stopcock and remove the syringe. 15 (b). 4.4.1.2. Purification. Altogether columna-cartucho enter the filtrate collected pursuant to point (15 (b). 4.3) and drain by gravity. Wash with 5 ml of colroformo (15 (b). 3.1) and then with 20 ml of methanol (15 (b). 3.2). Discard the eluates. During these operations, prevent the columna-cartucho set to stay dry. Elute aflatoxin B with 40 ml of the mixture acetone/water (15 (b). 3.5. 1) and collect the whole of the eluate in the round flask (150 ml) of the Rotary evaporator (4.4). Concentrate the eluate on the Rotary evaporator at a temperature of 40-50 ° C until stops the distillation of acetone.


Note: At that time remaining in the flask 0,5 ml of liquid approximately. It has been shown experimentally that continue the evaporation has no harmful consequences and that when 0,5 ml of liquid acetone this quantity is not significant. The presence of acetone residue could cause losses of aflatoxin B in C. Add 1 ml of methanol cartridge (15 (b). 2.2), shake the flask to dissolve aflatoxin B attached to your walls, add 4 ml of water and mix. Disconnect and discard the cartridge. Wash the glass column with water and retained to carry out the purification C.


15 (b). 4.4. 2 purification with SEP-PAK C.


15 (b). 4.4.2. 1 preparation of the whole columna-cartucho. Attach a wrench (15 (b). 2.18) to the shorter cartridge C branch ((15 (b). 3.10).) (Refer to figure 1 (b). 1.)


Purge cartridge and remove the air by moving quickly with a syringe (15 (b). 2.19) 10 ml of methanol (15 (b). 3.2) for the key through the cartridge. (Cartridge air bubbles are visible in the form of spots of light on a gray background). Take 10 ml of water and pass 8 ml through the cartridge (avoid introducing air when switching from methanol to water). Join the longest branch of the cartridge to a glass column and insert into the column through the cartridge the remaining 2 ml of water. Close the stopcock and remove the syringe.


15 (b). 4.4.2. 2 purification. Quantitatively transfer to column (15 (b). 2.17) the extract collected in the point ((15 (b). 4.4.1. 2), washing twice the flask with 5 ml of the mixture of water and methanol (15 (b). 3.5. 2) and drain by gravity.) During these operations, prevent the columna-cartucho set to stay dry. (If air bubbles are formed in the narrowing next to the cartridge, stop the flow and tap the top of the glass column to remove the bubbles. Immediately, resume operations.) Elute with 25 ml water / methanol mixture (15 (b). 3.5. 2). Discard the eluates. Elute aflatoxin B with 50 ml of water / acetone mixture (15 (b). 3.5. 3) and collect the whole of the eluate in a volumetric flask of 50 ml. make up to volume with water up to 50 ml and mix, the obtained test solution is used for chromatography (15 (b). 4.5).


Attention: Normally it is not necessary to filter the final extract prior to HPLC. When it is necessary to filter, must be avoided cellulose filters, given that they can result in loss of aflatoxin teflon filters B. can be used.


15 (b). 4.5 high resolution liquid chromatography. (See Figure 2 for the installation of the unit.)


Allow enough time for the instruments to heat up and stabilize before use.


Note 1: The flows mentioned for HPLC mobile phase and the post-column reagent are purely indicative. It may be necessary to make an adjustment on the basis of the characteristics of the HPLC column.


Note 2: The response of the detector to the aflatoxin B depends on the temperature, so the compensation should be by drift (see Figure 3). The injection of an amount fixed reference of aflatoxin B pattern ((15 (b). 3.13. 3) at regular intervals (e.g. every three injections) allows to correct, using the mean response, the values of the peaks of the aflatoxin B between these reference standards, provided that the difference between responses of consecutive reference standards is very small (R 10 per 100).) Therefore injections must be done without interruptions. If an interruption is required, the last injection before interruption and the first after this must be injections of the reference standard (15 (b). 3.13. 3). Since the calibration curve is linear and passes through the origin, the quantities of aflatoxin B present in the sample extracts are determined directly by reference to the adjacent patterns.


15 (b). 4.5. HPLC pump setting 1. Set the HPLC pump (15 (b). 2.5) so as to obtain a flow of 0,5 or 0,3 ml/min for a 5 xm or 3 xm HPLC column (15 (b). 2.6), respectively, using the mobile phase (15 (b). 3.5).


15 (b). 4.5. 2 setting of the pump for the post-column reaction. Set the pump (15 (b). 2.7) so as to obtain a flow rate of 0, 2-0, 4 ml/min of aqueous saturated iodine solution (15 (b). 3.7). Information as an indication: recommended 0,4 or 0,2 ml/min flow rates, approximately, 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) at a wavelength of 365 nm and emission of 435 nm excitation (filter unit: T 400 nm). Adjust the detector attenuator so 80 per 100 approximately the maximum travel of the pen register corresponds to 1 ng of aflatoxin b


15 (b). 4.5. 4 injector. For all solutions injected 250 xl amounts according to the instructions of the manufacturer of the device.


15 (b). 4.5. 5 check of chromatographic separation. Inject the chromatographic solution (15 (b). 3.14. 1).


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



15 (b). 4.5. 6 checking the stability of the system. Before each series of analyses, perform repeated injections of the reference standard (15 (b). 3.13. 3) until the areas of the peaks to stabilize.


Note: The peaks caused by aflatoxin B between consecutive injections required differences less than 6 per 100. Proceed immediately to carry out the check of linearity (15 (b). 4.5. 7).


15 (b). 4.5. 7 verification of the linearity. Inject the aflatoxin B for calibration solutions (15 (b). 3.13. 1) and (15 (b). 3.14. 4). Use the reference standard (15 (b). 3.13. 3) at intervals of three injections, to correct the drift in the answers.


Note: Responses of the peaks of the reference standard must submit differences below 10% in 90 minutes: correcting drift by applying the formula indicated in point 15 (b). 6. The calibration graph should be linear and pass through the origin, within the limits of 2 times the value of the standard deviation of the estimate of and. The found values must differ by less than 3% of the nominal values. If these conditions are met, continue the operations immediately. If not, identify and correct the cause 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 reference standard (15 (b). 3.13. 3) after two injections of summary of sample according to the following sequence: pattern of reference, extract, extract, reference, extract, extract, reference, etc. pattern pattern...


15 (b).5 confirmatory test.


15 (b). 5.1 further treatment of the extract (15 (b). 4.4.2. 2). Add 5 ml of 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 funnel (15 (b). 2.24. 1). Pour the combined chloroform extracts in a 10 ml test tube through 1 g approximately of sodium sulfate (15 (b). 3.15. 2). (May be a small funnel (diameter 4 cm) by placing a cotton covered approximately 1 g of sodium sulphate in narrowing).


Wash the coat of sulfate of sodium with a few ml of chloroform and collect the washings in the same test tube. Evaporate in the segment of chloroform extract to dryness using the heat source (15 (b). 2.24. 2) and dissolve in 1 ml of chloroform.


15 (b). 5.2 preparation of derivatives and thin layer chromatography. See 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: contained aflatoxin B lg/kg = m · V contained aflatoxin B lg/kg = m · V contained aflatoxin B lg/kg = m · VV content lg/Kg aflatoxin B = V · M · V contained aflatoxin B lg/kg = m · VV is: m = quantity in ng of aflatoxin B, represented by the B peak in the sample, calculated in the following way: m = P (sample) · 2 r (st) P (st) + P (st) P (sample) = area of the peak of aflatoxin B of the sample.


P (st1) = area of the peak of aflatoxin B from the previous reference pattern (15 (b). 3.13. 3).


P (st2) = area of the peak of the next reference standard aflatoxin B (15 (b). 3.13. 3).


r (st) = injected amount of reference pattern (15 (b). 3.13. 3), expressed in ng.


V = volume of the injected sample extract in ml.


V = final volume of the extract of the sample in ml (15 (b). 3.13. 3).


M = mass of the sample in g.


V = volume of filtrate transferred to 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 applies, the formula is reduced a: content of aflatoxin B, lg/kg = 20 x m.


15 (b). 6. (1) the calculation of the results can also be made by measuring the height of the peaks.


15 (b).7 repeatability. See item 15 (b). 9.1 (observations).


15 (b).8 reproducibility. See item 15 (b). 9.1.


15 (b).9 observations.


15 (b). 9.1 Precision.


Table 1 shows the results of repeatability and reproducibility in a collaborative trial (1) on compound feedingstuffs conducted internationally. 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. According to the standard ISO 3534-1977, 2.35 (2) and decision 89/610/EEC of the Commission (3), both r and R are also listed in table 1 in the form of coefficients of variation.


TABLE 1 repeatability r and reproducibility (R) expressed in differences and coefficients of variation 15. Labs level (lg/kg) / r / R / CVt (*) percentage / CVr percentage 8-14 / 1.4 / 1.7 / 11 / 18 (*) CV = coefficient of variation.


(1) Egmond, H.P. van. Heisterkamp, S.H. and Paulsch, W.E. (1991), Food Additives and Contaminants 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 characteristics of absorption of the florisil cartridge may be modified if you are using a different ethanol stabilizer. This must be verified pursuant to paragraph 15 (b). 9.3 when the chloroform described is not available.


15 (b). 9.3 accuracy. The correct application of the method must be checked by carrying out measurements with certified reference materials. If these materials are not available, the validity of the method must be checked through samples blank recovery experiments. The difference between the average and the actual value must not exceed the limits of - 20 to 10 per 100 of the real value.


15 (b).10 references. Directive 92/95/EEC. «Official Journal of the European communities» number L 327, 9 November 1992.


(FIGURES OMITTED) 16. Index of peroxides this determination shall apply to those fats which are used as raw material.


The procedure shall be as set out in the method number 21 of the official methods of analysis of oils and greases approved by order of 31 January 1977 («Official Gazette» of 14 July).


In the event that it is necessary to prepare the sample, follow the procedure in number 1 of the aforementioned order method.


17. total phosphorus (method spectrophotometrically) 17.1 principle. Determination of phosphorus in a mineral sample, through the transformation of its compounds phosphorus in ortofosforados.


Mineralization occurs via dry (calcination) and subsequent dissolution in acid, or by acid digestion.


Measurement of absorbance at 430 nm of the complex formed with the reagent nitro-molibdo-vanadate.


17.2 material and devices.


17.2.1 spectrophotometer, capable of performing readings of 430 nm, having cells of 10 mm light path with.


17.2.2. incineration of quartz or porcelain crucibles.


17.2.3 test tubes of 25 to 30 ml with mouth of frosted glass.


17.2.4. pipettes of double diffuser of 5, 10, 15, 20, 25 and 50 ml.


17.2.5 Matraces Kjeldahl 250 to 500 ml of capacity.


17.2.6. graduated flasks of 100, 250, 500 and 1000 ml.


17.3 reagents.


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 d = 1,84 g/ml.


17.3.6. concentrated ammonia of d = 0.910 g/ml.


17.3.7 ammonium heptamolybdate solution. Dissolve in water Hot 100 g of ammonium heptamolybdate tetrahydrate (NH) MoO · 4HO. add 10 ml of ammonia (17.3.6), is transferred to a volumetric flask of 1000 ml and cold once is complete with water up to the mark.


17.3.8 solution of ammonium metavanadate. Dissolve 2.35 g of metavanadate ammonium (NHVO) in a 500 ml conical flask with 400 ml of hot distilled water. Add slowly and shaking 20 ml of a solution containing 7 ml of nitric acid (17.3.3) and 13 ml of distilled water. Lead to a volumetric flask of 1000 ml and, once cold, make up to volume with distilled water.


17.3.9 nitro-molibdo-vanadate solution. In litre volumetric flask, mix 200 ml of ammonium heptamolybdate solution (17.3.7) with 200 ml of ammonium metavanadate (17.3.8) and 134 ml of nitric acid (17.3.3). Fill with distilled water to the mark.


17.3.10 solution pattern of phosphorus containing 1 mg of phosphorus per ml. Dissolve in litre volumetric flask 4,387 g of monopotassium phosphate (KHPO) (previously dried in oven 100 ° C until constant weight) in distilled water and bring volume.


17.4 procedure.


17.4.1 curve of calibration.


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


In six Erlenmeyer flask or test (17.2.3) tubes take with double diffuser 10 ml of each solution pattern of phosphorus pipettes. Add to each of them, also with pipette double diffuser, 10 ml of reagent of nitro-molibdovanadato (17.3.9), shake to mix and allow to stand 10 minutes at 20 ° C.


Carry out photometric readings at 430 nm, using 10 mm light path cuvettes, using white phosphorus as reference solution solution.



Graphically represent the absorbances obtained against the micrograms/ml 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 organic substances free from phosphates resulting insoluble products the incinerated).


Weigh 2.5 g of the sample, with accuracy of 1 mg, capsules of quartz or porcelain.


Mix with 1 g of carbonate of calcium (17.3.1.). Put in muffle at 550 ° C - 5 ° C until white or grey ash (a small part of coal does not interfere). Transfer the ash to a glass of 150 ml. add 10 ml of water washing the crucible with hydrochloric acid (17.3.2) until effervescence ceases. Add other 10 ml of hydrochloric acid (17.3.2). Evaporate hydrochloric acid in boiling sand bath until dry. Cool and dissolve residue with 10 ml of nitric acid (17.3.4), boil in sand bath for five minutes, without drying. Filter through paper over 500 ml graduated flask, wash with distilled water the vessel and furring strips once cold.


17.4.2.2 mineralization by acid digestion (for mineral compounds and liquid feedingstuffs). Weigh 1 g of sample, with accuracy of 1 mg, and bring a flask Kjeldahl (17.2.5), add 20 ml of acid sulphuric (17.3.5), shake the flask circularly to prevent the sample from sticking to the walls and boil for ten minutes. Let cool slightly and add 2 ml of nitric acid (17.3.3), heat and bring again to the boiling point. Repeat this procedure until the discoloration of the solution. Cool, add a little water and transfer the liquid quantitatively, filtering if necessary, to flask 500 ml volumetric, wash the Kjeldahl flask with hot water, and join the washing fluids if necessary through the filter used initially. Once cold, add water up to the mark.


17.4.3 the color development and measurement of absorbance. Dilute an aliquot of the filtering problem, to achieve a concentration of not more than 40 micrograms/ml match.


Transfer 10 ml of this solution to the Erlenmeyer flask or test tube (17.2.3) and add 10 ml of the reagent nitro-molibdovanadato (17.3.9). The two solutions taken with two flushes 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 a solution of 10 ml of solution white with 10 ml of the reagent nitro-molibdo-vanadate as a reference.


Always make a test with the reagents used, following the same procedure exprerimental. Use this as a reference in each Spectrophotometric reading.


17.5 calculations. Determine the concentration of phosphorus in the diluted aliquot portion of the solution to be analysed or the micrograms/ml of such solution, by reference to the curve of calibrated, calculate the percentage by weight of phosphorus in the sample.


% P = S · C · C · ... C


G · 10,000 · · · ... Be: G = weight of the sample in grams.


C C... C = dilutions to which aliquots were in milliliters.


TO A... To = aliquots taken for successive dilutions in milliliters.


S = micrograms/ml of phosphorus measured of the problem solution in relation to the curve of calibration.


17.6 observations. The difference between the results of two successive determinations should not exceed: 3 per 100 (relative value), with contents of less than 5 per 100 match (m/m).


0.1 per 100 (absolute value) for content match 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, 20 December.


18. soluble in hydrochloric acid and pepsin 18.1 crude protein principle. Method makes it possible to determine the fraction of crude protein dissolved by pepsin and hydrochloric acid under the conditions given. It is applicable to animal flours.


The sample is subjected to digestion for 48 hours at 40 ° C by pepsin hydrochloride solution. Suspension is filtered or centrifuged and determined the nitrogen content of the filtrate or supernatant, according to the official method number 3.


18.2 material and devices.


18.2.1. water bath or range of incubation set at 40 ° C ± 1 ° C.


18.2.2. carbon and Distiller Kjeldahl.


18.3 reagents.


18.3.1 hydrochloric acid (d = 1,125).


18.3.2. solution of hydrochloric acid 0, 075N.


18.3.3 pepsin 2.0 U/mg. The activity must be checked according to the official method number 19.


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


18.3.5 Defoamers.


18.3.6 appearing in 3.3.


18.4 procedure. Weigh to the nearest 1 mg, 2 g of sample. Enter the sample flask 500 ml and Add 450 ml of pepsin (18.3.4), previously heated to 40 ° C. hydrochloride solution Shake so 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 incubation (18.2.1) stove and keep it for 48 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), cool to 20 C, make up to volume with water and filter flask.


Take 250 ml of the filtrate and place in the Kjeldahl digestion flask and proceed as in the official method number 3. Carry out a blank test.


18.5 calculations. HCL-soluble protein content and pepsin for 100 g of sample shall be given by the following formula: protein soluble/100 g sample = 2 · 1.4 · 6.25 (a'n VN V ') P being: P = weight, in g, of the sample.


V = the volume, in ml, of sulphuric acid.


N = normality of the sulphuric acid.


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


N' = normality of sodium hydroxide solution.


18.6 observations. Products with a fat content exceeding 10 per 100 must be previously defatted 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 19.1 pepsin activity principle. The method used to check the activity of the pepsin used in the determination of pepsin and hydrochloric acid-soluble protein.


Hemoglobin is treated in accordance with pepsin and hydrochloric acid. The non-hydrolyzed fraction of proteins is precipitated by trichloroacetic acid. Filtering solution of sodium hydroxide added to it are and Folin-Ciocalteu reagent. The absorbance of this solution is measured at 750 nm and the number of corresponding tyrosine is read on a curve pattern.


The unit of pepsin is defined as the quantity of that enzyme which liberates per minute, in the conditions of the method, a number of composite hidroxiarilos whose coloration by the Folin-Ciocalteu reagent has an absorbance corresponding to the one of 1 mole of tyrosine, under the same conditions.


19.2 material and devices.


19.2.1 water bath, regulated at 25 ° C ± 0.1 ° C for the ultratermostato.


19.2.2. spectrophotometer.


19.2.3. Stopwatch; accuracy: 1 second.


19,2,4 pH meter.


19.3 reagents.


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) of trichloroacetic acid.


19.3.5, 0 sodium hydroxide solution 5N.


19.3.6. Folin - Ciocalteu reagent. Place 100 g of dihydrate sodium tungstate (NaWO · 2HO), 25 g of dihydrate sodium molybdate (NaMO · 2HO) and 700 ml of water in a 2-litre grinding closing round-bottomed flask. Add 50 ml of phosphoric acid (d = 1.71) and 100 ml of concentrated hydrochloric acid (d = 1.19), fit a reflux condenser to the flask, bring to the boil and keep the solution in boiling soft for 10 hrs. leave to cool, detach the reflux condenser, add 175 g of lithium sulphate dihydrate (smooth · 2HO), 50 ml of water and 1 ml of bromine. To boil for 15 minutes to eliminate excess bromine.


Stop cooling, quantitatively transfer the solution into a 1 litre volumetric flask, make up to volume with water, homogenize and filter. Retrieved reagent should not have color verduzca. Before use, dilute 1 volume of the reagent with two volumes of water.


19.3.7 haemoglobin solution: weigh a quantity of haemoglobin, a protein substrate according to Anson (2 g approximately) corresponding to 354 mg of nitrogen and place in a flask of 200 ml of ground closure. Determine the content of nitrogen through a semi-microkjeldahl (theoretical content: 17.7 per 100 of nitrogen). Add a few ml of acid hydrochloric (19.3.2) connect the flask to the vacuum pump and shake until complete dissolution of hemoglobin. Stop making vacuum and add, stirring constantly, HCL (19.3.2) to complete to 100 ml. prepare immediately before use.


19.3.8. standard tyrosine solution: dissolve 181,2 mg of tyrosine in the hydrochloric acid (19.3.2) and make up to 1 litre with the same acid hydrochloric.


19.3.9. working standard solution: take 20.0 ml of the solution (19.3.8) and dilute to 100 ml with hydrochloric acid (19.3.1). 1 ml of this solution contain 0.2 micromoles of tyrosine.


19.4 procedure.



19.4.1. preparation of the solution (see 19.6.1). Dissolve 150 mg of pepsin in 100 ml of hydrochloric acid (19.3.2). Take pipette 2 ml of the solution through, place it in a 50 ml graduated flask and make up to volume with hydrochloric acid (19.3.3). PH, controlled by the pH-meter, must be 1.6 ± 0.1. Maintain the flask in the water bath at 25 ° C (19.2.1).


19.4.2 hydrolysis. Into by pipette 5,0 ml test tube solution of hemoglobin (19.3.7) bring 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 a glass rod widened at one end, using approximately 10 moves back and forth. Keep the test tube in the water bath at 25 ° C for 10 minutes exactly, counted from the time of the addition of the pepsin solution (duration and temperature must be fully respected). Then add 10.0 ml of trichloroacetic acid (19.3.4) previously carried at 25 ° C. Homogenize and filter through a dry filter.


19.4.3 development of coloration and measurement of absorbance. Take by pipette 5,0 ml of the filtrate, place it in a 50 ml Erlenmeyer flask, add 10.0 ml of (19.3.5) sodium hydroxide solution, stirring continuously, 3.0 ml of dilute Folin-ciocalteu reagent (19.3.6). After 5 to 10 minutes, determine the absorbance of the solution at 750 nm in cells 1 cm thick, using as white water.


19.4.4. blank test. For each determination, proceed with a test blank as shown below. Enter using pipette 5,0 ml test tube 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 carried at 25 ° C, mix thoroughly and then add 1.0 ml of the pepsin obtained in 19.4.1 solution. Mix with a glass rod support and keep the test tube exactly ten minutes in bath water (19.2.1) to 25ºc. Homogenize and filter through a dry filter.


Continue the operating method as described in 19.4.3.


19.4.5 curve of calibration. Insert into 50 volumes of 1.0 ml Erlenmeyer; 2.0; 3.0; 4.0; 5.0 ml of standard tyrosine solution (19.3.9) respectively corresponding to amounts of tyrosine 0.2; 0.4; 0.6; 0.8; and 1.0 micromoles. Complete the series by a tyrosine-free control. Take the volumes to 5.0 ml with hydrochloric acid (19.3.1) support. Add 10.0 ml of sodium (19.3.5) hydroxide solution. and, shaking constantly, 3.0 ml of the diluted reagent Folin-ciocalteu reagent (19.3.6). Measure the absorbance as indicated in the last sentence of point (19.4.3). Plot the curve of calibrated by placing the concentrations of tyrosine against the absorbances obtained.


19.5 calculations. Read about the curve of calibrated amount of tyrosine in micromoles absorbance of the colored, corrected of the blank test solution.


The activity of the pepsin in micromoles of tyrosine, mg and per minute at 25 ° C, is given by the following formula: units per mg (U/mg) = 0.32 · to P being: to = quantity of tyrosine in micromoles read on the standard curve.


P = weight in mg of the quantity of pepsin added in 19.4.2.


19.6 observations.


19.6.1. the quantity of pepsin that need to be put in the solution must be such that to obtain a final absorbance of 0.35 ± 0.035.


19.6.2. two units per mg obtained by this method correspond to 3.64 millionth units Anson/mg (micromoles of tyrosine/mg min at 35.5 C) or 36,400 commercial units / g, (micromole of tyrosine/g in 10 minutes at 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 20.1 total casein principle. It is based on precipitate (after reconstituted milk in warm water), the casein to their isoelectric point. Per wash contains pure casein eliminating serum and impurities. Via Kjeldahl determines its level of protein N x 6.38 and this value deducted the corresponding target is expressed as casein.


This method is applicable to raw milk (whole, low-fat or nonfat milk powder).


20.2 material and devices.


20.2.1. material needed for determination of crude protein.


20.2.2. water bath with thermostat to 40 ° C.


20.2.3 centrifuge capable of reaching 4,000 rpm.


20.3 reagents.


20.3.1. necessary reagents for determination of crude protein.


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


20.3.3. dilution of 1N sodium acetate.


20.3.4 number 240 Albet filter paper or similar.


20.4 procedure. Weigh 5 g of the sample, with a precision of 1 mg. dissolve in 100 ml of deionized water heated to 40-45 ° C. Centrifuge at 4000 rpm 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 that you precipitate during 10 minutes.


Add 5 ml of sodium (20.3.3) acetate solution and wait 10 minutes to precipitate casein evolves and is bind. Filter through filter (20.3.4) paper.


Wash with water acidulated with a few drops of acetic acid to remove the rest of milk soluble proteins or not, until a curdled pure casein.


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


20.5 calculation. The casein content will be given in percentage of weight according to: casein % = N x 6.38 being: N = total nitrogen of the precipitate, expressed as a percentage of the sample.


20.6 observations. Washing the curd should be acidified water and with great care, on one side so no they remaining in her serum, other proteins so as not lose casein by tampering.


20.7 references. Officers of milk analysis methods. Method number 3.


21 urea 21.1 principle. Clarification of the sample and measure the absorbance at 420 nm of the compound formed by the addition of p-dimethylaminobenzaldehyde.


21.2 material and devices.


21.2.1 mixer or stirrer able to do 35 to 40 turns per minute.


21.2.2 test tubes of approximately 160 x 16 mm with screw caps.


21.2.3. spectrophotometer.


21.3 reagents.


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


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


21.3.2 above solution Carrez I.


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


21.3.3 solution Carrez II.


Dissolve 10.6 g of hexacyanoferrate (II) trihydrate potassium (KFe (CN) 3HO ·) in distilled water and make up to 100 ml.


21.3.4. active carbon that does not absorb urea.


21.3.5. standard of urea solution to the 0.1 per 100 (w/v).


21.4 procedure.


21.4.1 curve of calibration. Take volumes 1, 2, 4, 5, and 10 ml of the solution pattern of urea (21.3.5) 100 ml volumetric flasks and make up to volume with distilled water. Take 5 ml of each solution, carry tubes (21.2.2) and add 5 ml of the DMAB (21.3.1) respectively. Mix and put tubes in a water bath at 20 ° C for 15 minutes. Measure the absorbance of each solution at 420 nm against a blank retrieved by taking 5 ml of distilled water and following the same procedure. Obtain the curve of calibration.


21.4.2. preparation of the sample. Weigh to approximation of 1 mg, 2 g of the sample and place it in a 500-ml graduated flask and add 1 g of active carbon (21.3.4). Add 400 ml of distilled water, 5 ml of Carrez solution I and 5 ml of Carrez II solution. Place the flask on the shaker (21.2.1) for 30 minutes. Make up to volume with distilled water, stir and filter.


21.4.3 determination. Take 5 ml of the filtrate and carry tubes (21.2.2). Add 5 ml of the DMAB (21.3.1) and continue as in (21.4.1).


21.5 calculations. Determine the content of urea when comparing the absorbances obtained against the curve of calibration. Express the result as a percentage of the sample.


21.6 references. First Commission directive of 15 June 1971 (71/250/EEC). «Official Journal of the European communities» number L 155, 12 July 1973.


22 22.1 hydrocyanic acid principle. The sample is suspended in water. The hydrocyanic acid is released under the action of enzymes, creeps by distillation into water vapor and is collected in a given volume of acidified silver nitrate solution. Silver cyanide is separated by filtration and excess silver nitrate is titrated with a solution of ammonium thiocyanate.


The method enables you to determine hydrocyanic acid free and combined in the form of glycosides in certain feedingstuffs and, in particular, products of flax seed, of flour of cassava and legume species.


22.2 material and devices.


22.2.1 stove provided with a thermostat set at 38 ° C.


22.2.2 distillation apparatus in current of water vapour provided a refrigerant with curved extension.


22.2.3 stoppered 100 ml flat-bottomed flasks.


22.2.4. oil bath.


22.2.5. burette graduated in 1/20 ml.


22.3 reagents.


22.3.1 suspension of Sweet Almonds: crush 20 almonds candy 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 paper picro-sodium or carrying out a blank test as described in the last paragraph of 22.4.



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


22.3.3 emulsion of anti-foam (silicone, for example).


22.3.4 nitric acid, d = 1.40.


22.3.5. solution of nitrate of silver 0, 02N.


22.3.6 0 ammonium thiocyanate solution, 02N.


22.3.7 saturated solution of ammonium ferric sulphate.


22.3.8 ammonia d = 0.958.


22.4 procedure. Weigh, with precision of 5 mg, 20 g of the sample, place in a flask of 1 litre flat-bottomed and add 50 ml of water and 10 ml of suspension of almond sweet (22.3.1). Cover the flask and keep it for sixteen hours in the oven at 38 ° C. Then cool to room temperature and add 8 ml of water and 10 ml of solution of sodium acetate (22.3.2) and a drop of emulsion anti-foam (22.3.3).


Connect the flask to the steam distillation apparatus and place it in a bath of oil previously brought to a temperature slightly above 100 ° C. Distil 200 to 300 ml of liquid by passing a strong current of steam into the flask and gently heating the oil bath. Collect the distillate in an Erlenmeyer flask located protected from light and contains exactly 50 ml solution of silver nitrate 0, 02N (22.3.5) and 1 ml of nitric acid (22.3.4). Make sure that the extension of the refrigerant is submerged in the solution of silver nitrate.


Quantitatively transfer the contents of the Erlenmeyer flask to a 500 ml graduated flask, make up to volume with water, stir and filter. Take 250 ml of the filtrate, add 1 ml approximately of ferric sulfate solution ammonium (22.3.7) and rating in reverse the excess silver nitrate with the solution of ammonium thiocyanate 0, 02N (22.3.6.) supplied by the burette graduated in 1/20 ml.


Be in the case that required a blank test applying the same operative method to 10 ml of suspension of almond sweet (22.3.1), in the absence of the sample to analyze.


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


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


22.6 observations. If the sample contains a significant amount of sulphides (e.g., beans), a black precipitate of silver sulfide seeping is formed with the sediment of cyanide of silver. The formation of this precipitate entails a loss of solution of silver nitrate 0, 02N whose volume must subtract the volume taken into consideration for the calculation of the hydrocyanic acid content. To this end, proceed as indicated below: treat the sediment on the filter with 50 ml of ammonia (22.3.8) to dissolve the silver cyanide. Wash the residue in dilute ammonia and proceed to the determination of its content in silver. Convert the value obtained into ml of 0 silver nitrate solution, 02N.


Hydrocyanic acid of the sample can be determined also 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, 12 July.


23 determination of ash insoluble in hydrochloric acid 23.1 principle. The method enables you to determine the content of mineral substances insoluble in hydrochloric acid of feed and its raw materials. Will provide for two procedures depending on the nature of the sample.


23.1.1. procedure a applicable organic raw materials and most of the compound feedingstuffs.


23.1.2 procedure B. mineral mixtures, as well as compound feedingstuffs whose content of insoluble hydrochloric, determined according to the procedure A, greater than 1 per 100.


23(1)(3) procedure to. Sample, is ash the ashes are treated by boiling hydrochloric acid and the insoluble residue filtered and weighed.


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


23.2 material and devices.


23.2.1. heating plate.


23.2.2. electric oven, with thermostat.


23.2.3 crucibles of incineration of Platinum or alloy of Platinum and gold (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 other material crucibles can withstand a temperature of 700 ° C.


23.3 reagents.


23.3.1 3N hydrochloric acid.


23.3.2 20 per 100 solution (w/v) of trichloroacetic acid.


23.3.3 solution 1 for 100 (w/v) of trichloroacetic acid.


23.4 procedure.


23.4.1 procedure a. incinerated the sample according to the official method described for the determination of crude ash. The obtained ash be used equally to that determination. Transfer the ash to a 250 to 400 ml beaker with 75 ml of hydrochloric acid 3N (23.3.1). Carefully bring liquid to a soft boil and keep it for 15 minutes. Filter the solution hot on an ashless filter paper and wash the residue with water hot until the disappearance of acid reaction. Dry the filter containing the residue and ash in a crucible set to a temperature of 550 ° C minimum and 700 ° C maximum. Cool in the desiccator and weigh.


23.4.2 procedure B. weigh to the nearest 1 mg, 5 g of the sample and pour them into a beaker of 250 to 400 ml. Add successively 25 ml of water and 25 ml of hydrochloric acid 3N (23.3.1), mix and wait at the end of the effervescence.


Add 50 ml of hydrochloric acid 3N (23.3.1). If there is a new release of gases, waiting for their final and then place the beaker in a boiling water bath and keep it there for thirty minutes or more, if necessary, in order to completely hydrolyze starch that may be present. Filter in hot on ashless filter and wash the filter with 50 ml of hot water (23.7), place the filter containing the residue in a crucible of incineration, dry and incinerated at a temperature of 550 ° C minimum and 700 ° C maximum. Continue as described in 23.4.1, second paragraph.


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 observations. If filtering is difficult, return to begin the definition by replacing the 50 ml of hydrochloric acid 3N per 50 ml of trichloroacetic acid to 20 per 100 (23.3.2) and washing the filter with the help of a hot solution of trichloroacetic acid to 1 per 100 (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, 12 July 1971.


24 24.1 mustard essence principle. The sample is suspended in water. The essence of mustard are released under the action of enzymes, crawl by distillation in the presence of ethanol and collected in dilute ammonia. The solution is hot with a certain volume of silver nitrate solution, cooled and filtered. Excess silver nitrate is titrated with a solution of ammonium thiocyanate.


The method enables you to determine content in essence of mustard draggable by water vapor, expressed as allyl isothiocyanate, of Brassica and Sinapis species and cake of compound feedingstuffs that contain it.


24.2 material and devices.


24.2.1. 500 ml flat-bottomed and stoppered flasks.


24.2.2 distillation apparatus fitted with a condenser and a device allowing to avoid liquid carryover.


24.3 reagents.


24.3.1 white mustard (Sinapis alba).


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


24.3.3 emulsion of anti-foam (silicone, for example).


24.3.4. ammonia, d = 0.958.


24.3.5 solution of nitrate of silver 0, 1N.


24.3.6 0 ammonium thiocyanate solution, 1N.


24.3.7 nitric acid, d = 1.40.


24.3.8 saturated solution of ammonium ferric sulphate.


24.4 procedure. Weigh, with precision of 1 mg, 10 g of the sample, place in a 500 ml flat-bottomed flask and add 2 g of white mustard finally crushed (source of ferment) (24.3.1) and 200 ml of water at 20 ° C. Cover the flask and keep it for two hours approximately 20 ° c by shaking it frequently. Add 40 ml of ethanol (24.3.2) and a drop of emulsion anti-foam (24.3.3). Distil about 150 ml and collect the distillate in a 250 ml flask containing 20 ml of ammonia (24.3.4) paying attention that the extremity of the refrigerant are immersed in the liquid. Add to the ammoniacal solution 50 ml of solution of nitrate of silver 0, 1N (24.3.5) (or more if necessary), place a small funnel over the volumetric flask and heat the mixture for one hour in a boiling water bath. Leave to cool, make up to volume with water, shake and filter. Take 100 ml of clear filtrate, add 5 ml of nitric acid (24.3.7) and 5 ml of solution of sulphate ferric ammonium (24.3.8). Titrate the excess silver nitrate with the solution of 0, 1N (24.3.6) ammonium thiocyanate.


Carry out a blank test applying the same method to 2 g finally crushed white mustard.



24.5 calculations. Subtract the volume of silver nitrate 0, 1N consumed in the blank test of the consumed by the sample solution. The value obtained gives the number of ml of 0, 1N consumed silver nitrate solution by the essence of mustard of the sample analyzed. 1 ml of nitrate of silver 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, 12 July 1971.


25 lactose 25.1 principle. The method enables you to determine the lactose content of feedingstuffs containing more than 0.5 per 100 of this product.


Sugars are dissolved in the water. The solution is subjected to fermentation by the Yeast Saccharomyces cerevisiae, which leaves the lactose intact. Previous clarification and filtration the lactose of filtering content is determined by the Luff-Schoorl method.


25.2 material and devices. Water bath fitted with thermostat, regulated from 38 to 40 ° C.


25.3 reagents.


25.3.1 suspension of Saccharomyces cerevisiae. To suspend 25 g of fresh yeast in 100 ml of water. Suspension a week maximum is preserved in the refrigerator.


25.3.2 Carrez I solution. dissolve in water 24 g of zinc acetate dihydrate Zn (CHOOO) · 2HO and 3 ml of glacial acetic acid. Make up to 100 ml with water.


25.3.3 solution Carrez II. Dissolve in water 10.6 g of hexacyanoferrate (II) potassium KFe (CN) · 3HO. make up to 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. solution of sodium carbonate. Dissolve 143.8 g of anhydrous soda ash in 300 ml approximately of water hot. Leave to cool and make up to 300 ml.


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


25.3.7. Luff - Schoorl reagent. Carefully pour the citric acid solution (25.3.4) on the solution of soda ash (25.3.5). Then add the copper sulphate solution (25.3.6) and make up to 1 litre with water. Let stand overnight and filter. Check the normality of the reagent retrieved (Cu 0.1 N;) NaCO 2N). The solution's pH should be approximately 9.4.


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


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


25.3.10 6N sulfuric acid solution.


25.3.11 0 Sodium Thiosulphate solution, 1N.


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


25.4 procedure. Weigh to the nearest 1 mg, 1 gram of sample into a volumetric flask of 100 ml. Add 25 to 30 ml of water. Place the flask for 30 minutes in a boiling water bath and cool to 35 ° C approximately. Add 5 ml of yeast suspension (25.3.1) and mix thoroughly. Leave the flask to stand for two hours in a water bath, the temperature of 38 to 40 ° C. Then refrigerate up to 20 ° C approximately.


Add 2.5 ml of Carrez I (25.3.2) and stir for thirty seconds, add 2.5 ml of Carrez II and shake, complete with 100 ml water, mix and filter. Take pipette a volume of the filtrate which does not exceed 25 ml and containing between 40 to 80 mg of lactose and fit in a 300 ml conical flask. If it is necessary to make up to 25 ml with water.


Carry out a blank test containing 5 ml of yeast suspension in the same way.


Add 25 ml exactly, the Luff-Schoorl reagent (25.3.7) two granules of pumice (25.2.8). Heat, waving hand, on one called free half-height and bring the liquid to a boil for two minutes approximately, immediately place the Erlenmeyer flask on a metallic fabric fitted with a screen of asbestos with a 6 cm diameter hole, under which a flame is lit previously. This is regulated in such a way that just warms the bottom of the Erlenmeyer flask. Then adapt a refrigerant to reflux over the Erlenmeyer flask. From now on, make boil for ten minutes exactly, cool immediately in cold water and after approximately five minutes, rating as follows: add 10 ml of solution (25.3.9) potassium iodide and immediately after and carefully (because of the risk of abundant foaming) 25 ml of sulphuric acid 6N (25.2.10). Rating below with the solution of Sodium Thiosulphate 0, 1N (25.3.11) until the appearance of a light yellow colour, add the indicator (25.3.12) and complete the assessment.


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


25.5 calculations. Using the attached table establish the amount of lactose in mg which corresponds to the difference between the results of two evaluations expressed in ml of 0 sodium thiosulfate, 1N.


Express the results in parts of lactose anhydrous percent each sample.


25.6 observations.


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


25.6.2 perform alongside an essay with a known amount of lactose monohydrate.


Table of values for 25 ml of Luff-Schoorl reagent, Na2S2O30, 1N: ml / glucose fructose C6H12O6 inverted sugars: 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, 12 July 1971.


26 potassium 26.1 principle. Sample ash and ashes are collected in the hydrochloric acid solution. The content of potassium of the solution is determined by flame photometry in the presence of aluminium nitrate and cesium chloride. The addition of such substances eliminated, to a wide extent, the interference of disturbing elements.


The method enables you to determine the content of potassium in feeds.


26.2 material and devices.


26.2.1 crucibles of incineration of Platinum, quartz or porcelain, if provided with lids.


26.2.2 oven with a thermostat.


26.2.3. flame photometer.


26.3 reagents.


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


26.3.2 p.a. caesium chloride


26.3.3 nitrate aluminum nono-hidrato to the (non-) · Chemically pure 9HO.


26.3.4 p.a. anhydrous potassium chloride.


26.3.5. buffer solution. Dissolve in water 50 g of caesium chloride (26.3.2) and 250 g of aluminium (26.3.3) nitrate, make up to 1 litre with water and homogenise. Store in plastic bottles.


26.3.6 standard solution of potassium. Dissolve in water 1,907 g of chloride of potassium (26.3.4), add 5 ml of hydrochloric acid (26.3.1), make up to 1 litre with water and homogenise. Store in plastic bottles. 1 ml of this solution contains 1,00 mg of potassium.


26.4 procedure.


26.4.1 determination. Weigh accurately 10 mg, about 10 g of the sample into a crucible of incineration and ash at 450 ºc for three hours. After cooling, quantitatively transfer residue incineration with the help 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 have ceased the possible release of carbon dioxide, heat the solution and keep it for two hours at a temperature close to 90 ° C, shaking from time to time. Let cool to room temperature, make up to volume, shake and filter. Insert into a 100 ml graduated flask an aliquot part of the filtrate containing at least 1.0 mg of potassium, add 10.0 ml of buffer (26.3.5) make up to volume with water and mix thoroughly. For high potassium content, dilute the solution to be analyzed in the proper proportion, prior to the addition of the buffer solution. The table below is given as an indication for a test sample of 10 g approximately.


Content in potassium percentage K sample course / dilution Factor / aliquot part of the solution up to 0.1 ml /-/ 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 measure by flame photometry at a wavelength of 768 nm. Calculate the result using the curve of calibration.



26.4.2 curve of calibration. Introduce 10 ml exactly the solution pattern (26.3.6) in a 250 ml graduated flask, make up to volume with water and mix thoroughly. Place in 100 ml graduated flasks exactly: 0, 5, 10, 15, 20 and 25 ml of this solution, corresponding respectively to amounts of potassium of 0; 0.2; 0.4; 0.6; 0.8; and 1.0 mg. Add to each flask 10.0 ml of buffer (26.3.5) make up to volume with water and mix thoroughly. Taking the measurements as described in 26.4.1. The path of the curve of calibration is generally linear up to a concentration in potassium of 1 mg in 100 ml of solution.


26.5 calculations. The potassium content is expressed in percentage of sample.


26.6 observations. The addition of buffer (26.3.5) to eliminate the interference of disturbing 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, 12 July 1971.


27 sodium 27.1 principle. Sample ash and ashes are collected in the hydrochloric acid solution. The sodium content of the solution is determined by flame photometry in the presence of aluminium nitrate and cesium chloride. The addition of these substances eliminated, to a wide extent, the interference of disturbing elements.


Method enables you to determine the content of sodium in feedingstuffs.


27.2 material and devices.


27.2.1 crucibles of incineration of Platinum, quartz or porcelain, if provided with lids.


27.2.2. electric oven, with thermostat.


27.2.3. flame photometer.


27.3 reagents.


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


27.3.2. caesium chloride, p.a.


27.3.3 nitrate aluminum nonahydrate, to the (non-) · 9HO, chemically pure.


27.3.4 p.a., anhydrous sodium chloride.


27.3.5. buffer solution. Dissolve in water 50 g of caesium chloride (27.3.2) and 250 g of aluminium nitrate (27.3.3) make up to 1 litre with water and homogenise. Store in plastic bottles.


27.3.6. standard sodium solution. Dissolve in water 2,542 g of sodium chloride (27.3.4) by adding 5 ml of hydrochloric acid (27.3.1), make up to 1 litre with water and homogenise. Store in plastic bottles. 1 ml of this solution contains 1,00 mg of sodium.


27.4 procedure.


27.4.1 determination. Weigh, to the nearest 10 mg, about 10 g of the sample into a crucible of incineration (27.2.1) and ash at 450 ºc for three hours. Avoid inflammation and projection. After cooling, transfer quantitatively the residue of incineration in 250-300 ml of water, and then 50 ml of hydrochloric acid (27.3.1) a 500-ml graduated flask.


After have ceased the possible release of carbon dioxide, heat the solution and keep it for two hours at a temperature close to 90 ° C, shaking from time to time. Let cool to room temperature, make up to volume with water, shake and filter. Enter into a 100 ml graduated flask an aliquot part of the filtrate containing 1.0 mg of sodium, add 10.0 ml of buffer (27.3.5), make up to volume with water and mix thoroughly. For higher contents of sodium, dilute the solution to be analyzed in the proper proportion, prior to the addition of the buffer solution. The following table is given for information purposes, a test sample of 10 g for approximately.


Content course of sample solution percentage Na / dilution Factor / aliquot part of the solution up to 0.1 ml /-/ 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 carry out the measure by flame photometry at a wavelength of 589 nm.


Calculate the result using the curve of calibration.


27.4.2 curve of calibration. Enter exactly 10 ml of the solution pattern (27.3.6) in a 250 ml graduated flask, make up to volume with water and mix. In 100 ml graduated flasks, introduce exactly 0, 5, 10, 15, 20 and 25 ml of this solution, corresponding respectively to amounts of sodium of 0; 0.2; 0.4; 0.6; 0.8; and 1.0 mg. Add to each flask 10.0 ml of buffer (27.3.5) make up to volume with water and mix thoroughly. Taking the measurements as indicated in 27.4.1. The path of the curve of calibration is generally linear up to a concentration of sodium 1 mg in 100 ml of solution.


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


27.6 observations.


27.6.1. for products with sodium content greater than the 4 by 100, it is preferable to incinerate the substance for two hours in a crucible with a lid. After cooling, add water, a waste to suspend with the help of a thread of Platinum, dry and ash again for two hours in the crucible with its lid.


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


27.7 references. First Commission directive of 15 June 1971 (71/250/EEC). «Official Journal of the European communities» number L 155, 12 July 1971.


28 magnesium 28.1 principle. Sample incineration and treatment of the ashes with dilute hydrochloric acid. If the sample contains no organic substances, dissolves directly in hydrochloric acid. The solution is diluted and the magnesium content determined by spectrophotometry of atomic absorption at 285,2 nm, by comparison with solutions of calibration.


It is particularly suitable for determining magnesium contents of less than 5 per 100 in feedingstuffs.


28.2 material and devices.


28.2.1. incineration of Platinum, quartz or porcelain crucibles.


28.2.2 oven or muffle on thermostat.


28.3 reagents.


28.3.1 hydrochloric acid d = 1.16, p.a.


28.3.2 hydrochloric acid d = 1.19, p.a.


28.3.3 magnesium Ribbon, yarn or heptahydrate magnesium sulfate, p.a. (Mg SO7HO) dried in vacuum temperature environment.


28.3.4 solution (chloride or nitrate) strontium salt p.a. to 2.5 per 100 (w/v) strontium, p.a. (76,08 g of ClSr 6 HO/litre, or 60,38 g (NO) Sr/litre).


28.3.5 solution of calibrated magnesium: weighing with accuracy of 1 mg, 1 g of magnesium (28.3.3), separating previously and carefully the oxide film, or a corresponding amount of magnesium (28.3.3) sulphate. Place it in a flask of 1000 ml add 80 ml of HCL (28.3.1) dissolve and make up to 1000 ml with deionized HO. 1 ml of this solution contains 1000 mg of magnesium.


28.4 procedure.


28.4.1. preparation of the sample.


(28.4.1.1 feed consisting exclusively of mineral substances: to) weigh with precision of 1 mg, 5 g of the sample and place in a 500 ml flask with 250-300 ml of deionized water. Add 40 ml of hydrochloric acid (28.3.1), bring to a slow boil for 30 minutes. Cool, make up to volume with deionized water, mix and filter into a beaker with folded filter. Delete the first 30 ml of the filtrate.


(b) in the presence of silica, treat 5 g of the sample with one sufficient (between 15-30 ml) of hydrochloric acid (28.3.2) and evaporate to dryness on a water bath. Continue such and shown in (28.4.1.2) from the third phase.


28.4.1.2 feed consisting essentially of minerals. Weigh to 1 mg, 5 g of the sample into a crucible and ash at 550 ºc in the muffle furnace until ashes free from carbonaceous particles is obtained.


To remove silica add to ashes enough (between 15-30 ml) of hydrochloric acid (28.3.2) and evaporate to dryness on a water bath. Dry then one hour in the oven at 105 ° C. Pick up residue with 10 ml of hydrochloric acid (28.3.1) and transfer with the help of hot deionized water into a graduated flask of 500 ml. bring to a boil, allow to cool and make up to volume with deionized water. Homogenize and filter through a pleated filter in glass. Delete the first 30 ml of the filtrate.


28.4.1.3 feed consisting essentially of organic substances. Weigh to 1 mg, 5 g of the sample into a crucible (28.2.1) and ash in a muffle oven until ashes free from carbonaceous particles is obtained. Treat the ash with 5 ml hydrochloric acid (28.3.2), evaporate to dryness on a water bath and then dry for one hour in an oven at 105 ° C for insolubilizar the silica.


Dissolve the residue with 5 ml of hydrochloric acid (28.3.1), transfer quantitatively using hot deionized water to a volumetric flask of 250 ml. bring to a boil, allow to cool and make up to volume with deionized water. Homogenize and filter through a folded filter into a glass. Delete the first 30 ml of the filtrate.


28.4.2. measurement of atomic absorption.


28.4.2.1 curve of calibration. Prepare, diluting the calibration (28.3.5) solution with deionized water, at least five solutions references of increased concentrations, chosen on the basis of the area of optimal measuring range of the spectrophotometer. Add to each solution 10 ml strontium salt solution (28.3.4) and make up to 100 ml with de-ionised water volume.


28.4.2.2 sample preparation. Dilute an aliquot part of the filtrate obtained in 28.4.1.1, 28.4.1.2 and 28.4.1.3 so as to obtain a concentration in magnesium in the concentration limits of the reference solutions with deionized water. The concentration of hydrochloric acid of this solution must not exceed 0, 4N. Add 10 ml of the solution of salt of strontium (28.3.4) and make up to the volume of 100 ml with distilled water.


Measure the absorption of the test solution and the reference solutions at a wavelength of 285,2 nm.



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


28.6 observations. The difference between the results of two parallel determinations carried out on the same sample should 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 March 30, 1973.


29 iron 29.1 principle. Determination of the trace element iron in feedingstuffs and raw materials. The lower limit of determination is 20 mg/kg. The sample is dissolved in hydrochloric acid solution, previous destruction of organic matter. The iron is determined, diluted proper, by atomic absorption spectrophotometry.


29.2 material and devices.


29.2.1 oven muffle, adjustable temperature.


29.2.2 resistant borosilicate glass material. He is recommended to use a material that serves exclusively for the dosage of the trace element.


29.2.3 capsules Platinum and, eventually, Quartz.


29.2.4 atomic absorption spectrophotometer, which responds to the demands of method, with regard to the sensitivity and the required accuracy within the limits of the utility that is used.


29.3 reagents.


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


29.3.2 6N hydrochloric acid.


29.3.3 hydrochloric acid 0, 5N.


29.3.4 hydrofluoric acid 38-40 per 100 (v/v), with a content of less than 1 mg/l iron and whose residue of evaporation is less than 10 mg/l (as sulphate).


29.3.5 p.a. sulphuric acid (d = 1,84).


29.3.6. oxygenated water of some 100 volumes of oxygen (30 per 100 by weight).


29.3.7 iron (1,000 micrograms of iron/ml) standard solution: dissolve 1 g of iron wire p.a., in 200 ml of 6N hydrochloric acid (29.3.2) add 16 ml of hydrogen peroxide (29.3.6) and make up to 1 litre with water.


29.3.8. (100 micrograms of iron/ml) working standard solution: take 10 ml of the solution (29.3.7) and make up to 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 make up to 1 litre with water.


29.4 procedure.


29.4.1 displays containing organic compounds.


29.4.1.1 incineration and preparation of the sample to be analyzed.


(i) place 5 to 10 g of the sample heavy with an accuracy of 0.2 mg in a quartz or Platinum (29.2.3) capsule (see 29.6.6), dry in an oven at 105 ºc and introduce the capsule in the oven muffle (29.2.1) cold. Close the oven (see 29.6.7) and gradually raise the temperature to reach 450 to 475 ºc in ninety minutes approximately. Maintain this temperature for four to six hours (e.g. overnight) to remove carbonaceous matter, then open the oven and leave to cool (see 29.6.8).


Moisten the ash with water, transfer them to a beaker of 250 ml. Rinse the cartridge with 5 ml of hydrochloric acid (29.3.1) and transfer, slowly and carefully, rinsing the beaker of solution (may cause a violent reaction due to carbon dioxide formation). Then add Dropwise hydrochloric acid (29.3.1), waving at the same time the contents of the beaker until the fizz stops. Evaporate to dryness periodically, stirring with a glass rod.


Add 15 ml of 6N hydrochloric acid (29.3.2) and approximately 120 ml of water to the residue. Mix using a glass rod, leave it in the beaker, and cover with a watch glass. Put liquid in soft boiling and keep boiling until apparently already ashes do not dissolve. Filter using an ashless filter paper and collect the filtrate in a volumetric flask of 250 ml. wash the beaker and filter with 5 ml of hydrochloric acid (29.3.2) hot 6N and twice with boiling water. Complete with water up to make up (the concentration of hydrochloric acid will be 0, 5N approximately).


(ii) if the residue that is left in the filter appears black (carbon), place it back in the oven and incinerate 450 to 475 ºc. The incineration, which will only require a few hours (three to five hours approximately), will be completed when the ashes are white or almost white. Dissolve the residue in approximately 2 ml of hydrochloric acid (29.3.1). Evaporate to dryness and add 5 ml of 6N HCL (29.3.2) acid. Heat, filter the solution into the volumetric flask and fill with water make up (the concentration of hydrochloric acid will be 0, 5N approximately).


29.4.1.2 preparation of the solutions pattern. Prepare a range of solutions from the solution pattern work (29.3.8) pattern so each standard solution has a concentration in hydrochloric acid 0, 5N approximately and a concentration of lanthanum chloride that corresponds to 0.1 per 100 of lanthanum (w/v) (29.3.9). The trace element concentrations selected must be in the area of sensitivity of the spectrophotometer used. The following table will give, for example, the types of composition of standard solution; According to the type and sensitivity of the spectrophotometer used, it may be necessary to select other concentrations: iron Fe/ml xg / 0 / 0.5 / 1 / 2 / 3 / 4 / 5 ml of standard solution of work (29.3.8). / 0 / 0.5 / 1 / 2 / 3 / 4 / 5 (1 ml = 100 Fe xg) + ml HCL 6N (29.3.2). / 7 / 7 / 7 / 7 / 7 / 7 / 7 + 10 ml of lanthanum chloride (29.3.9) complete with water to 100 ml if the sample had a weight ratio Ca + Mg - T 2, the addition of lanthanum (29.3.9) chloride solution P may be omitted.


29.4.1.2.2. preparation of the solution to be analysed. Introduce pipette an aliquot portion of the solution prepared according to (29.4.1.1) in a 100 ml volumetric flask; Add 10 ml of lanthanum (29.3.9) chloride solution. Complete with hydrochloric acid 0, 5N (29.3.3).


If the sample had a weight ratio Ca + Mg - T 2, the addition of lanthanum (29.3.9) chloride solution P may be omitted.


29.4.1.2.3. blank test. Carry out a blank test covering all the stages prescribed operating procedure, without the presence of the sample. Pattern 0 do not use the solution for the blank test.


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


29.4.2 mineral compounds. In the absence of organic matter, it will be useless after incineration. Apply the operating procedure from the 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 analyzed by a curve of calibration and express the result in milligrams of trace element per kg of sample (ppm).


The difference between the results of two parallel determinations carried out on the same sample by the same analyst should not exceed:-5 mg/kg, in absolute value, for contents in the trace element up to 50 mg/kg.


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


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


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


29.6 observations.


29.6.1. the water used for the preparation of reagents and solutions required for analysis must be exempt from the cation to determine. Either by double distillation in a borosilicate or quartz apparatus, is obtained either by double permutation in ion-exchange resin.


29.6.2 reagents shall be, at least, quality analysis (p.a.). The absence of the element to be determined, should be checked by a blank test. If necessary, reagents will undergo a more thorough purification.


29.6.3. you can substitute solutions pattern described by solutions commercial pattern provided that the same are guaranteed and controlled before use.


29.6.4 green fodder (fresh or dried) may contain large amounts of vegetable silica that can hold the trace element and which should be removed. Samples of such feedingstuffs shall undergo the following treatment: carry out the operation (29.4.1.1) until the phase of filtration. Wash the filter paper containing the insoluble residue twice with boiling water and place it in a capsule of Platinum (29.2.3). Incinerate (29.2.1) muffle oven at a temperature below 550 ºc until all carbonaceous matter has disappeared completely. Let cool, add a few drops of water and 10 to 15 ml of hydrofluoric acid (29.3.4). Evaporate to dryness at 150 ° C approximately. If the waste contains even silica, dissolve it in several millilitres of hydrofluoric acid (29.3.4) and evaporate to dryness. Add five drops of sulphuric acid (29.3.5) and heat until the disappearance of white fumes. Add 5 ml of 6N hydrochloric acid (29.3.2) and 30 ml approximately of water, heat, filter the solution into a 250 ml graduated flask and make up to make up (the concentration of hydrochloric acid will be 0, 5N approximately). Continue the procedure starting from point (29.4.1.2).


29.6.5 determining the trace element will be opportune to draw attention in terms of the risk of contamination. Therefore, the instruments used for the preparation of samples must be exempt from the metal.



To reduce the risk of contamination should be working in dust-free atmospheres, with a rigorously clean material and carefully washed glass apparatus.


29.6.6 calculate the weight of the sample as a function of the content in the feed and the trace element to assess the sensitivity of the spectrophotometer used. For certain the trace element-poor feed 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 incinerated in a furnace closed 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 July 29, 1978.


30 copper 30.1 principle. Determination of the trace element copper in feed and its raw materials. The lower limit of determination is 10 mg/kg. The sample is dissolved in hydrochloric acid solution, previous destruction of organic matter. The copper is determined, diluted proper, by atomic absorption spectrometry.


30.2 material and devices. Ditto 29.2.


30.3 reagents. Ditto 29.3. until 29.3.6. inclusive.


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


30.3.8. standard of work (10 micrograms of copper/ml) solution. Take 10 ml of the standard solution (30.3.7) and make up to 1,000 ml with water.


30.4 procedure. Ditto 29.4. up to 29.4.1.2.


30.4.1.2. Spectrophotometric determination.


30.4.1.2.1 preparation of the solutions pattern from the work (30.3.8) in a way that each pattern solution solution has a concentration of hydrochloric acid 0, 5N approximately. The trace element concentrations selected must be in the area of sensitivity of the spectrophotometer used.


The following table will give, by way of information, the types of composition of standard solution; According to the type and sensitivity of the spectrophotometer used, it may be necessary to select other concentrations: copper xg Cu/ml / 0 / 0.1 / 0.2 / 0.4 / 0.6 / 0.8 / 1.0 ml of standard solution of work (30.3.8). / 0 / 1 / 2 / 4 / 6 / 8 / 10 (1 ml = 100 xg Cu) + ml HCL 6N (29.3.2). / 8 / 8 / 8 / 8 / 8 / 8 / 8 complete with water to about 100 ml 30.4.1.2.2 preparation of the solution to be analysed. Solution prepared according to (29.4.1.1) may, as a rule, be used directly. If necessary, take your concentration to the concentrations of the solutions range pattern, you can introduce pipette an aliquot in a 100 ml volumetric flask and complete with hydrochloric acid 0, 5N (29.3.3) to make up the volume.


30.4.1.2.3. blank test. Ditto 29.4.1.2.3.


30.4.1.2.4 measured by atomic absorption.


Measure the absorbance of the solutions pattern and the solution to be analyzed using an oxidizing air-acetylene flame, with a wavelength of 324,8 nm. Perform four times each.


30.4(2) mineral compounds. Ditto 29.4.2.


30.5 calculations. Ditto 29.5.


30.6 observations. Ditto 29.6.


30.7 references. Ditto 29.


31 manganese 31.1 principle. Determination of trace manganese in feed and its raw materials. The lower limit of determination is 20 mg/kg. The sample is dissolved in hydrochloric acid solution, previous destruction of organic matter. Manganese is determined, diluted proper, by atomic absorption spectrophotometry.


31.2 material and devices. Ditto 29.2.


31.3 reagents. Ditto 29.3. until 29.3.6. inclusive.


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


31.3.8 standard of work (10 micrograms Nm/ml) solution: take 10 ml of the solution (31.3.7) and make up to 1,000 ml with water.


31.3.9 Idem 29.3.9.


31.4 procedure. Ditto 29.4 to 29.4.1.2.3, inclusive, with the exception of the composition of the solution pattern types box.


Manganese Mn/ml xG / 0 / 0.1 / 0.2 / 0.4 / 0.6 / 0.8 / 1.0 ml of standard solution of work (31.3.8). / 0 / 1 / 2 / 4 / 8 / 8 / 10 (1 ml = 10 Mn xg) + ml HCL 6N (29.3.2). / 7 / 7 / 7 / 7 / 7 / 7 / 7 + 10 ml of lanthanum (29.3.9) chloride solution.


Make up with water to 100 ml 31.4.1 measured by atomic absorption. Measure the absorbance of the solutions pattern and of the solution to be analyzed, using one called oxidizing air-acetylene 279,5 nm wave length. Perform four times each.


31.4.2 Idem 29.4.2.


31.5 calculations. Ditto 29.5.


31.6 observations. Ditto 29.6.


31.7 references. Ditto 29.7.


32 zinc 32.1 principle. Determination of the trace element zinc in feedingstuffs and raw materials. The lower limit of determination is 20 mg/kg. The sample is dissolved in hydrochloric acid solution, previous destruction of organic matter. Zinc is determined, diluted proper, by atomic absorption spectrophotometry.


32.2 material and devices. Ditto 29.2.


32.3 reagents. Ditto 29.3 until 29.3.6, inclusive.


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


32.3.8 standard of work (10 micrograms Zn/ml) solution: take 10 ml of the solution (32.3.7) and make up to 100 ml with water.


32.3.9 Idem 29.3.9.


32.4 procedure. Ditto 29.4 to 29.4.1.2.3, inclusive, with the exception of the composition of the solution pattern types box.


Zinc Zn/ml xG / 0 / 0.05 / 0.1 / 0.2 / 0.4 / 0.6 / 0.8 ml of standard solution of work (32.3.8). / 0 / 0.05 / 1 / 2 / 4 / 6 / 8 (1 ml = 10 xg Zn) + ml HCL 6N (29.3.2). / 7 / 7 / 7 / 7 / 7 / 7 / 7 + 10 ml of lanthanum (29.3.9) chloride solution. Make up with water to 100 ml 32.4.1 measured by atomic absorption. Measure the absorbance of the solutions pattern and of the solution to be analyzed, using one called oxidizing air-acetylene with 213.8 nm wavelength. Perform four times each.


32.4.2 Idem 29.4.2.


32.5 calculations. Ditto 29.5.


32.6 observations. Ditto 29.6.


In addition to note (29.6.5) that the determination of zinc is especially sensitive to contamination from equipment glass, reagents, dust and other disruptive elements.


32.7 references. Ditto 29.7.


33 moisture fats and oils animal and vegetable 33.1 principle. The sample is subjected to drying at 103 ° C until constant weight.


This method enables you to determine the moisture content (water and other volatile matter) of animal and vegetable oils and fats.


33.2 material and devices.


33.2.1. containers of flat-bottomed, resistant to corrosion with 8-9 cm diameter and height of approximately 3 cm.


33.2.2 thermometer mercury, strengthened bulb and expansion Chamber at the top end, graduated from 80 to 110 ºc minimum and a length of 10 cm approximately.


33.2.3 bath sand or electric hot plate.


33.2.4. desiccator containing an efficient drying agent.


33.2.5. analytical balance.


33.3 procedure. Weigh to the nearest 1 mg, 20 g of the homogenized sample, in the container (33.2.1), dry and set, containing the thermometer (33.2). Heat on bath sand or hotplate (33.2.3), stirring constantly with the help of the thermometer, so that the temperature reaches 90 ºc in seven minutes approximately.


Raise the temperature slowly, without exceed 105 ° C, shaking with the thermometer until the bubbles cease.


To ensure the complete elimination of moisture, repeat several times to 103 ºc ± 2 ºc warming, cool then in desiccator (33.2.4) to room temperature and weigh. Repeat this operation until the difference between two successive weighings does not exceed by 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 global warming.


P = weight, in grams, of the container with its contents, after the warm-up.


The results below the 0.05 per 100 should be expressed as less than the 0.05 per 100.


33.5 observations.


33.5.1. an increase of the weight of the sample after repeated heating indicates an oxidation of the fat. In this case do the calculation from the weighing carried out immediately before this increase in weight.


33.5.2. the difference between the results of two parallel determinations carried out on the same sample should not exceed the 0.05 per 100 in absolute value.


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


34 (a). Starch (polarimetric method) 34 (a).1 principle. The method comprises a double determination. In the first, the sample is treated in hot with dilute hydrochloric acid. Previous clarification and filtration, the rotating power of the solution is measured using a polarimeter.


In the second, the sample is extracted with ethanol to 40 per 100. After acidification of the filtrate by HCL, clarification and filtration, Rotary power in the same conditions as in the first determination is measured.


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



The method enables you to determine the content of starch and its degradation products of high molecular weight in feedingstuffs, except those that contain peelings, pulp, leaves or necks dried beet, pulp, dehydrated yeast, products rich in inulin (e.g. peelings and Tiger nut flour) or Greaves.


34 (a).2 Material and devices.


34 (a). 2.1 conical flask of 250 ml of ground mouth with reflux condenser.


34 (a). 2.2 polarimeter or saccharometer.


34 (a).3 reagents.


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


34 (a). 3.2 hydrochloric acid to the 1,128 per 100 (w/v). The concentration must be checked by titration with the help of a solution of sodium hydroxide 0, 1N in the presence of 0.1 per 100 methyl red (w/v) in ethanol to 94 per 100 (v/v) (10 ml = 30.94 ml of NaOH 0, 1N).


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


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


34 (a). 3.5. 40 per 100 ethanol (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 entirely through a 0,5 mm mesh sieve


34 (a). 4.2 determination of the total rotational power (P or S) (see observation (34. ())) ((a). 6.1).


Weigh to the nearest 1 mg, 2.5 g of ground sample and insert into a volumetric flask of 100 ml. Add 25 ml of hydrochloric acid (34 (a). 3.2), shake to obtain a good distribution of the sample and again add 25 ml of hydrochloric acid (34 (a). 3.2). Immerse the flask in a boiling water bath and, for the first three minutes following shake vigorously and regularly to prevent the formation of agglomerates. The amount of water in the bathroom should be sufficient to allow you to keep the bath at boiling point when the flask is immersed in it. This may not be withdrawn bath throughout the turmoil. 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 I solution (34 (a). 3.3) and shake for one minute. Then add 5 ml of Carrez II solution (34 (a). 3.4) and shake again for one minute. Make up to volume with water, homogenize and filter. If the filtrate is not perfectly clear (which is rare), to start the analysis using one greater quantity of Carrez I and II, for example 10 ml solutions again.


Then measure the rotating power of the solution in a 200 mm using a polarimeter or a saccharometer tube.


34 (a). 4.3 determination of rotational power (P' or S') of substances soluble in ethanol to 40 per 100.


Weigh, with precision of 1 mg, 5 g of the sample, place in a 100 ml graduated flask and add about 80 ml of ethanol (34 (a). 3.5) (see observation 34 (a). 6.2). Leave the flask to stand for 1 hour at room temperature, throughout this period of time, proceed five times to a vigorous agitation so that the test sample is well mixed with the ethanol. Then bring to volume with ethanol (34 (a). 3.5), homogenize and filter.


Enter using pipette 50 ml of the filtrate (= 2.5 g of the sample) into a conical flask of 250 ml. Add 2.1 ml. Add 2.1 ml of hydrochloric acid (34 (a). 3.1) and shake vigorously. Fit a condenser to reflux to the Erlenmeyer flask and immerse it in a boiling water bath. After 15 minutes, remove the Erlenmeyer flask from the bath, transfer the contents to a 100 ml volumetric flask by rinsing with a little cold water, and cool to 20 ° C. Then defecate with help of the Carrez I solution (34 (a). 3.3) and Carrez II (34 (a). 3.4), make up to volume with water, homogenize, filter and measure the as shown in 34 (a). 4.2, second and third subparagraphs.


34 (a).5 calculations. The content of starch in percentage of sample is obtained by the following formulas: 34 (a). 5.1 measurements made with a polarimeter.


% of starch = 2000 (P - P') % of starch = 2000 (P - P') % of starch = (u) being: P = total in degrees of arc Rotary power.


P' = power Rotary in degrees of arc for the ethanol-soluble substances to 40 per 100.


(u) = specific Rotary power of pure starch. Conventional values supported for this factor are the following: + 185,9 °: rice starch.


+ 185.4 °: potato starch.


+ 184,6 °: corn starch.


+ 182.7 °: wheat starch.


+ 181.5 °: barley starch.


+ 181.3 °: oat starch.


+ 184.0 °: other types of starch, as well as mixtures of starches of compound feedingstuffs.


34 (a). 5.2 measurements carried out by saccharometer.


% of 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) is: S = total degrees saccharometers Rotary power.


S' = Rotary power in degrees sacarimetricos given by the ethanol-soluble substances to 40 per 100.


N = weight, in g, of sucrose in 100 ml of water, under 200 mm thick, giving a rotary power of 100 ° sacarimetricos.


16.29 for the French saccharometers.


26,00 for the German saccharometers.


20.00 for the mixed saccharometers.


(u) = power specific Rotary of pure starch (34 (a). 5.1).


The difference between the results of two parallel determinations carried out on the same sample should not exceed the 0.4 in absolute value, for starch contents of less than 40 per 100; 1.1 per 100 in relative value, for starch contents equal to or greater than 40 per 100.


34 (a).6 observations.


34 (a). 6.1 when the sample contains more than 6 per 100 calculated carbonate in calcium carbonate, they must be destroyed by treatment with the help of an exactly appropriate quantity of sulphuric acid, diluted, before the determination of the total rotational power.


34 (a). 6.2 for products high in lactose, such as the powder of whey or skim milk, proceed as follows after adding 80 ml of ethanol (34 (a). 3.5). Fit a reflux condenser to the flask, immerse the flask for 30 minutes in a water bath at 50 ° C. Then cool and continue the analysis as indicated in (34 (a). 3.5).


34 (a).7 references. Third Commission directive of 27 of April 1972. (72/199/EEC). «Official Journal of the European Communities' number L 123, 29 of May 1972.


34 (b). Starch (method of pancreatin) 34 (b).1 principle. The sugars present in the sample are removed by extraction with ethanol. Starch extraction residue is sacarificado by pancreatin. Sugars are hydrolyzed by hydrochloric acid and the formed glucose is valued by the Luff-Schoorl method. The amount of glucose obtained multiplied by a constant factor gives the content in starch in the sample.


The method enables you to determine the content of starch and its degradation products of high molecular weight in feedingstuffs containing peelings, pulp, leaves, or dried necks of beet, pulp, dried yeasts, products rich in inulin (e.g. fractions and Tiger nuts or potato flour). The assessment should be carried out only when the microscopic examination indicates the presence in the sample of significant amounts of starch.


34 (b).2 Material and devices.


34 (b). 2.1 Extractor (see Figure 34 (b).1) which is made up of: 34 (b). 2.1. 1 flask 500 ml of long-necked Erlenmeyer flask.


34 (b). 2.1. 2 refrigerant reflux adapted to the erlenmeyer flask.


34 (b). 2.1. 3 sliding Rod located in the central of the refrigerant pipe fitted with a hook on its lower extremity and a clip to fasten the rod.


34 (b). 2.1. 4 metal basket, designed to be suspended from the hook on the rod (34 (b). 2.1. 3) and to support the filter Crucible (34 (b). 2.1. 5).


34 (b). 2.1. filter Crucible, maximum pore size 5 Crucible: 90-150 micrometers (e.g. G1), approximately 30 ml of capacity.


34 (b). 2.1. 6 paper filters of format appropriate to the filter Crucible (34 (b). 2.1. 5).


34 (b). 2.2 range of incubation set at 38 ° C.


34 (b). 2.3 flasks volumetric 200 ml with ground mouth normalized and refrigerant of reflux.


34 (b). 2.4 flasks aforados 100 ml with refrigerant and standard ground mouth of reflux.


34 (b).3 reagents.


34 (b). 3.1 ethanol 90 per 100 (v/v) neutral to phenolphthalein.


34 (b). 3.2 n-Amyl Alcohol p.a.


34 (b). 3.3-toluene p.a.


34 (b). 3.4 solution buffer. Dissolve in water 9,078 g of monopotassium phosphate KPOH and 11,876 g phosphate disodium dihydrate NaPOH. 2HO. make up to one litre with water.


34 (b). 3.5 sodium chloride solution: 0, 2N.


34 (b). 3.6 solution of Carrez I. dissolve in water 21.9 g of zinc acetate dihydrate Zn (CHCOO). 2HO and 3 g of glacial acetic acid. Make up to 100 ml with water.


34 (b). 3.7 Carrez II solution. Dissolve in water 10.6 g of potassium ferrocyanide trihydrate K4 (Fe (CN)). 3HO. make up to 100 ml with water.


34 (b). 3.8 1N hydrochloric acid.


34 (b). 3.9 acid hydrochloric p.a. approximately 8N (d = 1,125).


34 (b). 3.10 p.a. approximately 10N sodium hydroxide solution (d = 1.33).


34 (b). 3.11. Indicator. 0.1 per 100 methyl Orange solution (w/v).


34 (b). 3.12 Pancreatin powdery to responding to the requirements given in (34 (b). 6.3). Store in closed containers, protected from light and moisture.



34 (b). 3.13. Luff - Schoorl reagent. Pour carefully stir the citric acid solution (34 (b). 3.13. 2) into the sodium carbonate solution (34 (b). 3.13. 3). Then add the copper sulphate solution (34 (b). 3.13. 1) and make up to 1 litre with water. Let stand overnight and filter. Check the normality of the reagent thus obtained (Cu, NaCO 2N 1N 0). The pH is approximately 9.4.


34 (b). 3.13. 1 copper sulphate solution. Dissolve 25 g of copper pentahydrate p.a. sulphate CuSO. 5HO in 100 ml of water.


34 (b). 3.13. 2 solution of citric acid. Dissolve 50 g of citric acid monohydrate for CHO. HO in 50 ml of water.


34 (b). 3.13. 3 solution of sodium carbonate. Approximately dissolve 143.8 g of anhydrous p.a. sodium carbonate in 300 ml of hot water. Leave to cool.


34 (b). 3.14 granules of pumice stone boiled in hydrochloric acid washed with water and dried.


34 (b). 3.15 to 30 per 100 solution (w/v) potassium p.a. iodide


34 (b). 3.16 approximately 6N sulphuric acid (d = 1,18).


34 (b). 3.17 0 Sodium Thiosulphate solution, 1N.


34 (b). 3.18 starch solution. Add a mixture of 5 g of starch, soluble in 30 ml of water to 1 liter of boiling water. Boil for three minutes and then allow to cool. Prepare immediately before use.


34 (b).4 procedure.


34 (b). 4.1 sample preparation. Grind the sample so that pass completely through a 0,5 mm mesh sieve


34 (b). 4.2 extraction. Weigh, with precision of 1 mg, 2 g of the sample and place in a filter Crucible (34 (b). 2.1. 5), which has been previously coated with filter paper (34 (b). 2.1. 6) moistened with ethanol ((34 (b). 3.1).) Introduce into flask (34 (b). 2.1. 1)) 55 ml of ethanol (35 (b). 3.1) and a few granules of pumice stone (34 (b). 3.14). Place the filter crucible in the metal basket (34 (b). 2.1. 4) and suspend it the rod hook (34 (b). 2.1. 3). Place the refrigerant on the erlenmeyer flask and lowering the rod so that the bottom of the Crucible protrude from the surface of the ethanol. Fasten the rod at this point with the help of the clamp. Bring the ethanol to a boil and keep it for three hours. Leave to cool then and lift rod (34 (b). 2.1. 3) in such a way that rises the Crucible as high as possible in the erlenmeyer flask. Open it carefully and drop 45 ml of water along the wall of the Erlenmeyer flask. Replace the coolant on the flask and keep the filter Crucible 10 cm from the level of the liquid. Bring the liquid to a boil and keep it that way three hours. Cool immediately, open the flask and remove the Crucible from the basket.


34 (b). 4.3 saccharification or hydrolysis. Place the filter crucible in an empty container and dried by vacuum. Deposit the residue extraction in a mortar and crush finely. Transfer quantitatively the dust in 60 ml of water approximately to a round 200 frosted ml standard flask and add a few drops of Amyl alcohol (34 (b). 3.2). Fit a reflux condenser to vessel. Heat to boiling and keep that way for an hour. Then cool down and disconnect the coolant. Add 25 ml of buffer solution (34 (b). 3.4) and 250 mg of pancreatin (34 (b). 3.12) 2.5 ml of sodium chloride solution (34 (b). 3.5) 10 drops of toluene. Stir for two minutes, place the container on the stove of incubation (34 (b). 2.2) and hold for twenty-one hours, stirring occasionally. Then let cool to room temperature.


Add 5 ml of Carrez I solution (34 (b). 3.6) and shake for one minute. Then add 5 ml of Carrez II (34 (b). 3.7) shake again for one minute. Make up to volume with water, homogenize and filter. Pipette 50 ml of filtrate and bring them into a graduated container 100 ml. Add a few drops of indicator (34 (b). 3.11) and acidify with hydrochloric acid 8N (34 (b). 3.9) until they turn red. Then add 6.25 ml of acid hydrochloric 8N, excess (12.5 ml if it acts on 100 ml of filtrate). Fit the reflux condenser to the container, put the solution to a boil and keep it for an hour. Let cool, neutralise with sodium hydroxide 10N solution (34 (b). 3.10) until they turn yellow indicator. Acidify then slightly adding a bit of 1N hydrochloric acid (34 (b). 3.3), make up the volume with water and mix. Determine the content of glucose by the Luff-Schoorl method as shown in (34 (b). 4.4).


34 (b). 4.4 rating according to Luff Schoorl. Pipette 25 ml of Luff-Schoorl reagent (34 (b). 3.13) and lead to a 300 ml Erlenmeyer flask; Add 25 ml, exactly measured, of the solution obtained in (34 (b). 4.3) containing a maximum of 60 mg of glucose. Add two pieces of pumice (34 (b). 3.14), heat, stirring by hand, over a free half-height flame, until the liquid is subjected to boiling in 2 minutes approximately. Immediately place the Erlenmeyer flask on a wire netting with disk in asbestos which has been made an opening of approximately 6 cm in diameter, below which has been previously lit a flame. This is regulated in such a way that only the bottom of the flask is heated. Then fit a reflux condenser to the flask. From this moment subjecting it to boil for 10 minutes exactly. Cool immediately in cold water and after five minutes, approximately, rating as follows: add 10 ml of potassium iodide solution (34 (b). 3.15) and immediately with caution (because of the risk of abundant foaming) add 25 ml of sulphuric acid 6N (34 (b). 3.16). Then Titrate with 0 Sodium Thiosulphate solution, 1N (34 (b). 3.17), until the appearance of a soft yellow color; Add the starch solution as indicator (34 (b). 3.18) and perform the valuation until the end.


Make the same assessment of an accurately measured mixture of 25 ml of Luff-Schoorl (34 (b). 3.13) and 25 ml of water, after adding 10 ml of potassium iodide solution (34 (b). 3.15) and 25 ml of sulphuric acid 6N (34 (b). 3.16) without boiling.


34 (b). 4.5 test blank. Carry out a blank test applying the operating mode described in 34 (b). 4.3 and 34 (b). 4.4 in the absence of the sample.


34 (b).5 calculations. With the help of the attached table establish the amount of glucose in mg corresponding to the difference between the results of two evaluations (expressed in ml of 0 sodium thiosulfate, 1N) involving both the sample and blank test.


The content in starch expressed as a percentage of the sample is given by the following formula: 0.72 (a - b) where: a = mg of glucose in the sample.


b = mg of glucose in the blank (see note 34 (b). 6.2.).


34 (b).6 observations.


34 (b). 6. (1) the simultaneous presence in the sample of starch partially dextrinated lactose can give rise to a result with an excess of 0.5 to 3 per 100 of starch. En_este_caso, the actual starch content is obtained as follows: a) to determine the content of reducing sugars of retrieved ethanolic extract in (34 (b). 4.2) and express the result as the percentage of glucose.


(b) determine the content of reducing sugars soluble in water sample and express the result as the percentage of glucose.


((c) subtract the result obtained in a.) of the retrieved in b) and multiply the difference by 0.9.


d) subtract the value obtained in c) content in starch obtained by the application of the method and calculate it as follows in (34 (b).5).


34 (b). 6.2 the amount of glucose in the blank is usually 0.25 mg and can not be greater than 0.50 mg.


34 (b). 6.3 regarding requirements to pancreatin: appearance: white yellow amorphous powder.


Glucose content: the amount of glucose in the blank (see 34 (b). 4.5) is usually 0.25 mg. Greater than 0.50 results indicate that pancreatin can not be used.


Control of iodine consumption: to suspend 62.5 mg of pancreatin in 50 ml of water and lead at 25-30 ° C. Add 1 ml of solution of iodine 0, 1N. Stir for two minutes. Titrate with a solution of 0 sodium thiosulfate, 1N (34 (b). 3.17) in the presence of starch indicator. The consumption of iodine by pancreatin solution should not exceed 0.5 ml.


Control of the activity amilolítica: mix 100 ml of starch solution (34 (b). 3.18), 5 ml of the buffer solution (34 (b). 3.4), 0.5 ml of sodium chloride solution (34 (b). 3.5) and 62.5 mg of pancreatin. The mixture is heated to 25-30 ° C and stir for two minutes. Then add 1 ml of solution of iodine 0, 1N. The blue color must disappear before the fifteen minutes following the addition of the iodine solution.


34 (b).7 references. Fifth Directive of the Commission of March 25, 1974. (74/203/EEC). «Official Journal of the European communities» number L 108/7, on April 22, 1974.


Table of values for 25 ml of reagent according to Luff-Schoorl ml of Na2S20301N, two-minute warm-up, ten minutes of boiling NaS2030, 1N: ml / glucose, fructose, inverted sugar, 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 / (OMITTED FIGURE) 35. Products derived from soya 35.1 urease activity principle. The test to determine the activity of products derived from soybean urease and accordingly highlight the inadequate cooking of these products.


The urease activity is determined by the amount of ammoniacal nitrogen liberated per 1 g of product in a minute, at 30 ° C from a solution of urea.


35.2 material and devices.


35.2.1 potentiometric or very sensitive pH titration apparatus (0,02 pH) with magnetic stirrer.


35.2.2. water bath fitted with thermostat set at 30 ºc exactly.


35.2.3 test tubes of 150 x 18 mm, with ground plugs.


35.3 reagents.


35.3.1 hydrochloric acid 0, 1N.


35.3.2. 0 sodium hydroxide solution, 1N.


35.3.3. 0,05 M phosphate buffered solution containing 4,45 g of hydrogen phosphate dihydrate sodium (NaHPO 2HO ·) and 3.40 g of dihydrogen phosphate potassium (KHPO) in 1000 ml.


35.3.4 solution buffered urea, prepared recently, containing 30,0 g of urea per 1000 ml of buffer solution; pH 6, 9-7, 0.


35.4 procedure. Approximately grind 10 g of the sample, so that it passes through a mesh of 0.2 mm. In a tube (35.2.3) weigh to the nearest 1 mg, 0.2 g of ground sample and add 10 ml of the solution (35.3.4). Close immediately and shake vigorously. Take the tube to bath water (35.2.2) and leave it for 30 minutes exactly. Immediately after, add 10 ml of hydrochloric acid 0, 1N (35.3.1), cool rapidly to 20 ºc, transfer quantitatively the contents of the tube into a container of valuation, by washing twice with 5 ml of water. Rating immediately and rapidly by means of a solution of 0, 1N (35.3.2) by potentiometer sodium hydroxide, using glass electrode to pH 4.7. Carry out a blank test, operating as indicated below: enter quickly and successively in a tube (35.2.3) an aliquot sample of 0.2 g, heavy with accuracy of 1 mg, 10 ml of hydrochloric acid 0, 1N (35.3.1) and 10 ml of buffered solution of urea (35.3.4). Immediately cool the tube in cold water and leave it for 30 minutes. Then transfer conditions listed above, the contents of the tube to the vessel and rating the hydroxide solution of sodium 0, 1N (35.3.2) until a pH 4.7.


35.5 calculations. The urease activity is given by the formula: N 30 ° C = 1.4 mg (V - V) N 30 ° C = 1.4 mg (V - V) g min at 30 ° C = 30 x P being: V = the volume, in ml, of analysis 0, consumed 1N sodium hydroxide solution.


V = the volume, in ml, of the blank test by 0, consumed 1N sodium hydroxide.


P = weight of sample in g.


35.6 observations.


35.6.1. the method is suitable for a urease activity that capable of 1 mg of n/g / min at 30 ° C. For most active products, the aliquot of the sample can be reduced to 50 mg.


35.6.2 products with a fat content exceeding 10% must have been pickled previously cold.


35.7 references. First the Commission directive of 15 June 1971. (71/250/EEC). «Official Journal of the European communities» number L 155, 12 July 1971.


36 free and total gossypol 36.1 principle. The method enables you to determine the free gossypol, total gossypol and chemically similar Constitution substances derived from gossypol, seeds, flours and cakes of cotton, as well as feed containing them. The lower limit of the determination is 20 mg/kg.


The gossypol is extracted in the presence of 3-amino-1-propanol, either with a mixture of isopropyl alcohol and hexane, for the determination of free gossypol, either by dimethylformamide, for the determination of total gossypol. The gossypol is transformed by aniline into gossypol-dianiline, the absorbance is measured at 440 nm.


36.2 material and devices.


36.2.1 agitator (of swing). Around 35 oscillations per minute.


36.2.2. spectrophotometer.


36.3 reagents.


36.3.1 mix isopropanol-hexane. Mix 60 parts by volume of isopropanol with 40 parts by volume of n-hexane.


36.3.2 solvent a. deposited in a volumetric flask of 1 litre, 500 ml approximately mix isopropanol-hexane (36.3.1), 2 ml of 3-amino-1-propanol, 8 ml of glacial acetic acid and 50 ml of water. Make up the volume with the mixture of isopropanol-hexane (36.3.1). Reagent is stable for one week.


36.3.3 solvent B. deposit by pipette in a flask aforado 100 ml, 2 ml of 3-amino-1-propanol and 10 ml of glacial acetic acid. Cool to room temperature and make up to volume with N, N-dimethylformamide. Reagent is stable for one week.


36.3.4 aniline. If the absorbance of the blank test exceeds 0.022 distil the aniline over zinc dust eliminating the fractions first and last 10 per 100 of the distillate. This reagent bottle capping machines Topaz glass and in the refrigerator, it retains several months.


36.3.5. A standard gossypol solution. Placed in a volumetric flask of 250 ml, 27.9 mg of gossypol acetate. Dissolve and make up to volume with solvent A (36.3.2). Introduce pipette 50 ml of this solution into a 250 ml graduated flask and make up to volume with solvent A. The concentration of gossypol of this solution is 0.02 mg/ml. leave to stand for one hour at the temperature environment, before use.


36.3.6. B standard gossypol solution. Placed in a volumetric flask of 50 ml, 27.9 mg of gossypol acetate. Dissolve and make up to volume with solvent B (36.3.3). The concentration of gossypol of this solution is 0.5 mg/ml.


Preserved in the shelter of the light solutions pattern A and B of gossypol, remain stable for 24 hours.


36.4 procedure.


36.4.1 sampling. Sampling is related to the course content in the sample gossypol. It is preferable to operate on a small test sample and an aliquot of relatively important filtering, so that to obtain an amount of sufficient gossypol for precise photometric measurement. For the determination of gossypol-free seeds, flours and cakes of cotton, sampling must not exceed 1 g; for compound feedingstuffs may reach the 5 g. An aliquot of 10 ml of filtrate is suitable in most cases; It should contain 50 to 100 micrograms of gossypol. For the determination of total gossypol, sampling may vary from 0.5 to 5 g in order that an aliquot of 2 ml of filtrate contains from 40 to 200 micrograms of gossypol.


The analysis must be made at an ambient temperature near 20 ° c.


36.4.2 determination of free gossypol. Place the test sample in a flask of 250 ml, whose bottom is covered with glass beads frosted mouth. Add 50 ml of solvent A (36.3.2) pipette, recap the flask and mix for one hour in the mixer. Filter in dry and collect the filtrate in a small ground-necked flask. During filtration, cover the funnel with a watch glass. Insert with pipette respectively into two 25 ml (A) and (B) volumetric flasks identical aliquot parts of filtrate containing 50 to 100 micrograms of gossypol. Complete if necessary, the volume to 10 ml with solvent A (36.3.2) help make up then to volume the contents of flask (A) by mixing isopropanol-hexane (36.3.1). This solution will be used as a reference solution for the measurement of the sample solution.


Introduce pipette 10 ml of solvent A (36.3.2) respectively in other two volumetric flasks of 25 ml (C and D). Complete the contents of the flask to volume (C) using the mix isopropanol-hexane (36.3.1). This solution will be used as a reference solution for the measurement of the test solution in white.


Add 2 ml of aniline (36.3.4) respectively in the flasks (D) and (B). Heat for 30 minutes over a boiling water bath to develop the colour. Cool to ambient temperature, make up to volume using the mix isopropanol-hexane (36.3.1), homogenize and leave to stand for one hour.


Using the spectrophotometer at 440 nm in cells 1 cm to determine the absorbance of the blank test solution (D) by comparison with the reference solution (C) and the absorbance of the sample solution (B) compared to the reference (A) solution.


Subtract the absorbance value of the solution of the blank of the sample solution (corrected absorbance). From this value calculate the gossypol content free as indicated in 36.5.



36.4.3 determination of total gossypol. Place a sample containing 1 to 5 mg of gossypol in a 50 ml graduated flask and add 10 ml of solvent B (36.3.3). At the same time prepare a blank test by introducing 10 ml of solvent B (36.3.3) in another flask of 50 ml. heat the two flasks for 30 minutes over a boiling water bath. Cool to room temperature and make the contents of each flask to volume with mix isopropanol-hexane (36.3.1). Homogenize and leave to stand for 10 to 15 minutes; then filter and collect the filtrates in ground mouth flasks.


Lead by pipette 2 ml of the sample filtrate respectively to two volumetric flasks of 25 ml and 2 ml of the filtrate of the blank respectively to two other flasks of 25 ml. take a flask from each series and complete the respective content by mixing 25 ml isopropanol-hexane (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 30 minutes in boiling water bath to develop the colour. Cool to room temperature, make up to 25 ml with hexane – isopropanol (36.3.1) mixture, homogenize and leave to stand for one hour.


Determine the absorbance as indicated in 36.4.2 for free gossypol. From this value calculate the total gossypol content as indicated in 36.5.


36.5 calculations. The calculation of the results can be made from the absorbance specific (36.5.1) or referring to a curve calibrated (36.5.2).


36.5.1 starting from the specific absorbance. Under the described conditions, the specific absorbance are as follows: free gossypol: E free gossypol: E = 625.


Total gossypol: E total gossypol: E = 600.


The free or total gossypol of sample content is given by the following formula: · 1250 gossypol % = A · 1250 e E · p · to be: A = absorbance corrected, determined as described in 36.4.2.


p = weight of sample in g.


a = aliquot part of the filtrate in ml.


36.5.2 starting from a curve of calibration.


36.5.2.1. free gossypol. Prepare two series of five 25 ml graduated flasks. In each series, introduce by pipette in flasks respective volumes of 2,0 - 4,0 - 6,0-8,0 and 10,0 ml of the solution pattern of gossypol (36.3.5), make up to 10 ml with solvent a. complete each series by a target consisting of a 25 ml flask containing only 10 ml of solvent A (36.3.2).


Complete volume of the flasks in the first series (including white) by mixing 25 ml isopropanol-hexane (36.3.1) (reference series).


Add 2 ml of aniline (36.3.4) to each flask in the second series (including white). Heat for 30 minutes in a boiling water bath to develop the colour. Cool to room temperature, make up the volume by mixing isopropanol-hexane (36.3.1), homogenize and leave to stand for one hour (standard series).


In the conditions specified in 36.4.2 determine the absorbance of the solutions of the series pattern compared with the corresponding series of reference solutions. Graphically plot the curve of calibration by putting the absorbance in relation to quantities of gossypol (in micrograms).


36.5.2.2 total gossypol. Prepare six graduated 50 ml. place flasks in the first flask 10 ml of solvent B (36.3.3) and the other, respectively 2,0 - 4,0 - 6,0-8,0 and 10,0 ml of solution B pattern of gossypol (36.3.5). Make the contents of each flask up to 10 ml with solvent B (36.3.3) support. Heat for 30 minutes in a boiling water bath. Cool to room temperature, make up to volume using the mix isopropanol-hexane (36.3.1) and homogenize.


Place 2.0 ml of these solutions respectively in two series of six graduated flasks of 25 ml. make up to 25 ml the contents of the flasks in the first series using the mixture of isopropanol-hexane (36.3.1) (reference series).


Add 2 ml of aniline (36.3.4) to each flask in the second instalment. Heat for 30 minutes in a boiling water bath. Cool to room temperature, make up to volume using the mix isopropanol-hexane (36.3.1), homogenize and leave to stand for one hour (standard series).


In the conditions specified in 36.4.2 determine the absorbance of the solutions of the series pattern compared with the corresponding series of reference solutions. Graphically plot the curve of calibration by putting the absorbance in relation to quantities of gossypol in micrograms.


The difference between the results of two parallel determinations carried out on the same sample must not exceed:-15 per 100, in relative value, for contents in gossypol of less than 500 mg/kg.


-Of 75 mg/kg, in absolute value, for contents of between 500 and 750 mg/kg.


-10 per 100, in relative value, for contents of more than 750 mg/kg.


36.6 references. Third of Commission directive of 27 April 1972. (72/199/EEC). «Official Journal of the European Communities' number L 123, 29 of May 1972.


37 theobromine 37.1 principle. The method enables you to determine the transformation of cocoa by-products theobromine content. Theobromine is extracted by chloroform. The extract is evaporated to dryness, gets in aqueous solution and is treated with a certain volume of silver nitrate solution. The nitric acid liberated is entitled by a solution of sodium hydroxide.


37.2 material and devices.


37.2.1. 500 ml flat-bottomed and stoppered flasks.


37.3 reagents.


37.3.1 chloroform p.a.


37.3.2. ammonia, d: 0.958.


37.3.3 p.a., anhydrous sodium sulfate.


37.3.4. 0 sodium hydroxide solution, 1N.


37.3.5 solution of nitrate of silver 0, 1N.


37.3.6 1 per 100 solution in ethanol (w/v) phenol red.


37.3.7 diethyl ether.


37.4 procedure. Weigh, to the nearest 1 mg, an aliquot of 10 g Max, containing not more than 80 mg of theobromine. Introducrla in a flask of 500 ml flat bottom and ground glass stopper, add 10 ml of ammonia (37.3.2) and 270 ml of chloroform (37.3.1). Close the flask and shake vigorously for five minutes. Then add 12 g of sodium sulphate anhydrous (37.3.3), shake again and leave until the next day. Filter in a 500 ml Erlenmeyer flask and wash the residue with 100 ml of chloroform (37.3.1). Distill the solvent and eliminate the last traces in a boiling water bath. Collect the extract in 50 ml of water and bring to a boil.


Cool, neutralise exactly with the solution of sodium hydroxide (37.3.4) in the presence of 0.5 ml of solution of phenol (37.3.6) red. Titrate the nitric acid liberated with a solution of sodium hydroxide (37.3.4) until turning of indicator (pH 7.4).


37.5 calculations. 1 ml of NaOH 0, 1N corresponds to 18 mg of theobromine. Express the result as a percentage of the sample.


37.6 observations. Products with a fat content exceeding 8 per 100 must be previously degreased by extraction for six hours with light petroleum (Eb. 40 ° C).


37.7 references. First the Commission directive of 15 June 1971. (71/250/EEC). «Official Journal of the European communities» number L 155, 12 July 1971.


38 Retinol (vitamin A) 38.1 principle. The method makes it possible to determine the content of retinol (vitamin A) in feedingstuffs, concentrates and premixes. The lower limit of detection is 10,000 IU/kg (equal to 0.3 micrograms of retinol 1 IU) for strongly pigmented feeds and 4,000 IU/kg for other products. The products are classified according to their content in retinol into two groups: Group A: contents of less than 200,000 IU/kg.


Group B: content equal or superior to 200,000 IU/kg.


Sample is hydrolyzed in hot by potassium hydroxide in ethanolic medium and in the presence of an antioxidant or in nitrogen atmosphere. The mixture is subjected to extraction with 1, 2-dichloroethane. The extract is evaporated to dryness, and redissolved in petroleum ether. The solution is aluminium oxide column chromatography (for Group B products, chromatography is only required in certain cases). Retinol is determined by spectrophotometry at 610 nm after obtaining a coloured complex according to the CarrPrice reaction, for Group A products; by spectrophotometry UV at 325 nm in the case of products of the Group B.


38.2 material and devices.


38.2.1. water bath.


38.2.2. Rotary evaporator vacuum with round flasks of different capacities.


38.2.3 glass for chromatography columns (length: 300 mm; inside diameter: 13 mm approximately).


38.2.4. spectrophotometer with cuvettes of 10 mm thick (of quartz for measurements in the UV).


38.2.5 lamp UV 365 nm.


38.3 reagents.


(38.3. a) used for analysing products of groups A and B.


38.3. a. 1 the 96 per 100 ethanol (v/v).


38.3. a. 2 solution 10 per 100 (w/v) of sodium p.a. ascorbate or (38.3. a. 3).


38.3. a. 3 purified nitrogen.


38.3. a. 4 50 per 100 solution (w/v) Koh p.a.


38.3. a. 5 1N potassium hydroxide solution.


38.3. a. 6 0 potassium hydroxide solution 5N.


38.3. a. 7 1, 2-dichloroethane p.a.


38.3. a. 8 ether oil, pure, boiling range 30-50 ° C. If necessary, purify as follows: shake 1,000 ml of petroleum ether with lots of 20 ml of concentrated sulphuric acid until the acid remains colourless.



Eliminate the acid and wash the ether successively with 500 ml water, twice with 250 ml of a solution to the 10 per 100 of sodium hydroxide and three times with 500 ml of water. Remove the aqueous layer, dry ether for one hour on active carbon and anhydrous sodium sulphate, filter and distill.


38.3. a. 9 oxide aluminum standardized according to Brockmann. Burnt for eight hours at 750 ºc, cool in a desiccator and keep in a glass bottle amber, screw cap. Before use in chromatography moisten as follows: place in a flask of glass amber 10 g aluminium oxide and 0.7 ml water, seal, heat five minutes in a boiling water bath shaking vigorously. Cool by stirring. Check the activity thus prepared, aluminum oxide analyzing a known amount of retinol pattern according to 38.4.2 and 38.4.3 (38.3. b. 4).


38.3. a. 10 oxide basic aluminium, degree of activity 1.


38.3. a. 11 ether diethyl pure. Remove peroxides and traces of water by chromatography on a column of basic aluminium oxide (38.3. a. 10) (25 g of aluminum 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 solution of sodium sulfide 0.5 M at 70 by 100 Glycerin, prepared from p.a. sodium sulfide


(38.3. b) used exclusively for analysing Group A products.


38.3. b. 1 p.a. crystallizable benzene.


38.3. b. 2 chloroform p.a. remove the ethanol, phosgene and traces of water by chromatography on a column of basic aluminium oxide (38.3. a. 10) (50 g per 200 ml of chloroform aluminium oxide); result should be twice the 50 first ml of the eluate.


38.3. b. 3 according to Carr-Price reagent. Stir 25 g approximately of trichloride antimony p.a. (kept in a desiccator) with 100 ml chloroform (38.3. b. 2) until saturation of the solution. A small deposit of antimony trichloride does not alter. Add 2 ml of acetic anhydride p.a. store in freezer in an amber with screw cap glass bottle. This reagent as well preserved is stable for several weeks.


38.3. b. 4 Retinol pattern controlled by spectrophotometry.


(38.3. c) used exclusively for analysing Group b products


38.3. c. 1 Isopropanol for chromatography.


38.4 procedure.


38.4.1 hydrolysis and extraction. Take a portion of the sample finely divided, proportional to the presumed content of vitamin A: - 0,1-1,0 g for concentrates (contents greater than 20,000 IU/g).


-3,0-5,0 g for premixes (contents ranging from 400 to 20,000 IU/g).


-10 to 20 g for mineral mixtures.


-30 g for Group A products.


Immediately place it in a 500-ml stoppered flask.


Add 40 ml of ethanol on (38.3. a. 1), 2 ml sodium ascorbate solution (38.3. a. 2) 10 ml of solution of potassium hydroxide to 50 per 100 (38.3. a. 4) and 2 ml of 0.5 M sodium sulfide solution (38.3. a. 13). Heat for 30 minutes at 70-80 ° C under reflux coolant and cool to tap. Add 50 ml of ethanol (38.3. a. 1) and 100 ml (taken with a pipette) of 1, 2-dicloroetanol (38.3. a. 7).


Shake vigorously and decant the supernatant liquid on funnel and add 150 ml of 1N potassium hydroxide solution (38.3. a. 5), stir for 10 seconds and leave to stand until the separation of the phases. Pick up the lower phase of dichloroethane in another separating funnel, add 40 ml of 0 potassium hydroxide solution 5N (38.3. a. 6), stir for 10 seconds and leave to stand until the separation of the phases. Pick up the phase of dichloroethane in another separating funnel and wash six or eight times with lots of 40 ml of water until the absence of alkali in wash (phenolphthalein test). Pick up the phase of dichloroethane and remove the last traces of water through filter paper strips.


Evaporate to dryness to vacuum and in water bath at 40 ° C, an aliquot part of the solution. Redissolve quickly the residue with 5 ml of petroleum ether (38.3. a. 8).


For products of the Group A, chromatograph as shown in 38.4.2.1.


For Group B products, transfer the solution to a 50 ml volumetric flask, make up to volume with petroleum ether (38.3. a. 8), mix and measure the absorbance as indicated in 38.4.3.


38.4.2 chromatography.


38.4.2.1 products of the group a. fill the column for chromatography (38.2.3) up to a height of 200 mm oxide aluminium (38.3. a. 9), previously impregnated with petroleum ether (38.3. a. 8). Enter the solution obtained in 38.4.1 column and immediately add 20 ml of petroleum ether (38.3. a. 8). Elute successively with portions of 10 ml of solutions of light petroleum with 4, 8, 12, 16 and 20 per 100 of diethyl ether (38.3. a. 12) vacuum or partial pressure; Elution speed should be two to three drops per second.


The carotene is eluted first. (This fraction carotene content can be determined by measurement of absorbance at 450 nm; In = 2. 600). 38.4.2.2 products of the Group B. Chromatography must not be carried out unless the absorbance measurements obtained in 38.4.3.2 not comply with prescriptions given in 38.4.3.2.


If chromatography is required, enter in the chromatographic column an aliquot part of the solution in light petroleum obtained in 38.4.1 containing approximately 500 IU of retinol and chromatograph as shown in 38.4.2.1.


38.4.3 absorbance measurement.


38.4.3.1 evaporate to dryness, vacuum, the fraction chromatographic 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 Carr-Price reagent (38.3. b. 3). A blue color develops. Measure the absorbance in the spectrophotometer at 610 nm exactly 30 seconds from the start of the reaction.


Determine the content of retinol using a curve pattern obtained from bencenicas solutions of increased concentrations in retinol pattern treated by the Carr-Price reagent (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 Carr-Price reagent (38.3. b. 3).


The curve pattern should be checked regularly and at short intervals with a recently prepared Carr-Price reagent solution.


38.4.3.2 products of Group B. take an aliquot part of the solution in light petroleum obtained in 38.4.1, containing approximately 200 IU of retinol. Evaporate to dryness under vacuum and redissolve the residue with 25 ml of isopropanol (38.3. c. 1). Measure the absorbance in the spectrophotometer at 334, 325 and 310 nm. The absorption maximum is located at 325 nm. The solution retinol content is calculated as follows: X 18.30 = UI of retinol, however, relations between y/y and y/y absorbance should be 6/7 = 0,857.


If one of these relationships separates appreciably from this value (0,880 more or less of 0,830), absorbance measurement must be preceded by chromatography according to the procedure given in 38.4.2. If measurement of absorbance carried out after chromatography shows that the relationships before mentioned they are still significantly separated from the value 0,857 (0,880 more or less of 0,830), the determination must be carried out according to the procedure given for Group A products.


38.5 calculations. Calculate the sample retinol content taking into account the weight of starting and the dilutions carried out throughout the analysis.


Express the result in IU of retinol per kg. The difference between the results of two parallel determinations carried out on the same sample should not exceed:-20 per 100 in relative value, for retinol contents lower than 75,000 IU/kg.


-15,000 IU of retinol content between 75,000 and 150,000 IU/kg.


-10 per 100 in relative value, for contents of between 150,000 and 250,000 IU/kg.


-25,000 for contents of between 250,000 and 500,000 IU/kg.


-5 per 100 in relative value, for contents of more than 500,000 IU/kg.


38.6 observations.


38.6.1. all manipulations should be made in the absence of direct light and if possible in amber glassware.


38.6.2. the addition of ascorbate is not required if the hydrolysis is carried out in a stream of nitrogen (38.4.1).


38.6.3 to lactation feed products with a tendency to swell, double the quantity of the 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 keep the precautions due to the endangerment of the reagents (particularly benzene).


38.7 references. Fourth of the Commission directive of 5 December 1972. 73/46/EEC. «Official Journal of the European Communities' number L 83/21, of March 30, 1973.


39 thiamine (aneurine, vitamin B) 39.1 principle. The sample is hot with dilute sulfuric acid and is then hydrolyzed by enzyme via.


The solution obtained is subjected to alkaline oxidation. The tiocromo formed is extracted with isobutanol and determined by fluorimetry.


The method enables you to determine the thiamine (aneurine, vitamin B) in feedingstuffs, concentrates and premixes. The detection limit is 5 ppm.


39.2 material and devices.


39.2.1. water bath.


39.2.2. centrifuge (3,500 rpm) with 30 to 50 ml screw cap tubes.


39.2.3 fluorimeter.


39.3 reagents.


39.3.1. thiamine 100 micrograms/ml standard solution.



Dissolve 112.3 mg thiamine hydrochloride, quality pattern, previously dried at vacuum to constant weight, in 1000 ml of acid sulphuric 0, 2N (39.3.2). Cold and darkness this solution is stable for one month.


39.3.2 sulphuric acid 0, 2N.


39.3.3 NaSO pure Sodium Metabisulphite.


39.3.4 20 per 100 solution (w/v) of hexacyanoferrate (III) potassium p.a.


39.3.5 25 per 100 solution (w/v) Koh p.a.


39.3.6 oxidizing mixture: Mix 2 ml of solution of hexacyanoferrate (III) potassium (39.3.4) with 48 ml potassium (39.3.5) hydroxide solution. This mixture is not conserved more than four hours.


39.3.7 Isobutanol p.a.


39.3.8 2 sodium acetate solution, 5N.


39.3.9 preparation multienzimatica containing protease, phosphatase and amylase (for example clarasa).


39.3.10 ethanol of 96 per 100 (v/v).


39.4 procedure.


39.4.1. enzymatic hydrolysis. Enter in two volumetric flasks A and B of 250 ml, identical amounts of the finely ground sample, containing 100 micrograms of thiamine and 125 ml sulphuric acid (39.3.2). Add only to the flask to 1,0 ml of standard solution (39.3.1) (internal standard).


Shake vigorously, take flasks to a boiling water bath and keep them for 15 minutes shaking from time to time. Cool to 45 ° C approximately. Add to each flask 20 ml of solution of sodium acetate (39.3.8) 0.5 g of preparation multienzimatica (39.3.9), then leave for twenty minutes at room temperature. Add 20 ml of sodium acetate solution and make up to volume with water, homogenize and filter. Collect the filtrates A and B after you have removed the 15 first ml. prepare the following solutions: 39.4.1.1 solution witness T. Insert in a tube from centrifuge (39.2.2), 5 ml of the filtrate A and 10 mg approximately metabisulfit sodium (39.3.3). Dip tube for 15 minutes in a boiling water bath and then cool to room temperature.


39.4.1.2 solution (internal standard) and B (sample). Introduce 5 ml of filtrate A in a tube centrifuge (39.2.2) and 5 ml of the filtrate B in another tube centrifuge (39.2.2).


39.4.2 oxidation. Add to solutions T, A and B, 5 ml oxidant mixture (39.3.6) and after a minute, 10 ml isobutanol (39.3.7). Recap the tubes and shake vigorously for five seconds. Let stand for a minute and centrifuge to separate the phases. Take, each tube, 5 ml of the phase isobutanolica supernatant, place respectively in the 25 ml graduated flasks, make up to volume with ethanol (39.3.10) and mix thoroughly. This gives rise to the extracts T, A and B.


39.4.3. measurement of fluorescence. Taking the measurements to the wavelength at which the fluorimeter gives an optimal response to the fluorescence of the tiocromo. Irradiated at 365 nm about.


Adjust the instrument to zero with the extract T. measure the intensity of fluorescence of extracts A and B.


39.5 calculations. The content in micrograms of thiamine per kg of sample is given by the formula: d x b (a - b) x c being: a = intensity of fluorescence of extract to (internal standard).


b = intensity of fluorescence of extract B (sample).


c = weight, in grams, of the sample.


d = amount of thiamine in micrograms added to the sample (internal standard).


The difference between the results of two parallel determinations carried out on the same sample must not exceed a: - 10 per 100 in value relative to the contents of less than 500 ppm and - 5 per 100 in relative value, for contents equal to or greater than 500 ppm.


39.6 references. Fourth of the Commission directive of 5 December 1972. 73/46/EEC. «Official Journal of the European Communities' number L 83/21, of March 30, 1973.


40 Ascorbic acid and dehydroascorbic acid (vitamin C) 40.1 principle. The method enables you to determine the amount of Ascorbic acid and dehydroascorbic (vitamin C) in feedingstuffs, concentrates and premixes. The detection limit is 5 ppm. The products are classified into two groups according to their content in vitamin c: Group A: contents lower than 10 g/kg.


Group b: contents equal to or greater than 10 g/kg.


The sample, placed in suspension in a dilute solution of metaphosphoric acid, is subjected to extraction with chloroform. The aqueous phase is treated with a solution of 2, 6-dichlorophenol-indophenol to transform the Ascorbic acid into dehydroascorbic acid, and then by a solution of 2, 4-Dinitrophenylhydrazine. The hydrazone formed is extracted with a mixture of ethyl acetate, glacial acetic acid and acetone. The solution is silica gel column chromatography, the eluate is evaporated to dryness and the residue is dissolved in dilute sulphuric acid. The absorbance of the solution is measured in a spectrophotometer at 509 nm.


For Group A products, the effluent from chromatography on column undergoes a thin layer chromatography to isolate the hydrazone.


40.2 material and devices.


40.2.1. water bath regulated at the temperature of 20 ° C by thermostat.


40.2.2. centrifuge (3500 r.p.m.) with screw caps fitted with 40 to 50 ml tubes.


40.2.3. Rotary evaporator vacuum with 250 ml flasks.


40.2.4 glass for chromatography columns (length: 100 mm inside diameter: 20 mm) with a plate of sintered glass (eg Allihn tubes).


40.2.5 spectrophotometer or colorimeter of filters with trays of 10 mm thick.


40.2.6 material for layer chromatography thin silica H (40.3.12), layer thickness gel plates: 0.5 to 0.6 mm. Dry plates in two hours thirty minutes to three hours in an oven at 120-130 ° C. Allow to cool and store in a desiccator for twenty-four hours before use.


40.2.7 oven regulated at 120-130 ° C.


40.3 reagents.


40.3.1 standard solution 0,05 per 100 of L-Ascorbic acid: dissolve 50 mg of L-Ascorbic acid p.a. in 20 ml approximately metaphosphoric acid (40.3.2) and make up to 100 ml with water. Prepare immediately before use.


40.3.2 solution 10 per 100 of metaphosphoric acid: dissolve in water 200 g metaphosphoric acid p.a. crushed in a mortar, and make up to 2,000 ml with water. Store at 4 ° C. Renew within one week.


40.3.3 chloroform p.a.


40.3.4 0.5 per 100 solution (w/v) 2, 6-dichlorophenol-indophenol p.a. prepare immediately before use.


40.3.5 auxiliary filtration (S and S number 121 or equivalent).


40.3.6 acid solution to 2 per 100 (w/v) 2, 4-Dinitrophenylhydrazine: dissolve 2 g of 2, 4-Dinitrophenylhydrazine in 100 ml of dilute sulphuric acid (25 ml of sulphuric acid p.a. d = 1.84, diluted to 100 ml with water). This solution is preserved in cold for a week.


40.3.7 nitrogen or (40.3.8).


40.3.8 carbon dioxide.


40.3.9 mixture of glacial acetic p.a./ácido ethyl acetate / acetone p.a.: 96/2/2 in volume.


40.3.10 mixture of diclorometanol glacial acetic p.a./ácido: 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 dilute sulphuric acid: place 105 ml water in a 200 ml graduated flask, make up to volume with sulphuric acid p.a., d = 1,84.


40.3.14 solvent Elution for thin layer chromatography: mix 75 ml of ether diethyl p.a., 4.0 ml of acetic acid of the 96 per 100 w/v, and 25 ml of ethyl p.a., p.a. renew after two or three chromatographies.


40.4 procedure.


40.4.1 extraction. Enter in two volumetric flasks A and B, 250 ml, identical amounts of the finely ground sample, which contain approximately 200 micrograms of vitamin C., adding only A, 0.4 ml of solution pattern (40.3.1) and mix, shaking gently (internal standard). Add to each flask 30 ml chloroform (40.3.3) and 25 ml of the solution of metaphosphoric acid (40.3.2) at 4 ° C. Stir lightly and let stand for ten to fifteen minutes. Add 25 ml of water, cover in the flasks, shake vigorously for 10 seconds and leave to stand for 10 to 15 minutes in water bath (40.2.1). Centrifuge to separate the aqueous phase of the cloroformica phase. Carry out the following operations simultaneously on aqueous extracts A (internal standard) and B.


40.4.2. oxidation. Take by pipette 40 ml of the aqueous supernatant (slightly cloudy) obtained in (40.4.1), place it in a reaction of screw cap tube, add 0.5 to 1 ml of solution 2, 6-dichlorophenol-indophenol (40.3.4) and homogenize. A color that should persist fifteen minutes at least is formed. Then add 300 mg about auxiliary filtration (40.3.5), stir and filter through a dry pleated filter. Filtering should not be necessarily transparent.



40.4.3 reaction with 2, 4-Dinitrophenylhydrazine and extraction of the hydrazone. Take by pipette 10 ml of the filtrate obtained in (40.4.2), insert it into a tube of centrifugal (40.2.2), add 2 ml of 2, 4-dinitrofenil-hydrazine (40.3.6) solution and mix. Do pass quickly through the pipe a stream of nitrogen (40.3.7) or carbon dioxide (40.3.8), cover the tube and immerse it for fifteen hours approximately (one night) in the water bath (40.2.1). Add then 3 ml water, 20 ml of the mixture of glacial acetic acid/ethyl acetate / acetone (40.3.9) and approximately 800 mg filtration (40.3.5) Assistant. Cover tube, shake vigorously for 30 seconds and centrifuge. Place 15 ml of the supernatant phase in a flask of evaporation and evaporation at reduced pressure in the Rotary evaporator (40.2.3) to obtain an oily residue. Dissolve the residue in 2 ml of the mixture of glacial acetic acid/ethyl acetate / acetone (40.3.9) by heating to 50 ºc, leave to cool, add 10 ml of the dichloromethane/acid acetic glacial (40.3.10) mix.


40.4.4 column chromatography. Fill the column chromatographic (40.2.4) up to a height of 30 mm with mix glacial acetic dichloromethane/acid (40.3.10). Suspend (shaking vigorously) 5 g of silica gel (40.3.11) in 30 ml of the dichloromethane/acid acetic glacial (40.3.10) mixture and pour the suspension in the column. Leave deposit and compress under weak pressure of nitrogen (40.3.7).


Quantitatively transfer the solution obtained in 40.4.3 chromatographic column, wash the flask with a small amount of mixture of dichloromethane/acid acetic glacial (40.3.10) and transfer to the column, fill with mixture (40.3.10) and continue washing the column with the same mixture (3 or 4 servings 5 ml approximately), until a colourless eluate is obtained. Eliminate the fraction eluted colored yellow.


Elute the reddish area at the head of the column through the mix glacial acetic acid/ethyl acetate / acetone (40.3.9), collect the eluate and evaporate to dryness.


40.4.4.1. for Group A products (contents in vitamin C lower than 10 g/kg), dissolve the residue in 2.0 ml mix glacial acetic acid/ethyl acetate / acetone (40.3.9) and proceed immediately to the thin layer chromatography as described in 40.4.5.


40.4.4.2. for products of Group B (contents in vitamin C equal to or greater than 10 g/kg), redissolve the oily residue in 4.0 ml dilute sulphuric acid (40.3.13), shake vigorously to dissolve the residue completely and proceed to absorbance measurement as shown in 40.4.6.


40.4.5 thin layer chromatography. Carry out the operations indicated from here twice.


Deposit in form of a band on the layer chromatography plate fine (40.2.6) 0.5 ml of the solution obtained in 40.4.4.1. Developing for twenty minutes at least through the eluent (40.3.14) camera saturated until clear separation in the area of the hydrazone, colored pink. Allow to air dry. Delimit the zona rosa, scraping with the spatula and quantitatively transfer the powdery mass to a column chromatography (40.2.4).


Elute successively one once with 2 ml and twice with 1.5 ml of the mixture of glacial acetic acid/ethyl acetate / acetone (40.3.9). Collect the eluate in a flask small (the last fraction should be colorless). Evaporate to dryness, redissolve the oily residue in 4.0 ml of acid diluted sulphuric (40.3.13), shake vigorously to dissolve the residue completely and proceed to absorbance measurement.


40.4.6 the absorbance measurement. Measure the absorbance in a spectrophotometer at 509 nm from twenty to thirty minutes since the dissolution of the residue in dilute sulfuric acid (40.3.13).


The measurements to be made by comparison with dilute sulfuric acid (40.3.13).


40.4.7. blank test.


At the same time carry out a blank test by applying the same method of operation.


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: to = blank absorbance.


b = absorbance of the solution of internal standard.


c = 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 should not exceed 10 per 100 in relative value, for contents in vitamin C lower than 10 g/kg and 5 per 100 in relative value, for contents equal to or greater than 10 g/kg.


40.6 references. Fourth of the Commission directive of 5 December 1972. (73/46/EEC). «Official Journal of the European Communities' number L 83/21, of March 30, 1973.


41 Menadione (vitamin K) 41.1 principle. The sample is subjected to extraction with dilute ethanol. Mixture soils with solution of tannin and centrifuged. The extract is treated with sodium carbonate solution. The liberated menadione is extracted with 1, 2-dichloroethane. The extract in dichloroethane is, according to its content of menadione, directly or after evaporation, with 2, 4-dinitro-phenylhydrazine solution in ethanol acidified with hydrochloric acid. The obtained hydrazone gives rise, by adding an excess of ammonia, a complex of blue-green color whose absorbance is measured at 635 nm. The method enables you to determine the menadione (vitamin K) in feedingstuffs, concentrates and premixes. The lower limit of detection is 1 ppm.


41.2 material and devices.


41.2.1. mechanical Shaker.


41.2.2. centrifuge (3,000 to 5,000 rpm).


41.2.3 separating funnels of 100 and 250 ml, with ground glass stopper.


41.2.4. Rotary evaporator vacuum with 250 ml flasks.


41.2.5 water bath.


41.2.6 spectrophotometer with cuvettes of 10 mm thick.


41.3 reagents.


41.3.1. ethanol 96 per 100 (v/v).


41.3.2. ethanol (41.3.1) diluted to 40 per 100 with water.


41.3.3 10 per 100 solution (w/v) of tannin, obtained from pure tannin powder.


41.3.4 1,2 dichloroethane p.a.


41.3.5 10 per 100 solution (w/v) p.a. anhydrous sodium carbonate


41.3.6 hydrochloric acid at 37 per 100 (w/w), de = 1.19.


41.3.7 absolute ethanol.


41.3.8 reagent of 2, 4-dinotrofenilhidrazina: dissolve approximately 40 mg of 2, 4-Dinitrophenylhydrazine p.a. in 40 ml of absolute ethanol (41.3.7) boiling. Allow to cool and transfer to a volumetric flask of 50 ml. Add 1 ml of hydrochloric acid (41.3.6) and make up to volume with ethanol absolute (41.3.7). Prepare immediately before use.


41.3.9 hydroxide ammonium to 25 per 100 (w/w), d = 0.91.


41.3.10 ethanol ammoniacal solution: Mix one volume of ammonia (41.3.9) and a volume of ethanol (41.3.7).


41.3.11 standard of menadione solutions: dissolve 20 mg menadione (vitamin K) in 1, 2-dichloroethane (41.3.4) and complete a 200 ml. diluted aliquots of this solution using the 1, 2-dichloroethane (41.3.4) to obtain a series of solutions pattern whose concentrations in menadione are included between 2 and 10 micrograms per milliliter. Prepare immediately before use.


41.4 procedure.


41.4.1. extraction. Taking a portion of finely ground sample proportional to the presumed content of menadione:-0.1 to 5.0 g for concentrates and premixes.


-20-30 g for the feed.


Immediately place it in a 250 ml flask with ground glass stopper. Add exactly 96 ml of dilute ethanol (41.3.2) and mechanically stir for 15 minutes at room temperature. Add 4.0 ml of tannin (41.3.3) solution, mix, transfer the extract to a centrifuge tube, centrifuged (3,000 to 5,000 rpm) and decant.


Enter 20 to 40 exactly measured ml of the extract into a separating funnel of 250 ml, add by pipette 50 ml of 1, 2-dichloroethane (41.3.4), mix and add by pipette 20 ml of solution of soda ash (41.3.5). Shake vigorously for 30 seconds and collect then the phase of dichloroethane in a separating funnel 100 ml. add 20 ml of water, stir for 15 seconds, pick up the phase of the dichloroethane and eliminate traces of water through filter paper strips.


For concentrates and premixes, take an aliquot of the extract and dilute with 1, 2-dichloroethane (41.3.4) to obtain a concentration of menadione of 2 to 10 micrograms per milliliter. For feedingstuffs, evaporate to dryness at 40 ° C, under reduced pressure, and atmosphere of nitrogen, an aliquot of the extract. Redissolve the residue in the proper amount of 1, 2-dichloroethane (41.3.4) to obtain a solution containing from 2 to 10 micrograms of menadione per ml.


41.4.2. formation of the hydrazone. Take 2.0 ml of the extract in dichloroethane obtained in 41.4.1 into a graduated flask 10 ml and add 3.0 ml of reagent of 2, 4-Dinitrophenylhydrazine (41.3.8). Recap the flask tightly so as to prevent all evaporation and heat for two hours at 70 ° C on water bath. Leave to cool, add 3.0 ml of ammonia solution of ethanol (41.3.10), mix and make up to volume with ethanol absolute (41.3.7) mix again.


41.4.3. measurement of absorbance. Measure the absorbance of the blue-green colored complex in spectrophotometer at 635 nm by comparison with a reactive white retrieved trying to 2.0 ml of 1, 2-dichloroethane (41.3.4) as indicated in 41.4.2. Determine the amount of menadione curve of calibration set for each series of analyses.


41.4.4. curve of calibration. Try 2.0 ml of each standard solution of menadione (41.3.11) as shown in 41.4.2. Measure the absorbance as indicated in 41.4.3. Plot the curve of calibrated taking ordered values of absorbance and ordinates the corresponding quantities of menadione in micrograms.



41.5 calculations. Calculate the menadione of sample content taking into account the weight of the sample and the dilutions carried out in the course of the analysis. Express the result in milligrams of menadione per kg.


The difference between the results of two parallel determinations carried out on the same sample should not exceed:-20 per 100 in relative value, for contents in menadione of less than 10 mg/kg.


-2 mg in absolute value for the menadione contents between 10 and 14 mg/kg.


-15 per 100 in relative value, for contents of between 14 and 100 mg/kg.


-15 mg/kg, in absolute value, for contents of between 100 and 150 mg/kg.


-10 per 100 in relative value, for contents greater than 150 mg/kg.


41.6 observations. All manipulations must be made in the absence of direct light and if possible in amber glass material.


41.7 references. Fifth Directive of the Commission of March 25, 1974. 74/203/EEC. «Official Journal of the European Communities' number 108/7 of 22 April 1974.


42 Virginiamycin (by diffusion on agar) 42.1 principle. Method makes it possible to determine the Virginiamycin in feedingstuffs and premixes. The lower limit of determination is 2 mg/kg. 1 mg Virginiamycin is equivalent to 1,000 UK units.


The sample is subjected to extraction with Tween 80 methanolic solution. Extract opts or spin, and then diluted. Its antibiotic activity is determined by measuring the diffusion of the Virginiamycin on agar seeded with Micrococus medium luteus. Diffusion is manifested by the formation of microorganism inhibition zones. The diameter of these zones is directly proportional to the logarithm of the concentration of antibiotic for the range of concentrations used.


42.2 reagents.


42.2.1. micro-organism: «Micrococus luteus» ATCC 9341 (NCTC 8340, NCIB 8553).


42.2.1.1 maintenance of the vine-planting culture (42.2.2.1) in inclined tubes, with Micrococus medium luteus. Incubate for 24 hours at 30 ºc, store in a refrigerator at about 4 ºc and renew the sowing every fifteen days.


42.2.1.2 preparation of the bacterial suspension. Collect the bacteria from a tube of agar (42.2.1.1) recent preparation, using 2 to 3 ml of solution of chloride of sodium (42.2.2.3). Sow with this suspension 250 ml of the culture medium (42.2.2.1) in a Roux flask and incubate for 18 to 20 hours at 30 ° C. Collect the bacteria in 25 ml of (42.2.2.3) sodium chloride solution and mix thoroughly.


Dilute the suspension to 1: 10 with the help of (42.2.2.3) sodium chloride solution. The light transmission of the suspension, measured at 650 nm in 1 cm thick, by comparison with the solution of chloride of sodium (42.2.2.3), should be around 75 by 100. This suspension can be kept a week at 4 ° C. Other methods can be used provided that it is shown that produce similar bacterial suspensions.


42.2.2. culture media and reagents.


42.2.2.1 means of maintenance of strain and basis for the determination. Any means of commercial cultivation of similar composition can be used and give the same results: meat Peptone: 6,0 g.


Tryptone: 4.0 g.


Yeast extract: 3.0 g.


Meat extract: 1.5 g.


Glucose: 1.0 g.


Agar: 10,0 to 20,0 g.


1,000 ml water.


pH: 6,5 (after sterilization).


42.2.2.2 buffer phosphate pH 6: hydrogen phosphate of potassium KHPO: 2 g.


Dihydrogen phosphate potassium KHPO: 8 g.


Water up to 1,000 ml.


42.2.2.3 0.8 per 100 solution (w/v) of sodium chloride: dissolve 8 g of sodium chloride in water and dilute to 1000 ml and sterilize.


42.2.2.4 methanol.


42.2.2.5 mixture of buffer phosphate (42.2.2.2) and methanol (42.2.2.4): 80/20 (v/v).


42.2.2.6 solution in methanol to the 0.5 per 100 (w/v) Tween 80: dissolve 5 g Tween 80 in methanol (42.2.2.4) and dilute to 1000 ml with methanol.


42.2.2.7 reference substance: Virginiamycin of known activity.


42.3 procedure.


42.3.1. reference solutions. An amount exactly heavy substance of reference (42.2.2.7) in methanol (42.2.2.4) and dilute with methanol (42.2.2.4) to obtain a stock of 1,000 micrograms of Virginiamycin/ml solution. stored in bottle capping machines, at 4 ºc, this solution is stable for five days. Prepare this solution and through 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 extract and solutions.


42.3.2.1 extraction.


42.3.2.1.1 products with a Virginiamycin content does not exceed 100 mg/kg. Weigh a sample of 50 g. add 200 ml of the solution (42.2.2.6), shake for 30 minutes and leave then settle or centrifuge. Collect 20 ml of the supernatant solution and evaporate to about 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 a so-called concentration of 1 microgram/ml Virginiamycin (= U).


42.3.2.1.2 products with a Virginiamycin content greater than 100 mg/kg. Weigh out a quantity of sample not exceeding 10,0 g and containing 1 to 50 mg Virginiamycin. Add 100 ml of solution (42.2.2.6), shake for thirty minutes, and let then settle or centrifuge. Dilute the supernatant solution using the mixture (42.2.2.5) to obtain a concentration of 1 microgram/ml Virginiamycin (= U).


42.3.2.2 solutions of extract. Prepare, from solution U8 and by successive dilutions (1 + 1) using the mixture (42.2.2.5), the solutions U (so-called concentration = 0.5 microgram/ml), U (so-called concentration = 0.25 microgram/ml) and U (so-called concentration = 0.125 microgram/ml).


42.3.3. modalities of definition.


42.3.3.1 inoculation of the culture medium. Sown at about 50 ° C the basic medium for 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 quantity of bacterial suspension which allows for the different concentrations of Virginiamycin inhibition zones the most extensive possible while still being crisp.


42.3.3.2 preparation of boxes. Diffusion on agar is carried out in a box with the four concentrations of the solution of reference (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each case must necessarily receive four concentrations of extract and reference. So, we had to choose the dimensions of the boxes in such a way that can dig into the agar medium at least 8 cavities of 10 to 13 mm in diameter, whose centers distended not less than 30 mm. Glass plates can be used as boxes flat, topped by a ring of aluminum or plastic of 200 mm in diameter and 20 mm high.


Enter a quantity of the medium (42.2.2.1), planted in boxes as indicated in (42.3.3.1), which allows obtaining a layer about 2 mm thick (60 ml for a box of 200 mm diameter). Let solidify, excavate cavities and place in them exactly measured volumes of the extract and reference solutions (0,10-0,15 ml per cavity according to diameter). Repeat at least 4 times each concentration so that each determination understands the evaluation of 32 zones of inhibition.


42.3.3.3 incubation. Incubate the trays for sixteen to eighteen hours at 30 ° C ± 2 ° C.


42.4 calculations. Measure the diameter of the inhibition zones with an error not exceeding 0,1 mm. For each concentration, registration measures stockings on semi-log paper, transferring the logarithm of the concentrations in relation to the diameters of the zones of inhibition. Draw the most fitting straight lines for the reference solution and the extract, for example, by the following procedure: determine the most appropriate point of the lowest level of the solution of reference (SL) using the formula: 7S + 4S + S - 2S (a) SL = - 10 determine the most appropriate point of the highest level of the reference (SH) using the formula solution : 7S + 4S + S - 2S (a) SL = - 10 Similarly determine the most appropriate points of the extract to the lowest level (UL) and the highest level (UH) by substituting S, S, S and S in the formulas above for U, U, U and U.


Enter the SL and SH values on the same graph. By joining the two points, the tighter straight to the reference solution is obtained. It follows the same method for UL and UH, and gets the tighter line for the extract.


If there is no interference, the straights are considered parallel if (SH-SL) and (UH-UL) do not differ by more than 10 per 100 of its media.


If the lines are not parallel, can be removed U and S or U and S. Values SL, SH, UL and UH that can produce the most fitting straight lines are then calculated using the following formulas: 5S + 2S - 5S S + 2S - S (a) SL = - or - 6 6 5S + 2S - 5S S + 2S - S (b) SH = - or - 6 6 and similar formulas for UL and UH. The use of this alternative requires to comply with the same criteria of parallelism.


Analytical bulletin a result from three levels should be noted.


When the lines are considered to be parallel, should calculate the logarithm of the relative activity (log A) by using one of the following formulae, depending on the number of levels (4 or 3) used for the assessment of parallelism.


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 activity of sample extract = activity of relevant reference A, IE = S x A.


If the relative activity is not in the range of values between 0.5 and 2.0, has repeated the determination proceeding to the appropriate settings of the concentrations of the extract or, where appropriate, the reference solutions. When this activity may not quite fit into the range of the required values, the result shall be deemed approximate and thus shall be indicated in the analysis report.


When the lines are not considered to be parallel, the determination will be repeated. If not now is the parallelism, the determination shall be considered unsatisfactory.


Express the result in milligrams of Virginiamycin per kilogram of feedingstuff.


The difference between the results of two determinations carried out on the same sample by the same analyst should not exceed: 2 mg/kg, in absolute value, for contents of Virginiamycin of less than 10 mg/kg.


20 per 100 of the highest result for contents from 10 to 25 mg/kg.


5 mg/kg, in absolute value, for contents of 25 to 50 mg/kg.


10 per 100 of the highest result for contents above 50 mg/kg.


42.5 references.


Directive of the Commission of 20 December 1983 amending directives 71/393, 78/633, and 72/199 (84/4/EEC). «European Communities Official Journal» of 18 January 1984, number L 15/28, annex II.


43 Bacitracin zinc (by diffusion on agar) 43.1 principle. The method enables you to determine the bacitracin zinc in feedingstuffs and premixtures. The lower limit of determination is 5 mg/kg. 1 mg of bacitracin zinc (quality feed) is equivalent to 42 international units (IU).


The sample to pH 2 using a mixture of ethanol and water, hydrochloric acid and sodium sulfide solution is removed. Sodium sulfide can precipitate soluble copper salts that may clog the determination. Born at pH 6.5, the summary is concentrated (where necessary) and diluted. Its antibiotic activity is determined by the diffusion of the bacitracin zinc in a medium of agar, planted with Micrococus luteus (flavus). Diffusion manifests through formation of microorganism inhibition zones. The diameter of these zones is directly proportional to the logarithm of the concentration of antibiotic for the range of concentrations used.


43.2 reagents.


43.2.1. micro-organism: Micrococcus luteus (flavus) ATCC 10240.


43.2.1.1 maintenance of the strain.


Grow the culture medium (43.2.2.1) in inclined tubes, with Micrococcus luteus (flavus). Incubate for 24 hours at 30 ºc, store in a refrigerator at about 4 ºc and renew the sowing every fifteen days.


43.2.1.2 preparation of the bacterial suspension. Collect the bacteria from a tube of agar (43.2.1.1) preparation recently with the help of 2 to 3 ml of the solution of chloride of sodium (43.2.2.3). Sow with this suspension 250 ml of the culture medium (43.2.2.1) in a Roux flask and incubate for 18 to 20 hours at 30 ° C. Collect the bacteria in 25 ml of (43.2.2.3) sodium chloride solution and mix thoroughly.


Dilute the suspension to 1: 10 with the help of (43.2.2.3) sodium chloride solution. The light transmission of the solution, measured at 650 nm in 1 cm thick by comparison with the solution of chloride of sodium (43.2.2.3), should be around 75 by 100. This suspension can be kept a week at 4 ° C. Other methods may be used provided that it is shown that produce similar bacterial suspensions.


43.2.2. culture media and reagents.


43.2.2.1 means of maintenance of the strain.


Meat peptone: 6,0 g.


Tryptone: 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: 1000 ml.


pH: 6,5 to 6,6 (after sterilization).


Any means of commercial cultivation of similar composition, giving the same results may be used.


43.2.2.2 basic medium for the determination.


Tryptone: 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: 1000 ml.


pH: 6,5 (after sterilization).


Any means of commercial cultivation of similar composition, giving the same results may be used.


43.2.2.3 solution to the 0.8 per 100 w/v of sodium chloride: dissolve 8 g of chloride of sodium in water, dilute to 1000 ml and sterilize.


43.2.2.4 mixture of methanol/water/hydrochloric acid (43.2.2.6): 80/17,5/2,5 (v/v/v).


43.2.2.5 buffer phosphate pH 6.5.


Hydrogen phosphate potassium KHPO: 22.15 g.


Kihidrogeno phosphate potassium KHPO: 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 1 m sodium hydroxide solution


43.2.2.9 solution of 0.5 M sodium sulphide about.


43.2.2.10 the 0.04 per 100 bromocresol purple solution (w/v) dissolve 0,1 g of bromocresol purple in 18,5 ml of 0,01 M sodium hydroxide solution. Make up to 250 ml with water and mix.


43.2.2.11 reference substance: zinc bacitracin of known (in IU) activity.


43.3 procedure.


43.3.1. reference solutions. Weigh an amount of substance (43.2.2.11) corresponding to 1050 reference UI (according to the activity indicated). Add 5 ml of 0,1 M hydrochloric acid (43.2.2.7) and let stand 15 minutes. Add 30 ml of water, adjust the pH to 4,5 with phosphate buffer (43.2.2.5) (about 4 ml), make up to 50 ml with water and mix (1 ml = 21 UI).


Prepare by successive dilution with buffer solution and this solution phosphate pH 6.5 (43.2.2.5), the following solutions: 0.42 s IU/ml.


S: 0.21 IU/ml.


S: 0,105 IU/ml.


S: 0,0525 IU/ml.


43.3.2. preparation of extract.


43.3.2.1 extraction.


43.3.2.1.1 premixes and mineral proofreaders. Weigh a quantity of sample of 2,0 to 5,0 g, add 29.0 ml of the mixture (43.2.2.4) and 0.1 ml of the solution of sodium (43.2.2.9) sulfide; shake briefly. Check that the pH is about 2. Shake for 10 minutes, add 30 ml of buffer phosphate (43.2.2.5), shake for 15 minutes and centrifuge. Take an aliquot of the supernatant solution and adjust the pH to 6.5 with 1 M sodium hydroxide solution (43.2.2.8) using a pH meter or the solution of bromocresol indicator (43.2.2.10) purple.


Dilute phosphate buffer (43.2.2.5) to obtain phosphate buffer (42.2.2.5) to obtain a presumed 0.42 IU/ml bacitracin-zinc concentration (= U).


43.3.2.1.2 protein concentrates. Weigh a quantity of sample of 10,0 g, add 49,0 ml of the mixture (43.2.2.4) and 1,0 ml of sodium (43.2.2.9) sulfide solution; shake briefly. Check that the pH is about 2. Shake for 10 minutes, add 50 ml buffer phosphate (43.2.2.5), shake for 15 minutes and centrifuge. Take an aliquot of the supernatant solution and adjust the pH to 6.5 with the hydroxide solution of bromocresol indicator (43.2.2.10).


Evaporate to approximately half volume in a rotary evaporator at a temperature not exceeding 35 ° C. Dilute phosphate buffer (43.2.2.5) to obtain a so-called concentration of bacitracin zinc 0.42 IU/ml (= U).


43.3.2.1.3. other feedingstuffs. Weigh a quantity of sample of 10 g (20,0 or for an alleged concentration of bacitracin-zinc 5 mg/kg). Add 24,0 ml of the mixture (43.2.2.4) and 1,0 ml of sodium (43.2.2.9) sulfide solution; homogenize for 10 minutes. Add 25 ml buffer phosphate (43.2.2.5), shake for 15 minutes and centrifuge. Take 20 ml of the supernatant solution and adjust the pH to 6.5 with sodium (43.2.2.8) hydroxide solution, using a neutral pH or the solution of (43.2.2.10) as indicator bromocresol purple. Evaporate approximately up to 4 ml in a rotary evaporator at a temperature not exceeding 35 ° C. Dilute the residue with buffer phosphate (43.2.2.5) to obtain a so-called concentration of 0.42 IU/ml bacitracin-zinc (= U).


43.3.2.2 solutions of extract. Prepare the solution from U and by dilutions successive (1 + 1) with phosphate (43.2.2.5) buffer Solutions U (so-called concentration = 0.21 IU/ml), U (so-called concentration = 0,105 IU/ml) and U (so-called concentration = 0,0525 IU/ml).


43.3.3 modes.


43.3.3.1 inoculation of the culture medium. Sown at about 50 ° C the basic medium for the determination (43.2.2.2) with the bacterial suspension (43.2.1.2). By preliminary tests on plates with the medium (43.2.2.2), to determine the quantity of bacterial suspension which allows for different concentrations of bacitracin zinc zones of inhibition the most comprehensive while still being crisp.


43.3.3.2 preparation of boxes. Diffusion on agar is carried out in a box with the four concentrations of the solution of reference (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each case must necessarily receive four concentrations of extract and reference. So we must choose the dimension of the boxes so that can dig into the agar medium at least 8 cavities of 10 to 13 mm in diameter, whose centers distended not less than 30 mm. They can be used as boxes, glass plates flat, crowned by a ring of aluminum or plastic of 200 mm in diameter and 20 mm high.



Introduce number the medium (43.2.2.2), planted in boxes as indicated in 43.3.3.1, that allows obtaining a layer about 2 mm thick (60 ml for a box of 200 mm diameter). Let solidify, excavate cavities, and place in them exactly measured volumes of the extract and reference solutions (0,10-0,15 ml per cavity, according to the diameter). Repeat at least four times each concentration so that each determination is the evaluation of 32 zones of inhibition.


43.3.3.3 incubation. Incubate the trays for sixteen to eighteen hours at 30 ° C ± 2 ° C.


43.4 calculations.


Measure the diameter of the inhibition zones with an error not exceeding 0,1 mm. For each concentration, registration measures stockings on semi-log paper, transferring the logarithm of the concentrations in relation to the diameters of the zones of inhibition. Draw the most fitting straight lines for the reference solution and the extract, for example, by the following procedure: determine the most appropriate point of the lowest level of the solution of reference (SL) using the formula: (a) SL = 7S + 4S + S - 2S 10 determine the most appropriate point of the highest level of the reference (SH) using the formula solution : (b) SH = 7S + 4S + S - 2S 10 determine likewise points most suitable extract to the lowest level (UL) and the highest level (UH) by substituting S, S, S and S in the previous formulas by U, U, U and U.


Enter the SL and SH values on the same graph. By joining the two points, the tighter straight to the reference solution is obtained. If you follow the same method for UL and UH gets tighter line for the extract.


If there is no interference, the lines are parallel. In practice are considered parallel if (SH-SL) and (UH-SL) do not differ more than 10 per 100 of its media.


If the lines are not parallel, can be removed U and S or U and S. SL, SH, UL and UH values which allow to obtain more straight, are then calculated using the following formulas: 5S + 2S - 5S S + 2S - S (a) SL = - or - 6 6 5S + 2S - 5S S + 2S - S (b) SH = - or - 6 6 and similar formulas for UL and UH. The use of this alternative requires to comply with the same criteria of parallelism. Analytical bulletin a result from three levels should be noted.


When the lines are considered to be parallel, should calculate the logarithm of the relative activity (log A) by using one of the following formulae, depending on the number of levels (4 or 3) used for the assessment of parallelism.


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 activity of sample extract = activity of relevant 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, has repeated the determination proceeding to the appropriate settings of the concentrations of the extract or, where appropriate, the reference solutions. When this activity may not quite fit into the range of the required values, the result shall be deemed approximate and thus shall be indicated in the analysis report.


When the lines are not considered to be parallel, the determination will be repeated. If not now attaining the parallelism, the determination is deemed unsatisfactory.


Express the result in milligrams of zinc bacitracin per kilogram of feedingstuff.


The difference between the results of two determinations carried out on the same sample by the same analyst should not exceed: 2 mg/kg, in absolute value, for contents of less than 10 mg/kg zinc-bacitracin


20 per 100 of the highest result for contents from 10 to 25 mg/kg.


5 mg/kg, in absolute value, for contents of 25 to 50 mg/kg.


10 per 100 of the highest result for contents above 50 mg/kg.


43.5 references. Directive of the Commission of 20 December 1983 amending directives 71/393, 72/199 and 78/633 (Directive 84/4/EEC). «European Communities Official Journal» of 18 January 1984, number L 15/28, annex III.


44 Flavophospholipol (by diffusion on agar) 44.1 principle. The method makes it possible to dispense the flavophospholipol in feedingstuffs, concentrates and premixes. The lower limit of dosage is 1 mg/kg.


The sample is subjected to extraction diluted methanol by heating at reflux. The extract is centrifuged, purified, if necessary, in ion exchange resins and diluted. The antibiotic activity is determined by measurement of the diffusion of flavophospholipol in a medium of agar seeded with Staphylococcus aureus. The diffusion is made clear by the formation of microorganism inhibition zones. The diameter of these zones is directly proportional to the logarithm of the concentrations used.


44.2 reagents.


44.2.1. micro-organism: Staphylococcus aureus ATCC 6538 P.


44.2.1.1 maintenance of the strain. Grow the culture medium (44.2.2.1) in inclined tubes, with Staphylococcus aureus. Incubate for 24 hours at 37 ° C, keep in a refrigerator at 4 ° C approximately and renew the sowing every month.


44.2.1.2 preparation of the bacterial suspension. Take two tubes containing the mother (4.2.2.1) culture and renew the sowing every week. Incubate for 24 hours at 37 ° C and keep in a refrigerator at 4 ° C approximately.


Twenty-four hours before dosing, sown by these cultures, of two to four tubes inclined containing culture medium (44.2.2.1).


Incubate for 16 to 18 hours at 37 ºc. Then put the germs suspended in the solution of chloride of sodium (44.2.2.3). The light transmission of the suspension, measured at 578 nm under 1 cm thick, by comparison with the solution of sodium chloride, shall be 40 per 100 approximately. Other methods may be used if they show that they produce a similar bacterial suspension.


44.2.2. culture media and reagents.


44.2.2.1 means of maintenance of the strain.


Meat peptone: 6,0 g.


Tryptone: 4.0 g.


Yeast extract: 3.0 g.


Meat extract: 1.5 g.


Glucose: 1.0 g.


Agar: 15,0 g.


Water: 1000 ml.


pH = 6.5 after sterilization.


You can use any commercial medium of similar composition and give the same results, e.g. Oxoid Antibiotic Medium (327 CM) added to agar Oxoid No. 3 (L-13).


44.2.2.2 basic medium for dosage.


44.2.2.2.1 bottom layer.


Meat peptone: 6,0 g.


Yeast extract: 3.0 g.


Meat extract: 1.5 g.


Agar: 10,0 g.


Water: 1000 ml.


pH = 6.5 after sterilization.


You can use any commercial medium of similar composition and give the same results, e.g. Oxoid Antibiotic Medium 2 (335 CM) added to agar Oxoid No. 3 (L-13).


44.2.2.2.2 layer to grow. Measure (4.2.2.1) with 2 g silicone antifoam emulsion. For example is 2 Wacker Chemie Gmon, Munich.


44.2.2.3 the 0.4 per 100 solution (w/v) of sodium chloride: dissolve 4 g of p.a. sodium chloride in water and dilute to 1000 ml and sterilize.


44.2.2.4 pure methanol.


44.2.2.5 methanol 50 by 100 (v/v). Dilute 500 ml of methanol with 500 ml of water.


44.2.2.6 methanol at 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 1.5 per 100 methanolic solution (w/v) potassium chloride. Dissolve 1.5 g of chloride of potassium p.a. in 20 ml of water, make up to 100 ml with methanol (44.2.2.4).


44.2.2.9 cation exchanger: Dowex 50 WX8, 20-25 mesh, forms Na (cat, servo number 41600) or equivalent.


44.2.2.10 anion exchanger: Dowex 1 X 2, 50 100 mesh, form Cl/cat. Servo number 41010) or equivalent. Before using it, to maintain 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 indicator paper pH (pH 6-8, 6, 1).


44.2.2.13 Ascorbic acid.


44.2.2.14 chemical pattern. Flavophospholipol of known activity.


44.3 material and devices.


44.3.1 tube for chromatography, glass, inside diameter: 9 mm, length 150-200 mm, equipped with key at bottom narrowing and grinding standardized (for funnel joint) at the top.


44.3.2 funnel with a deposit of 250 ml, with ground key, standard.


44.3.3 Erlenmeyer of 250 ml, standard grinding.


44.3.4 reflux, standardized grinding coolant.


44.4 procedure.


44.4.1 standard solutions. Dissolve an accurately weighed amount of substance pattern (44.2.2.14) in methanol to 50 per 100 (44.2.2.5) and dilute to obtain a flavophospholipol of 100 micrograms/ml stock solution. preserved in capped vial at 4 ºc, this solution is stable for two months.


Prepare from this solution, and by successive dilutions using methanol to 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 summary.


44.4.2.1 extraction.



44.4.2.1.1 concentrates, premixes and mineral proofreaders. Weigh out a quantity of sample of 2-5 g and add about 150 mg of Ascorbic acid (44.2.2.13). Mix with 150 ml of methanol to 50 per 100 (44.2.2.5) in a conical flask (44.2.3.3) and adjust the pH to 8, 1-8, 2 by about 400 mg of tris (hydroxymethyl) aminomethane (44.2.2.7). Check the pH with indicator paper (44.2.2.12). Let marinate 15 minutes, readjust the pH 8, 1-8, 2, using tris (hydroxymethyl) aminomethane (44.2.2.7), and then boil for ten minutes with refrigerant to reflux (44.2.3.4), stirring constantly. Let cool, centrifuge and decant the extract.


44.4.2.1.2. other feedingstuffs. Weigh out a quantity of sample of 5 to 30 g containing at least 30 micrograms of flavophospholipol. Mix to 50 by 100 to 150 ml of methanol (44.2.2.5) in a conical flask (44.2.3.3) and adjust the pH to 8, 1-8, 2 400 mg approximately of tris (hydroxymethyl) aminomethane (4.2.2.7). Check the pH with indicator paper. Let marinate 15 minutes, readjust the pH 8, 1-8, 2 with tris (hydroxymethyl) aminomethane, then boil for ten minutes with (44.2.3.4) reflux condenser, stirring constantly. Let cool, centrifuge and decant the extract.


44.4.2.2 purification (you may omit this mode for concentrates, premixes and mineral concealers).


Mix 110 ml of the extract with 11 g of heat exchanger of cations (44.2.2.9), boil for one minute with refrigerant to reflux (44.2.3.4) shaking constantly. Separate the cation exchanger by centrifugation or filtration. Mix 100 ml of the extract with 150 ml of methanol (44.2.2.4) and leave the solution of twelve to fifteen hours at 4 ° C. Remove floculada by filtration cold matter.


Put (44.2.3.1) a glass (44.2.2.11)-wool plug at the lower end of a tube, pour 5 ml of (44.2.2.10) anion exchanger tube and wash the column with 100 ml of methanol to 80 per 100 (44.2.2.6). Then transfer to the column through funnel (44.2.3.2) a volume of filtering of 100 ml which is contains at least 16 micrograms of flavophospholipol (200 ml for a sample of 30 g of feedingstuff at 1 ppm). If necessary, dilute the filtrate before transfer it to the column with methanol at 80 per 100 (44.2.2.6) to obtain a presumed flavophospholipol from 16 micrograms in 100 ml. Regular concentration liquid outlet 2 ml flow approximately per minute. Delete all of the filtering. Then wash the column with 50 ml of methanol to 80 per 100 (44.2.2.6) and remove the filter.


Elute the flavophospholipol with the solution (44.2.2.8) potassium chloride methanolic keeping the drip rate approximately 2 ml per minute. Collect 50 ml of Elution in a flask, add 30 ml of water and mix thoroughly. This solution should have a content flavophospholipol of 0.2 micrograms/ml (= U).


44.4.2.3 solutions of extract. If necessary, (in particular in cases in which the purification had omitted), dilute the extract obtained in 44.4.2.1.1 with methanol to 50 by 100 (44.2.2.5) to obtain a presumed flavophospholipol of 0.2 microgram/ml concentration (= U).


Prepare from the solution or by successive dilutions (1 + 1) with methanol to 50 per 100 (44.2.2.5) solutions U (so-called concentration = 0.1 microgram/ml), U (so-called concentration = 0.05 microgram/ml) and U (so-called concentration = 0.025 microgram/ml).


44.4.3 modalities of the dosage.


44.4.3.1 inoculation of the culture medium. Inoculate at 50 ° C approximately the basic medium of dosing (44.2.2.2.2) with the bacterial suspension (44.2.1.2). Through preliminary tests in the middle layers (44.2.2.2.2) determine the quantity of bacterial suspension that allows for the various concentrations in flavophospholipol, zones of inhibition as extensive as possible and which still remain crisp (about 30 ml per liter).


44.4.3.2 preparation of boxes. Diffusion on agar is carried out in a box with the four concentrations of the solution pattern (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each case must necessarily receive four concentrations of extract and pattern. To this end, choose the dimension of the boxes so that can hollow out in the agar medium at least 8 cavities of 10 to 13 mm in diameter, whose centers are separated by less than 30 mm. It may be used as boxes, glass plates flat, crowned by a ring of aluminum or plastic of 200 mm in diameter and 20 mm high.


Enter a quantity of the medium (44.2.2.2.1) that allows to obtain a coating of 1.5 mm thick (45 ml for a box of 200 mm diameter) approximately in the boxes. Allow to solidify and add a quantity of the medium (44.2.2.2.2), planted as described in section 44.3.3.1, that allow to obtain a layer 1 mm thick (30 ml for a box of 200 mm diameter). Let solidify, hollow cavities and place in them exactly measured volumes of solutions pattern and extract (0,10-0,15 ml per cavity, according to diameter).


Make at least four replicates of each concentration so that each determination is subject to an evaluation of 32 inhibition zones.


43.4.3.3 incubation. Incubate the trays of sixteen to eighteen hours at 29-30 ºc.


44.5 calculations. Measure the diameter of the zones of inhibition with an approximation of 0.1 mm. For each concentration, record the measurements on semi-log paper scoring the logarithm of the concentrations against the diameter of the inhibition zones. Trace the lines better adjusted to the standard solution and the extract proceeding, for example, in the following way: determine the most appropriate of the lowest level of the solution point pattern (SL) using the formula: (a) SL = 7S + 4S + S - 2S 10 determine the most appropriate high level of solution point pattern (SH) using the formula : (b) SH = 7S + 4S + S - 2S 10 Similarly determine the most appropriate points of the extract to the lowest level (UL) and the highest level (UH) by substituting S, S, S and S in the formulas above for U, U and U.


Represent the SL and SH values on the same graph. Joining two points, will give the tighter line for the standard solution. Proceed similarly for UL and UH, will be the tighter line for the extract.


In the absence of any interference the lines should be parallel. In practice, it shall be deemed them as if they were parallel when (SH-SL) and (UH-UL) do not differ by more than 10 per 100 of its media.


If the lines are not parallel, you can remove either U and S or U and S. Values SL, SH, UL and UH that allow to obtain the more straight lines are then calculated using the following formulas: 5S + 2S - 5S S + 2S - S (a') SL = - or - 6 6 5S + 2S - 5S S + 2S - S (b) SH = - or - 6 6 and similar formulas for UL and UH. The use of such alternative should also be a verification as to the parallelism of the lines indicated above. A result from three levels should be mention in the analysis report.


When considering the straight lines as parallel, calculate the logarithm of the relative activity (log A), using one of the following formulae: (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 real activity = activity so-called x relative activity.


When the lines are not considered as non-parallel, repeat the determination. If it continued without reaching the parallel, calculate the logarithm of the activity on (log. A) by the formula (c). However the result obtained as approximate should be considered and will be coming to mention it in the analysis report.


The difference between the results of two determinations carried out on the same sample by the same analyst should not exceed:-0.5 mg/kg, in absolute value, for contents of flavophospholipol from 1 to 2 mg/kg.


-25 per 100 of the highest result for contents of more than 2 mg/kg and up to 10 mg/kg.


-20 per 100 of the highest result for contents of more than 10 mg/kg and up to 25 mg/kg.


-5 mg/kg, in absolute value, for contents of above 25 mg/kg and up to 50 mg/kg.


-10 per 100 of the higher result for contents above 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 July 29, 1978, page 43, pages 5 to 9 of the annex.


45 tylosin (by diffusion on agar) 45.1 principle. The method makes it possible to determine the tylosin in feedingstuffs, concentrates and premixes. The lower limit of determination is 2 mg/kg.


The sample is treated by buffer phosphate pH 8, previously led to 80 ° C, and is then subject to removal by methanol. After centrifuging, the extract is diluted and its antibiotic activity is determined by measurement of the diffusion of the tylosin in the midst of agar seeded with Sarcina lutea. The broadcast is indicated by the formation of inhibition zones in the presence of the micro-organism. The diameter of these zones is directly proportional to the logarithm of the antibiotic concentration.


45.2 reagents.


45.2.1. micro-organism: Sarcina lutea ATCC 9341 number.



45.2.1.1 maintenance of the strain. Sow Sarcina lutea in tube of agar slant with the culture (45.2.2.1), adjusted to pH 7.0. Incubate overnight at 35 ° C approximately. Keep the culture in a refrigerator and replant every month on inclined agar.


45.2.1.2 preparation of suspension of germs. Collect the bacteria from a tube of agar slant (45.2.1.1) recently prepared with 2 to 3 ml of physiological saline (45.2.2.4). With this suspension seed a Roux flask containing 250 ml of the medium (45.2.2.1), adjusted to pH 7.0. Incubate for 24 hours at 35 ° C, collect the bacteria in 25 ml of physiological saline (45.2.2.4). Homogenize, and dilute this suspension to obtain approximately a light transmission of 75 per 100 at 650 nm.


Preserved in refrigerator, this suspension is usable for a week.


By preliminary tests on plates with the basic medium for the determination (45.2.2.1), establish the quantity of inoculum which allow to obtain, for the different concentrations of tylosin used, zones of inhibition as extensive as possible are also sharp. Inoculation of the culture medium is made at 48-50 ° C.


45.2.2. culture media and reagents.


45.2.2.1 middle of the basis for the determination.


Glucose: 1.0 g.


Peptone peptone: 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 to 1000 ml.


Set at the time of use to pH 7.0 for maintenance of the strain and the preparation of suspension of germs and pH 2.0 for the determination.


Any commercial medium of similar composition and give the same results, can be used.


45.2.2.2 buffer phosphate pH 8.


Dihydrogen phosphate potassium KHPO p.a.: 0,523 g.


Hydrogen phosphate potassium KHPO p.a.: 16,730 g.


Distilled water to 1000 ml.


45.2.2.3 buffer phosphate pH 7.


Dihydrogen phosphate potassium KHPO p.a.: 5.5 g.


Hydrogen phosphate potassium KHPO p.a.: 13.6 g.


Distilled water to 1000 ml.


45.2.2.4 sterile physiological saline.


45.2.2.5 pure methanol.


45.2.2.6 methanol to 10 per 100 (v/v).


45.2.2.7 mixture of buffer phosphate (pure 45.2.2.2)/metanol (60/40 v/v).)


45.2.2.8 reference substance: tylosin of known.


45.3 procedure.


45.3.1. reference solutions. Dry the reference solution (45.2.2.8) for 3 hours at 60 ° C in a vacuum oven (5 mm of mercury). Weigh 10 to 50 mg into a graduated flask, dissolve in 5 ml of methanol (45.2.2.5) and dilute the solution through the buffer phosphate pH 7 (45.2.2.3) to obtain a tylosin-base of 1,000 micrograms/ml concentration. From this stock solution, prepare by diluting with the mixture (45.2.2.7) a solution of reference work containing 2 micrograms of tylosin base per ml.


Then prepare by successive dilutions (1 + 1) using 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. extraction. For highly concentrated among tylosin products take a portion of 10 g sample; for premixes and feedingstuffs, a portion of the sample of 20 g. Add 60 ml buffer phosphate pH 8 (45.2.2.2), previously heated to 80 C, and homogenize for 2 minutes (appliances Ultra-turrax etc.)


Let stand for 10 minutes, add 40 ml of methanol (45.2.2.5) and homogenize for 5 minutes. Centrifuge, take an aliquot part of the extract and dilute with the solution (45.2.2.7) to obtain a presumed tylosin of 2 micrograms/ml concentration (= U). Then prepare the concentrations U, U and U by successive dilutions (1 + 1) using the solution (45.2.2.7).


For contents of less than 10 mg/kg evaporates extract dry in a rotary evaporator at 35 C and dissolve the residue in methanol to 40 per 100 (45.2.2.6).


45.3.3. modalities of definition.


45.3.3.1 planting of the culture medium. Plant 48 - 50 ° C the basic medium for 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 boxes. Diffusion on agar is carried out in some boxes with the four concentrations of the solution of reference (S, S, S and S) and 4 concentrations of the extract (U, U, U and U). Each case must necessarily receive four concentrations of extract and reference.


To this end, choose the dimensions of the boxes so that at least eight cavities of 10 to 13 mm in diameter to practice in the agar medium. Calculate the amount of the seeded medium (45.3.3.1) that will be used so that a uniform coating of 2 mm can be obtained approximately thick. It is preferable to use as boxes, glass plates flat, fitted with a ring of aluminum or plastic material perfectly flat 200 mm in diameter and 20 mm high.


Insert into cavities by pipette amounts exactly measures of antibiotic solution between 0.10 and 0.15 ml, depending on diameter.


For each sample, do at least four repetitions of each concentration diffusion, so that each determination is subject to an evaluation of 32 inhibition zones.


45.3.3.3 incubation. Incubate the trays overnight at 35 ± 37 ° C.


45.4 calculations. Measure the diameter of the inhibition zones, preferably by projection. Represent the measures on semi-log paper, taking the logarithm of the concentrations in relation to the diameters of the zones of inhibition. Draw the straight lines of reference and extract solution. In the absence of interference, the two lines should 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 - real SActividad = supposed 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, on May 29, 1972.


46 Spiramycin (method by diffusion on agar) 46.1 principle. The method enables you to determine the Spiramycin in feedingstuffs and premixes. The lower limit of detection is 1 ppm (1 mg of Spiramycin is equivalent to 3,200 international units). The sample is subjected to extraction with a mixture of methanol and buffer phosphate-hydrogen carbonate to pH 8. Extract decant or spin, and then dissolves. The antibiotic activity of the extract is determined by measuring the diffusion of Spiramycin in agar, planted with Micrococus medium luteus. Broadcast is manifested by the formation of the microorganism inhibition zones. The diameter of these zones is directly proportional to the logarithm of the concentration of antibiotic for the range of concentrations used.


46.2 reagents.


46.2.1. micro-organism: Micrococus luteus ATCC 9341 (NCTC 8340, NCIB 8553).


46.2.2. maintenance of the strain. Grow the culture medium (46.2.4) in inclined tubes, with Micrococus luteus. Incubate for 24 hours at 30 ° C, keep in a refrigerator at 4 ° C approximately and renew the sowing every fifteen days.


46.2.3. preparation of the bacterial suspension (46.5.1). Pick up the germs in a tube of agar (46.2.2) recently prepared, using 2 to 3 ml of solution of chloride of sodium (46.2.6). Sow with this suspension 250 ml of culture medium (46.2.4) in a Roux flask and incubate for 18 to 20 hours at 30 ° C. Collect the bacteria in 25 ml of (46.2.6) sodium chloride solution and mix thoroughly. Dilute the suspension to 1/10 (46.2.6) sodium chloride solution using. The light transmission of the suspension, measured at 650 nm, a thickness of 1 cm compared with the solution of chloride of sodium (46.2.6) under, shall be 75 per 100 approximately. This suspension can be kept a week at 4 ° C approximately.


46.2.4 maintenance of strain (46.5.2) culture media.


Meat peptone: 6,0 g.


Tryptone: 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: 1000 ml.


pH 6,5 - 6,6 (after sterilization).


46.2.5 basis of the determination (46.5.2) culture medium.


Tryptone: 5,0 g.


Yeast extract: 4.0 g.


Meat extract: 3.0 g. Agar: 10,0 to 20,0 g.


Water: 1000 ml.


pH: 8,0 (after sterilization).


46.2.6 0.8 per 100 solution (w/v) of sodium chloride. Dissolve 8 g of sodium chloride in water, diluted in 1000 ml and sterilize.


46.2.7 buffer phosphate-hydrogen carbonate, pH 8.0.


KHPO potassium phosphate: 16.7 g.


KHPO potassium dihydrogen phosphate: 0.5 g.


Hydrogen carbonate sodium NaHCO: 20.0 g.


Water up to 1,000 ml.


46.2.8 mixture of methanol and buffer fosfato-bicarbonato (46.2.7). 50/50 (v/v).


46.2.9 chemical pattern. Spiramycin of known activity (IU).


46.2.10 standard solution. Dissolve an accurately weighed amount of the substance pattern (46.2.9) in the mixture (46.2.8) and dilute with the same mixture to obtain a stock solution of 1000 IU of Spiramycin/ml.


Preserved in flask with ground glass stopper at 4 ºc, this solution is stable for five days.


Prepare this solution and by successive dilutions using 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 out a quantity of sample of 20.0 g for feed and 1.0 to 20,0 g for premixes. Add 100 ml of the mixture (46.2.8) and shake for 30 minutes.


Centrifuge or decant, then dilute the solution that floats on the surface with the help of the mixture (46.2.8) to obtain a presumed Spiramycin of 1 IU/ml concentration (U).


For contents in Spiramycin less than 2.5 mg/kg of feed, perform extraction as follows. Despite a number of sample from 20.0 g. Add 100 ml of mixture (46.2.8), shake for 30 minutes, then centrifuge for a few minutes. Take 50 ml of the supernatant solution and evaporate to about 4 ml approximately under reduced pressure in a rotary evaporator at a temperature not exceeding 40 ° C. Dilute the residue with the help of the mixture (46.2.8) to obtain a presumed Spiramycin 1 IU/ml concentration (U).


On the basis of the solution U, prepare by successive dilutions (1 + 1), using the mixture (46.2.8), the solutions whose alleged 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. Planting approximately the basic medium for the determination (46.2.5) with the bacterial suspension (46.2.3) at about 50 ºc. By preliminary tests on plates with the medium (46.2.4), determine the quantity of bacterial suspension that allows for the different concentrations of Spiramycin, zones of inhibition the most extensive possible and that they are still clean.


46.3.3 preparation of the plates. Diffusion on agar is carried out in plates with the four concentrations of the solution pattern (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 extract. For this purpose, we had to choose the dimensions of the plates in such a way that it could be in the agar at least eight cavities of 10 to 13 mm in diameter, whose centers are situated at a distance of at least 30 mm. As plate glass plates can be used flat, topped by a ring of aluminum or plastic of 200 mm in diameter and 20 mm high.


Enter a quantity of the medium (46.2.5), planted on plates as shown in (46.3.2), which allows obtaining a layer of 2 mm thick (60 ml for a plate of 200 mm diameter). Let solidify, making the cavities and deposited exactly measured volumes of solutions of the pattern and the extract (0,10-0,15 ml per cavity, according to diameter). Perform at least four replicates of each concentration so that each determination is subject to an evaluation of 32 inhibition zones. Incubate the plates for 16 to 18 hours at 30 ° C ± 2 ° C.


46.4 calculations. Measure the diameter of the zones of inhibition with an accuracy of 0.1 mm. For each concentration, register the media on semi-log paper values, taking the logarithms of the concentration on the diameters of the zones of inhibition. Draw the most fitting straight lines for the standard solution and the extract proceeding, for example, as follows: determine the most appropriate point of the lowest level of the solution (SL) using the formula: (a) SL = 7s + 4s + s - 2s 10 determine the most appropriate high level of solution point pattern (SH) using the formula : (b) SH = 7s + 4s + s - 2s 10 determine likewise points most suitable extract to the lowest level (UL) and the highest level (UH) by substituting s, s, s and s in the formulas mentioned before for u, u, u and u. being lowercase letters "s" and "u" the diameters of the zones of inhibition. Take the SL and SH values to the same graph. Joining two points Gets the tighter line for the standard solution. Proceed similarly for UL and UH gets tighter line for the extract.


If there is no interference, the lines will be parallel. In practice, are considered parallel when SH-SL and UH-UL do not differ by more than 10 per 100 of its environment.


If the lines are not parallel, they can be removed either u and s or u and s. Values SL, SH, UL and UH that can produce the most fitting straight lines are then calculated using the following formulas: 5s + 2s - 5s s + 2s - s (a') SL = - or - 6 6 5s + 2s - 5s s + 2s - s (b') SH = - or - 6 6, and similar formulas for UL and UH. The use of this alternative requires that the same criteria of parallelism are respected. A result from three levels should be mentioned in the analysis report.


When the lines are parallel, calculate the logarithm of the relative activity (log. A) by using one of the formulas that follow, according to the number of levels (four or three) used for the assessment of parallelism.


For four 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 activity of sample extract = activity of the corresponding pattern (U - S x A) x if the relative activity is not the range of values between 0.5 and 2.0, repeat the determination proceeding to the appropriate settings of the concentrations of the extract or, where appropriate, solutions pattern. When this activity can not move to the range of the required values, the result should be considered approximate and this indication will be noted in the analysis report.


When the lines are considered to be non-parallel, repeat the determination. If parallelism is not never reached, the determination shall be deemed unsatisfactory.


Express the result in milligrams of Spiramycin base per kg of feed.


The differences in the results of two parallel determinations carried out on the same sample by the same person may not exceed:-2 mg/kg, in absolute value, for contents of Spiramycin base 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 contents of 25 to 50 mg/kg.


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


46.5 observations.


46.5.1 can be used other methods, provided that it is shown that produce similar bacterial suspensions.


46.5.2 any commercial medium of similar composition that gives the same results may be used.


46.6 references. Tenth directive community, of July 25, 1984, 84/425/EEC. «Official Journal of the European Communities' number 238 L/34, of 6 September 1984.


47 Monensin sodium (method by diffusion on agar) 47.1 principle. The method enables you to determine monensin sodium in feedingstuffs and premixes. 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 90 per 100. Applies to extract the appropriate according to monensin sodium content of sample treatments. Its antibiotic activity is determined by measuring the spread of monensin sodium in the midst of agar seeded with Bacillus subtilis. Diffusion is manifested by the formation of microorganism inhibition zones. The diameter of these zones is directly proportional to the logarithm of the concentration of antibiotic for the range of concentrations used. The sensitivity of this method is reduced in the presence of sodium ions.


47.2 material and devices.


47.2.1. Rotary evaporator, with 250 ml round-bottomed flask.


47.2.2 columns of glass for chromatography, internal diameter: 25 mm, length: 400 mm, with a diameter of 2 mm at the lower end.


47.2.3 column of glass for chromatography, internal diameter: 11 mm, length: 300 mm approximately, with a diameter of 2 mm at the lower end.


47.3 reagents.


47.3.1. micro-organism: Bacillus subtilis ATCC 6633 (NCIB 8054).


47.3.2 conservation of the strain.


Plant cultivation (47.3.4), in inclined tubes and environment with Bacillus subtilis. Incubate overnight at 30 ° C, store in refrigerator at 4 ° C and renew the sowing every month.


47.3.3 preparation of suspension of spores (47.6.1). Collect the bacteria from a tube of agar (47.3.2) recently prepared, using 2 to 3 ml of sterile water. Planting with this suspension 300 ml of culture medium (47.3.4) in a Roux flask and incubate for three to five days at 30 ° C. After controlling sporulation under a microscope, collect the spores in 15 ml of ethanol (47.3.6) and mix thoroughly. This suspension can be kept five months at 4 ° C approximately.


47.3.4 maintenance of strain (47.6.2) culture medium.


Tryptone: 10,0 g.


Yeast extract: 3.0 g.


Meat extract: 1.5 g.


Glucose: 1.0 g.


Agar (depending on quality): 10,0 to 20,0 g.


Water: 1000 ml.


pH: 6,5 (after sterilization).


47.3.5 medium basis for the determination.


Glucose: 10,0 g.


Yeast extract: 2.5 g.


Hydrogen phosphate of potassium (KHPO): 0.69 g.


(KHPO) potassium dihydrogen phosphate: 0,45 g.


Agar (depending on quality): 10,0 to 20,0 g.


Water: 1000 ml.


pH: 6,0 (after sterilization).


47.3.6 20 per 100 ethanol (v/v). Dilute 200 ml of ethanol with 800 ml of water.


47.3.7 anhydrous methanol.


47.3.8 methanol 90 per 100 (v/v). Dilute 900 ml of methanol (47.3.7) with 100 ml of water.


47.3.9 methanol 50 by 100 (v/v). Dilute 500 ml of methanol (47.3.7) with 500 ml of water.


47.3.10 granulated aluminium oxide (alcoa F, 20 mesh: Activated Alumina UG: F. Lancester and Co., or equivalent).



47.3.11 standard 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 accurately weighed amount of the substance pattern (47.3.11) in methanol (47.3.7), dilute to obtain a stock solution of monensin sodium of 800 micrograms/ml. keep in bottle capping at 4 ºc, this solution is stable for two weeks.


Prepare this solution and by successive dilutions with methanol to 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. Premixes. Despite 2,0 g of the sample, add 100 ml of methanol to 90 by 100 (47.3.8), mix thoroughly and then centrifuge for a few minutes. Dilute the supernatant solution with methanol to 50 per 100 (47.3.9) to obtain a concentration approximate monensin sodium of 8 micrograms/ml (U).


47.4.2 extraction. Feed in which the monensin sodium content is not less than 50 mg/kg.


Despite 10,0 to 20,0 g of the sample, add 100 ml of methanol to 90 by 100 (47.3.8), homogenize for 15 minutes and leave.


Insert a plug of cotton in the bottom of the glass (47.2.2) column, add oxide aluminium (47.3.10), giving light shocks to the column, until the filling has 75 to 80 mm high.


Decant the extract in the aluminium oxide column and collect the filtrate. Dilute 30 ml of the filtrate to 50 ml with water.


Then dilute with methanol to 50 per 100 (47.3.9) to obtain a concentration approximate monensin sodium of 8 micrograms/ml (U).


47.4.3 extraction. Feed in which the monensin sodium content is less than 50 mg/kg (up to the limit of 10 mg/kg).


Despite 10,0 to 20,0 g of the sample, add 100 ml of methanol to 90 by 100 (47.3.8), homogenize for 15 minutes. Centrifuge for a clean summary.


Take 40 ml of the supernatant to a sample with monensin sodium content of 20 mg/kg; 80 ml for a sample with a content of 10 mg/kg. Evaporate the vacuum with rotary evaporator to dryness (47.2.1) at a temperature not exceeding 40 ° C. Dissolve residue with 10 ml of methanol to 90 by 100 (47.3.8).


Insert a plug of cotton in the bottom of the glass (47.2.3) column, add oxide aluminium (47.3.10), giving light shocks to the column, until the filling has 75 to 80 mm high.


Decant the solution methanolic of residue on the aluminium oxide column and collect the filtrate. Wash the column with 10 ml of methanol to 90 by 100 (47.3.8) and join the previous filtering.


Evaporate the solution to the vacuum in a rotary evaporator to dryness (47.2.1) a temperature lower than 40 ° C. Dissolve residue with 10 ml of anhydrous methanol (47.3.7) and make up to 20 ml with water.


Centrifuge at 4000 rpm for five minutes as a minimum. Then dilute with methanol to 50 per 100 (47.3.9) to obtain a concentration approximate monensin sodium of 8 micrograms/ml (U).


47.4.4 solutions of extract. Prepare the solution from U and by successive dilutions (1 + 1) with methanol to 50 per 100 (47.3.9) solutions whose alleged concentrations are U (4 micrograms/ml), U (2 micrograms/ml) and U (1 microgram/ml).


47.4.5 modalities of definition. Inoculate at 50-60 ° C the culture medium base for determination (47.3.5) with the suspension of spores (47.3.3). By preliminary tests on plates with the medium (47.3.5), establish the quantity of inoculum which allows to obtain the broader inhibition zones which are also clear for the various concentrations of monensin sodium.


47.4.6 preparation of the plates. Diffusion on agar is carried out in plates with the four concentrations of the solution pattern (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each plate must necessarily receive four concentrations of pattern and extract. For this purpose, it is necessary to choose the size of the plates so can practice in the agar medium at least 8 cavities of 10 to 13 mm in diameter, whose centers not distended each other less of 30 mm. They can be used as plates, flat sheets of glass, topped with a ring of aluminum or plastic 200 mm in diameter and 20 mm high.


Enter a quantity of the medium (47.3.5), planted on plates as shown (47.4.5), so as to obtain a layer about 2 mm thick (60 ml for a plate of 200 mm diameter). Let it solidify, open cavities and deposit solutions exactly measured volumes pattern and extract (0,10-0,15 ml per cavity, according to diameter). Perform at least four replicates for each concentration so that each determination is subject to an evaluation of 32 inhibition zones.


47.4.7 incubation. Incubate the nine plates for eighteen hours approximately at 35 - 37ºc.


47.5 calculations.


Measure the diameter of the zones of inhibition with an accuracy of 0.1 mm. For each concentration, scoring average on semi-log paper values representing the logarithm of the concentrations against the diameter of the inhibition zones. Draw the most fitting straight lines for the standard solution and the extract proceeding, for example, as follows: determine the most appropriate of the lowest level of the solution point pattern (SL) using the formula: (a) SL = 7S + 4S + S - 2S 10 determine the most appropriate point of the highest level of the reference (SH) using the formula solution : (b) SH = 7S + 4S + S - 2S 10 equally determine points most suitable extract to the lowest level (UL) and the highest level (UH) by substituting S, S, S and S for U, U, U and U in the previous formulas.


Register the SL and SH values on the same graph. Joining two points Gets the tighter line for the standard solution. Proceed similarly for UL and UH gets tighter line for the extract.


In the absence of interference, the lines should be parallel. In practice are considered parallel when SH-SL and UH-UL does not differ more than 10 per 100 of its media.


If the lines are not parallel, can be removed U and S or U and S. Values SL, SH, UL and UH that can produce the most fitting straight lines are calculated using the following formulas: 5S + 2S - 5S S + 2S - S (a) SL = - or - 6 6 5S + 2S - 5S S + 2S - S (b) SH = - or - 6 6 and with similar formulas for UL and UH. The use of this alternative requires that the same criteria of parallelism are respected. A result from three levels should be mentioned in the analysis report.


Once considered the straight parallel, calculate the logarithm of the relative activity (log. A) by 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 real activity = supposed activity x relative activity.


If the relative activity is less than values included between 0.5 and 2.0, repeat the determination by making the appropriate concentrations of the extract settings or if it is necessary, the solution pattern. If the activity cannot be carried between the two previous values, the result should be considered approximate and this should be noted in the analysis report.


Given that the lines are not parallel, repeat the determination. If parallelism is not achieved, the determination should be considered unsatisfactory.


The difference between results of two determinations carried out on the same sample by the same person should not exceed:-20 per 100 of the highest result for contents of monensin sodium between 10 and 25 mg/kg.


-5 mg/kg, in absolute value, for contents of between 25 and 50 mg/kg.


-10 per 100 of the higher result for contents above 50 mg/kg.


47.6 observations.


47.6.1 other methods can be used to the extent that is proven to produce similar spore suspensions.


47.6.2 can use all means of commercial cultivation of similar composition and give the same results.


47.7 references. Ninth Commission directive of 31 of July 1981, 81/715/EEC. «Official Journal of the European communities» number L 257/9, on September 10, 1981.


48 Avoparcin (by diffusion on agar) 48.1 principle. The method allows the determination of Avoparcin in feedingstuffs and premixes. The lower limit of detection is 2 mg/kg. The polyether antibiotics interfere with 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 Avoparcin in a medium of agar, planted with Bacillus subtilis. Diffusion is manifested by the formation of microorganism inhibition zones. The diameter of these zones is directly proportional to the logarithm of the concentration of antibiotic for the range of concentrations used.


48.2 reagents.


48.2.1 microorganism. Bacillus subtilis ATCC 6633 (NCIB 8054).


48.2.2. conservation of the strain. Grow the culture medium (48.2.4), in inclined tubes, with Bacillus subtilis. Incubate overnight at 30 ° C, keep in a refrigerator at 4 ° C approximately and renew the sowing every month.



48.2.3. preparation of the spore suspension. (See 48.5.1). Collect the bacteria from a tube of agar (48.2.2) recently prepared, with 2 or 3 ml of sterile water. Planting with this suspension 300 ml of culture medium (48.2.4) in a Roux flask and incubate for three to five days at 30 ° C. After controlling sporulation under a microscope, collect the spores in 15 ml of ethanol (48.2.5) and mix thoroughly. This suspension can be kept for up to five months at 4 ° C approximately.


48.2.4. means of maintenance of strain (48.5.2).


Peptone: 6,0 g.


Tryptone: 4.0 g.


Yeast extract: 3.0 g.


Meat extract: 1.5 g.


Glucose: 1.0 g.


Agar: 15,0 g.


Water: 1000 ml.


pH: 6,5 (after sterilization).


48.2.5 20 per 100 ethanol (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 2 m sodium hydroxide solution


48.2.8 buffer phosphate 0.1 M. weigh 13.6 g of dihydrogen phosphate potassium (KHPO) and dilute to 1000 ml with water and adjusted to pH 4.5.


48.2.9 mixing acetone/water/acid hydrochloric (48.2.6): 65/32,5/2.5 (v/v/v).


48.2.10 chemical pattern. Sulphate of known activity Avoparcin.


48.2.11 standard solution. Dissolve 10 mg, exactly heavy, substance pattern (48.2.10) in buffer phosphate (48.2.8) and dilute with this buffer to obtain a solution of 100 micrograms/ml Avoparcin. preserve in capped vial at 4 ° C as a maximum seven days.


48.2.12 standard for premixes solutions. Prepare by successive dilution with buffer solution (48.2.11) and phosphate (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 patterns. Prepare by successive dilution with buffer solution (42.2.11) and phosphate (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 the solutions. Premixes. Weigh, with precision of 10 mg, a sample containing 10 to 100 mg Avoparcin, transfer to a 100 ml volumetric flask, add 60 ml of the mixture (48.2.9) and stir for 15 minutes with mechanical stirrer. Check the pH and adjust to pH = 2, if necessary, with hydrochloric acid (48.2.6). Make up to volume with the mixture (48.2.9) and mix thoroughly. Filter paper Whatman No. 1 part or similar and delete the first 5 ml. take an aliquot and adjust the pH to 4,5 with sodium (48.2.7) hydroxide solution. Dilute this solution with phosphate buffer (48.2.8) to obtain a presumed concentration 4 micrograms/ml (U) of Avoparcin.


Prepare this solution and by successive dilutions (1 + 1) with buffer phosphate (48.2.8), the so-called solutions U (2 micrograms/ml), U (1 microgram/ml) and U (0.5 micrograms/ml). 48.3.2 preparation of the extract and the solutions. Feed. Weigh a sample of 50 g, add 100 ml of mixture (48.2.9) and shake for 30 minutes with mechanical stirrer. Clarify the extract by centrifugation (in stoppered tubes), take an aliquot of the extract clean (see table) and adjusted to pH 4,5 with sodium (48.2.7) hydroxide solution. Dilute this solution with buffer phosphate (48.4.8) to get the solution U (see table).


TABLE content in Avoparcin (mg/kg) / 5 / 7.5 / 10 / 15 / 20 / 40 weight of sample in g (± 0,1 g) / 50 / 50 / 50 / 50 / 50 / 50 mix volume (ml) (48.2.9) / 100 / 100 / 100 / 100 / 100 / 100 volume of extract clean (ml) / 20 / 15 / 20 / 15 / 20 / 10 final volume (ml): U / 25 / 25 / 50 / 50 / 100 / 100 so-called concentration in micrograms/ml. / 2 / 2 approx. / 2 / approx. 2 / 2 / 2 prepare from this solution and by successive dilutions (1 + 1) with buffer phosphate (48.2.8) the so-called solutions U (1.0 microgram/ml), U (0.5 micrograms/ml), U (0.25 mcg/ml).


48.3.3 determination. Inoculation of the culture medium. Inoculate at 50-60 ° C the average basis of determination (48.2.4) with the suspension of spores (48.2.3). By preliminary tests on plates with the medium (48.2.4), determine the amount of inoculum to the broader areas of inhibition that are also sharp, for the different concentrations of Avoparcin.


48.3.4 determination. Preparation of the plates. Diffusion on agar is carried out in plates with the four concentrations of the solution pattern (S, S, S and S) and the four concentrations of the extract (U, U, U and U).


Each plate must necessarily receive four concentrations of extract and pattern. To do so, choose the dimensions of the plates in such a way that you can practice in the agar medium at least eight cavities of 10 to 13 mm in diameter, whose centers not distended each other less of 30 mm. They can be used as plates, flat sheets of glass, crowned an aluminium or plastic ring 200 mm in diameter and 20 mm high.


Insert into the plates a quantity of the medium (48.2.4), seeded as indicated in (48.3.3), that allows obtaining a layer about 2 mm thick (60 ml for a plate of 200 mm diameter). Let it solidify, open cavities and deposited exactly measured volumes of solutions pattern and extract (0,10-0,15 ml per cavity, according to the diameter). Perform at least four replicates for each concentration so that each determination is subject to an evaluation of 32 inhibition zones.


48.3.5 incubation. Incubate the plates between sixteen and eighteen hours at 30 ° C.


48.4 calculations. Measure the diameter of the zones of inhibition with an accuracy of 0.1 mm. For each concentration, registration measures stockings on semi-logaritmico paper, representing the logarithm of the concentrations against the diameter of the inhibition zones. Draw the most fitting straight lines for the standard solution and the extract proceeding, for example, as follows: determine the most appropriate of the lowest level of the solution point pattern (SL) using the formula: (a) SL = 7S + 4S + S - 2S (a) SL = 10 determine the most appropriate of the highest level of the solution point pattern (SH) using the formula : (b) SH = 7S + 4S + S - 2S (b) SH = 10 Similarly determine the most appropriate points of the extract to the lowest level (UL) and the highest level (UH) by substituting S, S, S and S in the formulas for U, U, U and U.


Take the SL and SH values on the same graph. Joining two points Gets the tighter line for the standard solution. Proceed similarly for UL and UH gets tighter line for the extract.


In the absence of any interference, the lines should be parallel when SH-SL and UH-UL does not differ more than 10 per 100 of its media.


If the lines are not parallel, can be eliminated well U and S or else U and S. Values SL, SH, UL and UH that can produce the most fitting straight lines are calculated according to: 5S + 2S - 5S S + 2S - S (a') SL = - or - 6 6 5S + 2S - 5S S + 2S - S (b') SH = - or - 6 6, and similar formulas for UL and UH. The use of this alternative requires that the same criteria of parallelism are respected. A result from three levels should be mentioned in the analysis report.


When the lines are 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 real activity = supposed activity x relative activity.


If the relative activity is below values between 0.5 and 2.0, repeat the determination proceeding to the appropriate settings of the concentrations of the extract or, where appropriate, solutions patterns. If this activity cannot be included among these values, the result should be considered approximate and this must appear in the analysis report.


When the lines are not parallel, repeat the determination. If parallelism is not achieved, the determination should be considered as unsatisfactory.


The difference between the results of two determinations carried out on the same sample by the same person should not exceed:-2 mg/kg, in absolute value, for contents of Avoparcin from 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 above 50 mg/kg.


48.5 observations.


48.5.1 other methods can be used to the extent that are proven to produce similar spore suspensions.


48.5.2 other means of commercial culture that has a similar composition, and give the same results may be used.


48.6 references. Ninth Commission directive of 31 of July 1981. 81/715/EEC. «Official Journal of the European Communities' number L 257/38, of 10 September 1981.


49 antibiotics of the tetracycline 49.1 detection and identification.


49.1.1 principle. The method makes it possible to detect and identify antibiotics of the tetracycline group in feedingstuffs containing at least 0.1 mg/kg of them, in concentrates and in premixes.



The sample is subjected to extraction with a mixture of methanol and hydrochloric acid. The summary is chromatography on paper via rising comparatively to its reference solutions. The antibiotics are detected and identified in comparison with their Rf values with the substances of pattern, either by fluorescence light UV (high antibiotic contents) or by bioautography on an agar medium sowing with B. cereus.


49.1.2 reagents.


49.1.2.1 buffer, pH 3.5. Citric acid monohydrate p.a.: 10,256 g.


Sodium Monohidrogenofosfato dihydrate (NaHPO.2HO) p.a.: 7,45 g.


Acetone p.a.: 300 ml.


Distilled water: up to 1,000 ml.


49.1.2.2 phosphate buffer: pH 5.5. (KHPO) p.a. potassium dihydrogen phosphate: 130,86 g.


Sodium Monohidrogenofosfato dihydrate (NaHPO.2HO) p.a.: 6,947 g.


Distilled water: up to 1,000 ml.


49.1.2.3 eluent I: mixture Nitromethane p.a./cloroformo p.a./alfa diclorhidrina: 20/10/1.5 in volume. Prepared at the time of their employment.


49.1.2.4 eluent II: Mixture nitrometrano p.a. / p.a./alfa methyl chloroform (20/10/3 by volume). Prepared at the time of their employment.


49.1.2.5 mix netanol p.a./ácido HCL (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 standard substances: chlortetracycline, oxytetracycline, tetracycline, whose activity is expressed in hydrochloride.


49.1.2.9 micro-organism: B. cereus ATCC number 11,778. Maintenance of strain, preparation of the spore suspension and inoculation of the culture medium. Implementing the provisions (49.2.2.1) and (49.2.2.2) method for the determination of chlortetracycline, oxytetracycline and tetracycline, by diffusion on agar, object of the part (49.2).


49.1.2.10 culture medium. (See 41.15.1): glucose: 1 g.


Peptone peptone: 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 their employment.


49.1.3 material.


49.1.3.1 equipment for ascending chromatography on paper (paper 25 cm height). Schleicher and Schüll paper 20040 b or 20043 b or equivalent.


49.1.3.2 centrifuge.


49.1.3.3 range of incubation, regulated at 30 ° C.


49.1.3.4. UV lamp for fluorescence detection.


49.1.3.5 20 x 30 cm glass plates approximately that allow mounting a box flat to the bioautobiografia.


49.1.4 procedure.


49.1.4.1 solutions for calibration.


49.1.4.1.1 standard solutions. Prepared from substances of calibration (49.1.2.8) and with the help of HCL (49.1.2.6) solutions with concentrations corresponding respectively to 500 micrograms of chlortetracycline-HCl or oxytetracycline-HCl and of tetracycline-HCl per ml.


49.1.4.1.2 reference in UV light detection solutions. Dilute the solutions (49.1.4.1.1) with phosphate buffer (49.1.2.2) to obtain solutions with concentrations corresponding to 100 micrograms of chlortetracycline-HCl or oxytetracycline-HCl and of tetracycline-HCl per ml.


49.1.4.1.3 solutions for detection by bioautography reference. Dilute the solutions (49.1.4.1.1) with phosphate buffer (49.1.2.2) to obtain solutions with concentrations corresponding to 5 micrograms of chlortetracycline-HCl, or oxytetracycline-HCl and of tetracycline-HCl per ml.


49.1.4.2 extraction. When the presumed antibiotic content is less than 10 mg/kg, you may use the homogenized sample or the finest fraction separated by sieving, given that antibiotics will be preferably in this fraction.


Place the sample suspended in the mixture (49.1.2.5) and centrifuge. Collect the supernatant liquid to use it as it is or diluted it, if necessary, by blending (49.1.2.5) to obtain a few concentrations of antibiotic 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 chromatography. Immerse the paper in the solution buffer, pH 3.5 (49.1.2.1). Eliminate excess fluid compressing the role between a few sheets of dry filter paper. Then place volumes of 0.01 ml of the reference (49.1.4.1.2 and 49.1.4.1.3) and (49.1.4.2 and 48.1.4.2.2) extract solutions on paper. To obtain a good will, the appropriate paper moisture content is critical; where appropriate, allow to dry slightly.


Develop by ascending chromatography. Use eluent I (49.1.2.3) for detection by bioautography, the eluent II (49.1.2.4) for detection in UV light. When the solvent front reaches 15 to 20 cm high (approximately one hour thirty minutes) stop chromatography and dry the paper.


49.1.4.3.2 detection of ultraviolet light. When the antibiotic content greater than 1 microgram/cm, fluorescent yellow or stains by irradiation under the lamp UV (49.1.3.4) previous treatment of the chromatogram using ammonia vapors (49.1.2.7) can be felt.


49.1.4.3.3 detection by bioautography. Pour the medium (49.1.2.10) previously seeded with B. cereus (49.1.2.9) on some plates of glass (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 place other than the culture medium where it will keep you during the period of incubation, incubate overnight in an oven at 30 ° C. The presence of an antibiotic of the tetracycline group is marked by clear zones of inhibition in the cloudy culture medium.


To fix the chromatogram, spray the solution (49.1.2.11) on the paper, after incubation.


49.1.4.3.4 identification. The RF values to antibiotics of the tetracycline group are given below. These values may vary slightly according to the quality of the paper and its moisture content: chlortetracycline (CTC): 0.60.


Tetracycline (TC): 0.40.


Oxytetracycline (OTC): 0.20.


4 epi - CTC: 0.15.


4 epi - TC: 0.13.


4 epi - OTC: 0.10.


"Epi" compounds have a lower than normal compounds antibiotic activity.


49.1.5 observations.


49.1.5.1 may be used any commercial medium of similar composition and give the same results.


49.2 chlortetracycline, oxytetracycline and tetracycline.


49.2. by diffusion on agar.


49.2. a. principle 1. The method enables you to determine chlortetracycline (CTC), oxytetracycline (OTC) and tetracycline (TC) in feedingstuffs, concentrates and premixes. The lower limit of determination is 5 mg/kg. Contents of less than 5 mg/kg may be estimated by graphic interpolation.


For contents equal to or less than 50 mg/kg, the sample shall be subjected to extraction by diluted formanida. For contents greater than 50 mg/kg, is subjected to extraction with a mixture of acetone, water and hydrochloric acid for the determination of CTC, and with a mixture of methanol and hydrochloric acid for the determination of OTC and TC.


The extracts are then diluted and their antibiotic activity is determined by measurement of the diffusion of the CTC, OTC or CT in the middle of agar seeded with B. cereus. The broadcast is indicated by the formation of zones of inhibition in the presence of the micro-organism. The diameter of these zones is directly proportional to the logarithm of the antibiotic concentration.


49.2. a. 2 reagents.


49.2. a. 2.1 microorganisms: B. cereus ATCC number 11,778.


49.2. a. 2.1.1 maintenance of the strain. Sow B. cereus in inclined agar consisting of the Middle tube culture (49.2. A. 3.1) Blue methylene and boric acid-free. Incubate overnight at 30 ° C approximately. Keep the culture in a refrigerator and replant every fourteen days on agar slant.


49.2. a. 2.1.2 preparation of the spore suspension. Collect the bacteria from a tube of sloped agar (49.2. A. 2.1.1) using 2 to 3 ml of physiological saline (49.2. A. 2.2.5). With this suspension seed a Roux flask containing 300 ml of culture medium (49.2. A. 2.2.1), free Blue methylene and boric acid, with a concentration in agar is 3 to 4 per 100. Incubate for three to five days at 28-30 ° C, then collect the spores in 15 ml of ethanol (49.2. A. 2.2.6), after checking sporulation under a microscope, and homogenize. This suspension can be kept in refrigerator for 5 months at least.


By preliminary tests on plates with the basic medium for the determination (49.2. A. 2.2.1), establish the quantity of inoculum which inhibition zones as long as possible to get to the different concentrations of antibiotic used, which are clear. This amount is usually 0.2 to 0.3 ml / 1000 ml. The planting of the culture medium is between 50 and 60 ° C.


49.2. a. 2 2 culture media and reagents.


49.2. A. 2.2.1. Basic medium for the determination (see 49.2. A. 5.1): glucose: 1 g.


Tryptic peptone: 10 g.


Meat extract: 1.5 g.


Yeast extract: 3 g.


Agar, according to quality: 10-20 g.


Tween 80: 1 ml.


Phosphate buffer, pH 5.5 (49.2. A. 2.2.2): 10 ml.


5 per 100 solution (w/v) boric acid: 15 ml.


Solution in ethanol to 0.5 per 100 of methylene Blue: 4 ml.


Distilled water: up to 1,000 ml.


Adjust to pH 5.8 before use.


49.2. a. 2.2.2 buffer phosphate pH 5.5. Dihydrogen phosphate of potassium (KHPO) p.a.: 130,86 g.


Sodium monohidrogeno-fosfato dihydrate (NaHPO.2HO) p.a.: 6,947 g.


Distilled water: up to 1,000 ml.


49.2. a. 2.2.3 buffer phosphate, pH 5.5, diluted to 1/10.


49.2. a. 2.2.4 buffer phosphate pH 8. Dihydrogen phosphate of potassium (KHPO) p.a.: 1,407 g.


Sodium monohidrogeno-fosfato dihydrate (NaHPO.2HO) p.a.: 57,539 g.



Distilled water: up to 1,000 ml.


49.2. a. 2.2.5 physiological saline sterile.


49.2. a. 2.2.6 to 20 per 100 ethanol (v/v).


49.2. a. 2.2.7 hydrochloric acid 0, 1N.


49.2. a. 2.2.8 formamide to 70 by 100 (v/v). Prepare fresh before use and adjust the pH to 4.5 using sulphuric acid 2N approximately.


49.2. a. 2.2.9 mixing acetone p.a./agua/ácido HCL (d = 1.19) (65/33/2 by volume).


49.2. a. 2.2.10 mix methanol hydrochloric p.a./ácido (d = 1.19) (98/2 v/v).


49.2. a. 2.2.11 patterns substances: CTC, OTC, TC whose activity is expressed in hydrochloride.


49.2. a. 3 procedure.


49.2. a. 3.1 solutions patterns.


49.2. a. 3.1.1 chlortetracycline. Prepare pattern from the substance (49.2. a. 2.2.11) and with the help of hydrochloric acid (49.2.. 2.2.7), a standard solution with a concentration corresponding to 500 micrograms/ml of chlortetracycline-HCl. such a solution remains a week in refrigerator.


From this solution prepare a solution of standard working S8 with a concentration corresponding to 0.2 micrograms/ml of chlortetracycline-HCl. Dilution is made with the help of the buffer phosphate, pH 5.5, diluted to 1/10 (49.2. a. 2.2.3), with addition of the 0.01 per 100 of amido black (see 49.2. A. 5.2).


Then prepare by successive dilutions (1 + 1) using the buffer solution (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 Oxytetracycline. Proceeding as indicated in (49.2. a. 3. 1.1) prepare from a standard solution with a concentration corresponding to 400 micrograms/ml of oxytetracycline-HCl, a standard solution work S8 of 1.6 mcg/ml of oxytetracycline-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 solution pattern with a concentration corresponding to 500 micrograms/ml of tetracycline-HCl, a standard solution work S8 of 1.0 mcg/ml of tetracycline-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 content equal to or less than 50 mg/kg. Treat sample by formamide (49.2. A. 2.2.8), according to the indications given in the box below. Shake for 30 minutes on Shaker. Dilute immediately after using the buffer phosphate (49.2. A. 2.3), according to the indications given below in the box, to obtain the concentration U. The concentration of formamide in this solution must not exceed 40 per 100. Centrifuge or let opt so as to obtain a clear solution.


Then prepare the concentrations U, U and U, by successive dilutions (1 + 1) using the phosphate buffer solution (49.2. A. 2.3).


Antibiotic / CTC / DTC / TC content in mg/kg. / 10 / 50 / 10 / 50 / 10 / 50 sample 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 buffer phosphate (49.2. A. 2.2.3). / 1/5 / 1/25 / 70 / 200 / 120 / 1/5 (a) / (b) / / / / (a) U concentration in micrograms/ml. / 0.2 / 0.2 / 1.6 / 1.6 / 1.0 / 1.0 (to) take 20 ml of extract and make up to 100 ml with buffer in a volumetric flask.


(b) pipette 4 ml of extract and make up to 100 ml with buffer in a volumetric flask.


49.2. a. 3.2.2 content higher than 50 mg/kg.


49.2. a. 3.2.2. 1 chlortetracycline. Treat, according to the presumed content of antibiotic of the sample or its guarantee of manufacture, a sample of 1 to 10 grams, 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 during extraction; If necessary adjust to pH 3 (for mineral compounds, with the help of acetic acid to 10 per 100). Take an aliquot part of the extract and adjust the pH to 5.5 using the buffer phosphate, pH 8 (49.2. A. 2.2.4), in the presence of the (turning from yellow to blue) bromocresol green. Dilute the buffer solution with phosphate, pH 5.5, diluted to 1/10 (49.2. A. 2.2.3) to obtain the concentration U (49.2. A. 3.2.1).


Then prepare the concentrations U, U and U by successive dilutions (1 + 1) using the buffer phosphate (49.2. A. 2.2.3).


49.2. a. 3.2.2. 2 Oxytetracycline and tetracycline. Proceed as described in (49.2. A. 3.2.2. 2) replacing the mixture (49.2.. 2.2) by mixing (49.2. a. 2.2.10).


49.2. a. 3.3 modalities of definition.


49.2. a. 3.3.1 seed of the culture medium. Sow at 50-60 ° C medium base for determination (49.2.. 2.2.1) with the spore suspension (49.2. A. 2.1.2).


49.2. a. 3.3.2 preparation of boxes. Diffusion on agar is carried out in boxes, with concentrations four pattern (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each case must necessarily receive four pattern and extract concentrations.


To this end, choose the dimensions of the boxes so that you can practice in the Middle gélosé at least 8 cavities of 10 to 13 mm in diameter. Calculate the quantity of inoculated culture medium (49.2. A. 3.3.1), to be used in such a way that you can get a uniform coating of 2 mm approximately thick. It is preferable to be glass plates fitted with a ring of aluminum or plastic flat perfectly plane, 200 mm in diameter and 20 mm high.


Pipette into the cavities amounts of antibiotic solution, exactly measures, between 0.10 and 0.15 ml, depending on the diameter.


For each sample, do at least four repetitions of each concentration diffusion, so that each determination is subject to an evaluation of 32 inhibition zones.


49.2. a. 3.3.3 incubation. Incubate the trays for eighteen hours approximately 28-30 ° c.


49.2. a. 4 calculations. Measure the diameter of inbicion zones, preferably by projection. Record the measurements on semi-log paper, representing the logarithm of the concentrations against the zonzas of inhibition diameters. Draw the straight lines of the solution of calibration and extract. In the absence of interference, the two lines should be parallel.


The logarithm of the relative activity is calculated by the following way: (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 real activity = supposed 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 observations.


49.2. a. 5.1 may be used any commercial medium of similar composition and give the same results.


49.2. a. 5.2 the amido black is used to characterize the zones of inhibition of solutions of calibration (blue rings).


49.2. B by turbidimetry.


49.2. B. 1 principle. The method enables you to determine chlortetracycline (CTC), oxytetracycline (OTC) and tetracycline (TC) in concentrations above 1 g/kg, insofar as no other substance interfering, giving rise to some shady excerpts. This method is quicker than diffusion on agar method.


The sample is subjected to extraction from mixture of acetone, water and HCL for the determination of CTC, and with a mixture of methanol and hydrochloric acid for the determination of OTC and TC.


The extracts are then diluted and their antibiotic effect is determined by measurement of the light transmission of a medium seeded with Staphylococcus aureus and antibiotic added. The light transmission is a function of the concentration of antibiotic.


49.2. B. 2 reagents.


49.2. B. 2.1 micro-organism: Staphylococcus aureus K 141. See (49.2. B. 5.1).


49.2. B. 2.1.1 maintenance of the strain. Sow S. aureus on inclined agar consisting of the Middle tube culture (49.2. B. 2.2.1), added 1.5 to 3 per 100 (depending on the quality). Incubate overnight at 37 ° C. Keep the culture in a refrigerator and replant four weeks on inclined agar. Simultaneously prepare a few subcultures for laboratory use.


49.2. B. 2.1.2 preparation of the safe. Twenty-four hours before use, replant a subculture on inclined agar and incubate overnight at 37 ° C. To suspend all of the cultivation of a tube of agar in 2 ml approx. of the basic medium (49.2. B. 2.2.1) and then transfer the suspension 100 ml sterile conditions approximately of the same basic medium (49.2. B. 2.2.1). Incubate in a water bath at 37 ° C until the growth of the strain enters its logarithmic phase (1 hour 30 minutes to 2 hours).


49.2. B. 2 2 culture media and reagents.


49.2. B. 2.2.1 average basis of 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.


Dihydrogen phosphate of potassium (KHPO) p.a.: 1.32 g.


Potassium (KHPO) p.a. monohidrogeno-fosfato: 3.68 g.


Distilled water: up to 1,000 ml.


pH after sterilization: 6.8 to 7.


49.2. B. 2.2.2 buffer phosphate pH 4.5. Dihydrogen phosphate of potassium (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 mixture acetone p.a./agua/ácido HCL (d = 1.19) (65/33/2 by volume).


49.2. B. 2.2.5 mixing methanol hydrochloric p.a./ácido (d = 1.19) (98/2 by volume).


49.2. B. 2.2.6 to 10 per 100 solution approximately (w/v) formaldehyde.


49.2. B. 2.2.7 substances pattern: CTC, OTC, CT, whose activity is expressed as hydrochloride.



49.2. B. 2.3 solution pattern. Prepare pattern from the substance (49.2. B. 2.2.7) and with the help of hydrochloric acid (49.2. B. 2.2.3) a pattern solution whose concentration is 400 to 500 micrograms/ml of CTC-HCL, OTC-HCL or TC-HCL. This solution is still a week in refrigerator.


49.2. B. 3 procedure.


49.2. B. 3.1 extraction.


49.2. B. 3.1.1 chlortetracycline. Place in a volumetric 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 30 minutes with agitator. Make up the volume with the buffer phosphate pH 4.5 (49.2. B. 2.2.2). Homogenize and leave that it has been deposited.


49.2. B. 3.1.2 Oxytetracycline and tetracycline. Enter a sample of 1 to 2 grams in a 200 to 250 ml graduated flask. Add approximately 100 ml of the mixture (49.2. B. 2.2.5) and shake for 30 minutes with agitator. Make up the volume with the phosphate buffer, pH 4.5 (49.2. B. 2.2.2). Homogenize and leave that it has been deposited.


49.2. B. 3.2 modes.


49.2. B. 3.2.1 preparation of series standard and extract. Diluted appropriately with the help of the phosphate buffer, pH 4.5 (49.2. B. 2.2.2) standard solution (49.2. B. 2.3) and extract (49.2. B. 3.1), to obtain a series of concentrations that allow you to set, for each determination, a curve of calibration, and interpolation on the curve of at least two values relating to the extract. The dilutions should be chosen according to the conditions of growth of the strain, which may vary from one laboratory to another. Proceed generally in the following way: 49.2. B. 3.2.1. 1 chlortetracycline. Dilute the standard solution (49.2. B. 2.3) with the phosphate buffer solution (49.2. B. 2.2.2) to obtain a standard solution with a concentration corresponding to 0.2 micrograms/ml of CTC-HCl. then prepare with a phosphate buffer solution (49.2. B. 2.2.2), as shown below, and determination for tubes, 6 dilutions with a repetition of each dilution.


Ml of working solution / Ml buffer phosphate (49.2. B. 2.2.2) / concentration of 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 diluted extract (49.2. B. 3.1.1) using the phosphate buffer solution (49.2. B. 2.2.2) to obtain a presumed CTC-HCl of 0.12 micrograms/ml concentration. introduce 1 ml of this solution into two tubes, and 0.75 ml (= 0.09 mcg) in other two tubes. Make the volume of the latter two tubes up to 1 ml with the phosphate buffer solution (49.2. B. 2.2.2).


49.2. B. 3.2.1. 2 Oxytetracycline and tetracycline. Dilute the solution of calibration (49.2. B. 2.3) with the phosphate buffer solution (49.2. B. 2.2.2) to obtain a working standard solution with a concentration corresponding to 0.6 micrograms/ml of OTC-HCl or of TC-HCl. then prepare the buffer solution with phosphate (49.2. B. 2.2.2) as shown below and on the definition for tubes, 7 dilutions with a repetition of each dilution.


Ml of working solution / Ml buffer phosphate (49.2. B. 2.2.2) / concentration of 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 diluted extract (49.2. B. 3.1.2) with the phosphate buffer solution (49.2. B. 2.2.2) to obtain a concentration presumed OTC-HCl or TC-HCl of 0.48 micrograms/ml. place 1 ml of this solution in two tubes, and 0.5 ml (= 0.24 micrograms) in other two tubes. Make the volume of the latter two tubes up to 1 ml with the phosphate buffer solution (49.2. B. 2.2.2).


49.2. B. 3.2.2 inoculation of the culture medium. Plant the basic medium for the determination (49.B.2.2.1) with the inoculum (49.2. B. 2.1.2) in such a way that a light transmission of 85 per 100 in a bucket of 5 cm or 92 per 100 in a bucket of 2 cm to obtain with the photometer at 590 nm, while the device regulated to 100 per 100 of transmission over the basic medium (49.2. B. 2.2.1) uninoculated.


49.2. B. 3.2.3 seeding. Insert in 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). Operation of filling of the tubes should be appropriate, but not necessarily in sterile conditions.


49.2. B. 3.2.4 incubation. Incubation should be mandatory in a bath of water whose temperature is maintained even at 37 ° C ± 0.1 ° C by stirring. The incubation time should be chosen in such a way that you can trace curves of transmission whose inclination is appropriate to some accurate measurements (usually two hours 30 minutes to 3 hours). Then block the growth by quickly introducing 1 ml of formaldehyde solution (49.2. B. 2.2.6) into each tube.


49.2. B. 3.2.5 measurement of growth. Measure the transmissions with the photometer at 590 nm, regulating the appliance at 100 by 100 transmission troubleshooting calibration more clear (which corresponds to the highest antibiotic content). Due to small differences in turbidity presented by the different tubes, it is recommended to use 2 cm trays at least, and preferably 5 cm.


49.2. B. 4 stones. Graphically plot the calibration envelope curve graph paper, putting the photometric transmissions in relation to antibiotic concentrations. Interpolate on the curve relating to extract transmissions. Calculate the antibiotic of the sample content.


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 observations.


49.2. B. 5.1 this strain, isolated by the LUFA at Kiel, accused a growth more rapidly than S. aureus ATCC 6538 P.


49.2. B. 5.2 can be used any means of commercial cultivation of similar composition and give the same results.


49.2. B. 6 references. Third Commission directive of 27 of April 1972. 72/199/EEC. «Official Journal of the European communities» number L, 123/6, from May 29, 1972.


50 oleandomycin (by diffusion on agar) 50.1 principle. The method enables you to determine the oleandomycin in feedingstuffs, concentrates and premixes, even in the presence of tetracyclines. The lower limit of the determination is 0.5 mg/kg.


The sample is subjected to extraction of tris (hydroxymethyl) aminomethane diluted methanolic solution. After centrifuging, the extract is diluted and its antibiotic activity is determined by measurement of the diffusion of the oleandomycin on seeded B. agar medium cereus. The broadcast will be indicated by the formation of zones of inhibition in the presence of the micro-organism. The diameter of these zones is directly proportional to the logarithm of the antibiotic concentration.


50.2 reagents.


50.2.1. micro-organism: B. cereus K 205 TR (see 50.5.1) (resistant to tetracyclines).


50.2.1.1 maintenance of the strain. Sow B. cereus on agar in tube tilted with the culture medium (50.3.2.2.1) with the addition of 100 micrograms of oxytetracycline for 5 ml. incubate overnight at 30 ° C approximately. Keep the culture in a refrigerator and replant every four weeks on inclined agar.


50.2.1.2 preparation of the spore suspension. Collect the bacteria from a tube of agar slant (50.2.1.1) with help of 3 ml approximately serum physiological (50.2.2.3). With this suspension seed a Roux flask containing 300 ml of culture medium (50.2.2.1) whose concentration is 3 to 4 per 100. Incubate for three to five days at 28-30 ° C, then collect the spores in 15 ml of ethanol (50.2.2.4) after checking sporulation under a microscope, and homogenize. This suspension can be kept in the refrigerator for at least five months.


By preliminary tests on plates with the basic definition (50.2.2.2) for medium establish the quantity of inoculum which inhibition zones as long as possible to get to the different concentrations of oleandomycin used, which are clear. This amount is usually 0.1 to 0.2 ml / 1000 ml. The planting of the culture medium will be at 60 ° C.


50.2.2. culture media and reagents.


50.2.2.1 medium maintenance of strain (see 50.5.2.)


Glucose: 1 g.


Peptone peptone: 10 g.


Meat extract: 1.5 g.


Yeast extract: 3 g.


Agar, according to quality: 10-20 g.


Distilled water: up to 1,000 ml.


Adjust the pH to 6.5 at the time of their employment.


50.2.2.2 average basis of definition (see 50.5.2). Medium (50.2.2.1) adjusted to pH 8.8.


50.2.2.3 sterile physiological saline.


50.2.2.4 20 per 100 ethanol (v/v).


50.2.2.5 methanol p.a.


50.2.2.6 the 0.5 per 100 solution (w/v) of tris (hydroxymethyl) aminomethane p.a.


50.2.2.7 extraction solution.


Pure methanol: 50 ml.


Distilled water: 50 ml.


TRIS (hydroxymethyl) aminomethane p.a.: 0.5 g.


50.2.2.8 standard substance: oleandomycin of known activity.


50.3 procedure.


50.3.1. standard solution. Dissolve substance pattern (50.2.2.8) in 5 ml of methanol (50.2.2.5) and dilute with the solution (50.2.2.6) to obtain an oleandomycin of 100 micrograms/ml concentration.


From the standard solution, prepared by diluting with the solution (50.2.2.6) a solution work S pattern containing 0.1 micrograms/ml of oleandomycin.


Then prepare by successive dilutions (1 + 1) using the solution (50.2.2.6), the following concentrations: 0.05 s micrograms/ml.


S: 0.025 micrograms/ml.


S: 0.0125 micrograms/ml.


50.3.2 extraction. Take according to the presumed content of oleandomycin of the sample, a quantity of 2 to 10 g, add 100 ml of the solution (50.2.2.7) and shake for 30 minutes with agitator.



Centrifuge, take an aliquot part of the extract and dilute with the solution (50.2.2.6) to obtain a presumed oleandomycin of 0,1 micrograms/ml concentration (U). Then prepare the concentrations U, U and U by successive dilutions (1 + 1) using the solution (50.2.2.6).


50.3.3 modalities of definition.


50.3.3.1 planting of the culture medium.


Inoculate at 60 ° C the basic medium for the determination (50.2.2.2) with the suspension of spores (50.2.1.2).


50.3.3.2 preparation of boxes. Diffusion on agar is verified in some boxes with the four concentrations of the solution of calibration (S, S, S and S) and the four concentrations of the extract (U, U, U and U). Each case must necessarily receive four concentrations of extract and calibrated.


To this end, choose the dimensions of the boxes so that at least eight cavities of 10 to 13 mm in diameter could be in the Middle gélosé. Calculate the amount of planting medium (50.3.3.1) that must be used to obtain a uniform coating of 2 mm approximately thick. It is preferable to be glass plates fitted with a ring of aluminum or plastic flat perfectly plane 200 mm in diameter and 20 mm high.


Insert the pipette in cavities accurately measured amounts of antibiotic solution between 0.10 and 0.15 ml according to the diameter.


For each sample, do at least four repetitions of each concentration diffusion, so that each determination is subject to an evaluation of 32 inhibition zones.


50.3.3.3 incubation. Incubate the trays for eighteen hours approximately 28-30 ° c.


50.4 calculations. Measure the diameter of the inhibition zones, preferably by projection. Record the measurements on semi-log paper, representing the logarithm of the concentrations against the diameter of the inhibition zones. Draw the straight lines of the solution of calibration and extract. In the absence of interference, the two lines should 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 + U + U - S - S - S - S) x0.602 U + U - S - S - U - U - S - S real activity = supposed 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 observations.


50.5.1 strain isolated by the LUFA at Kiel.


50.5.2 can be used any commercial medium of similar composition and give the same results.


50.6 references. Third Commission directive of 27 of April 1972. 72/199/EEC. «Official Journal of the European communities» number L, 123/6, from May 29, 1972. Annex II.


51 Amprolium 51.1 principle. The method enables you to determine the amprolium in feedingstuffs, concentrates and premixes. The lower limit of detection is 40 mg/kg.


The sample is subjected to extraction with diluted methanol. The extract is purified on a column of aluminium oxide and is treated by a 2, 7-dihidroxinaftaleno, hexaciano ferrato methanolic solution (III) potassium, potassium cyanide, and sodium hydroxide. A purple color develops. Amprolium is determined by spectrophotometry at 530 nm.


51.2 material and devices.


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 for analitical column (inside diameter: 9 mm, length: 400 to 500 mm).


51.2.5 centrifugal, with 25 ml screw cap tubes.


51.2.6 spectrophotometer with cuvettes of 10 mm thick.


51.3 reagents.


51.3.1 methanol p.a.


51.3.2. diluted methanol.


Mixing two volumes of methanol (51.3.1) and one volume of water.


51.3.3 the 0.2 per 100 solution (w/v) of hexacyanoferrate (III) potassium (K Fe (CN)) p.a.


This solution is stable for two weeks.


51.3.4 1 per 100 solution (w/v) potassium p.a. said cyanide solution is stable for two weeks.


51.3.5 the 1,125 per 100 solution (w/v) sodium p.a. hydroxide


51.3.6. methanolic sodium hydroxide solution.


Take 15 ml of the solution (51.3.5) and make up to 200 ml with methanol (51.3.1).


51.3.7 the 0,0025 per 100 solution (w/v) 2, 7-dihidroxinaftaleno. Dissolve 25 mg 2, 7-dihidroxinaftaleno p.a. in methanol (51.3.1) and make up to 1000 ml with methanol (51.3.1).


51.3.8 staining reagent. Enter 90 ml of solution 2, 7-dihidroxinaftaleno (51.3.7) in a 250 ml (51.2.1) Erlenmeyer flask, add 5 ml of hexanocianoferrato (III) potassium (51.3.3) and mix thoroughly. Then add 5 ml of solution of cyanide of potassium (51.3.4), cover and mix thoroughly. Let stand for thirty to thirty-five minutes, add 100 ml of solution methanolic hydroxide sodium (51.3.6), homogenize and filter through a melting pot filter (51.2.3). Use said reagent in the seventy-five minutes following filtration.


51.3.9 oxide of aluminium for column chromatography. Before use, shake for thirty minutes, 100 g of oxide of aluminium with 500 ml of water, filter, wash three times the sediment on the filter with 50 ml of methanol (51.3.1) each time, dry suction, let stand overnight and dry for two hours at 100 ° C in a vacuum oven. Leave to cool in a desiccator. Control activity analyzing, starting point (51.4.2), a certain amount of solution (51.3.11). The recovery rate of the amprolium should be 100 per 100 ± 4 per 100.


51.3.10 chemical pattern. Pure Amprolium that responds to the characteristics defined below:-(decomposition) melting point: 248 ° C.


-Coefficient of molecular extinction at 265-235 nm in the distilled water: 11.0 x 103.


51.3.11 standard solution. Weigh, with precision of 0,1 mg, 50 mg of substance pattern (51.3.10). Dissolve with diluted methanol (51.3.2) in a 500 ml graduated flask, make up to volume with the same solvent and mix. Pipette 10,0 ml make up to 50 ml with methanol diluted (51.3.2) in a volumetric flask and mix 1 ml of this solution contains 20 micrograms of amprolium.


51.4 procedure.


51.4.1 extraction and purification.


51.4.1.1 feed and premixes. 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. Enter the sample in a 250 ml Erlenmeyer flask (51.2.1) and add 100 ml of diluted methanol (51.3.2) exactly. Shake for 60 minutes and filter. Dilute, if necessary using diluted methanol (51.3.2) to obtain a solution containing 5 to 15 micrograms of amprolium per ml. insert into a column for chromatography (51.2.4) previously provided in your lower limb of a plug of cotton, 5 g of aluminium oxide (51.3.9) and then 25.0 ml of extract. Let pass the liquid, remove the 5 first ml and collect the following 12 ml into a graduated cylinder.


51.4.1.2 concentrates. Weigh to the nearest 1 mg, 0.5 g of sample finely divided and homogenized, place in a 500 ml (51.2.1) Erlenmeyer flask, add 250 ml of diluted methanol (51.3.2), shake for 60 minutes and filter. Take 5.0 ml of the filtrate and make up to 200 ml with methanol diluted (51.3.2) in a volumetric flask.


51.4.2 development of 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 insert it into a tube of centrifugal (51.2.5). Introduce 5,0 ml of methanol diluted (51.3.2) in a tube from centrifuge B (51.2.5). Add in each tube 10.0 ml of reagent for staining (51.3.8), recap the tubes, homogenize and leave to stand for eighteen minutes. Then centrifuged for three minutes to obtain clear solutions and decant solutions A and B in the Erlenmeyer flask of 50 ml (51.2.1).


Measure the absorbance of the solution immediately in the spectrophotometer at 530 nm, using targeted solution B. determine the amount of amprolium referring to calibration (51.4.3) curve.


51.4.3 curve of calibration. Insert tubes centrifuge (51.2.5) in respective volumes of 1, 0-2, 0-3, 0-4, 0 and 5,0 ml of standard solution (51.3.11). Make the volume of the first four tubes up to 5.0 ml with diluted methanol (51.3.2). Add five tubes 10.0 ml of reagent for staining (51.3.8), recap the tubes, homogenize and leave to stand for eighteen minutes. Then centrifuged for three minutes and decant the Erlenmeyer flask 50 ml (51.2.1) solutions.


Measure immediately in the spectrophotometer at 530 nm the absorbance of the solutions, using a mixture of 5 ml of diluted methanol (51.3.2) and 10 ml of reagent for staining (51.3.8) targeted. Plot the curve of calibrated taking the ordinate the abscissa and the absorbance values the corresponding quantities of amprolium in mg.


51.5 calculations.


51.5.1 feed and premixes. Content in mg of amprolium per kg of sample is given by the formula: X F x 20,000 H 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 (effected if the case should arise in 51.4.1.1.).


51.5.2 concentrates. The content of amprolium in percentage of sample is given by the formula: X 200 X 200 D x 200 being: A = quantity, in mg, of amprolium determined by Spectrophotometric measurement.


P = weight, in g, of the test sample.



The difference between the results of two parallel determinations carried out on the same sample must not exceed:-10 mg/kg in absolute value for amprolium contents lower than 100 mg/kg.


-10 per 100, in relative value, for contents of between 100 and 5,000 mg/kg.


-500 mg/kg, in absolute value, for contents of between 5,000 and 10,000 mg/kg.


-5 by 100, in relative value, for contents greater than 10,000 mg/kg.


51.6 references. Fifth Directive of the Commission of March 25, 1974. (74/203/EEC). «Official Journal of the European communities» number L, 108/7 of 22 April 1974.


52 Etopabato (methyl-4-acetamido-2-etoxibenzoato) 52.1 principle. The method enables you to determine the etopabato in feedingstuffs, concentrates and premixes. The lower limit of detection is 2 mg/kg.


The sample is subject to removal by diluted methanol. The solution is acidified and extracted with chloroform. Cloroformico extract is first washed with an alkaline solution and then with water. The purified extract is concentrated, the etopabato is hydrolyzed by dilute hydrochloric acid. The derivative aminated thus formed is diazota and copulate with N-(1-naphthyl)-ethylenediamine. The colored complex is extracted by butanol and the absorbance of the solution is measured at 555 nm.


52.2 material and devices.


52.2.1 Erlenmeyer of 250 ml, with screw cap.


52.2.2 separating funnels of 100 ml, with ground glass stopper.


52.2.3 agitator.


52.2.4. Rotary evaporator vacuum with 250 ml flasks.


52.2.5. water bath.


52.2.6 centrifuge tubes 15 and 50 ml of standard grinding.


52.2.7 refrigerant air, ground mouth.


52.2.8 spectrophotometer with cuvettes of 10 mm thick.


52.3 reagents.


52.3.1 methanol p.a.


52.3.2 methanol 50 by 100 (v/v). Mix equal volumes of methanol (52.3.1) and water.


52.3.3 p.a. hydrochloric acid (d = 1.19).


52.3.4 hydrochloric acid diluted to 1/10. Take 10,0 ml of hydrochloric acid (52.3.3), make up to 100 ml with water.


52.3.5 hydrochloric acid 0, 3N approximately. Take 25.0 ml of hydrochloric acid (52.3.3), make up to 1000 ml with water.


52.3.6 chloroform p.a.


52.3.7 the 4 by 100 solution (w/v) of sodium carbonate. Dissolve 40 g of p.a. anhydrous sodium carbonate in water and make up to 1,000 with water.


52.3.8 the 0.2 per 100 solution (w/v) of sodium nitrite.


Dissolve 100 mg of nitrite of sodium p.a. water and make up to 50 ml with water in a graduated flask. Prepare immediately before use.


52.3.9 the 1.0 per 100 solution (w/v) of ammonium sulfamate. Dissolve in water 500 mg of p.a. ammonium sulfamate, and make up to 50 ml with water in a graduated flask. Prepare immediately before use.


52.3.10 solution to the 0.2 by (w/v) of N-(1-naphthyl)-ethylenediamine. Dissolve in water 100 mg of N-(1-naphthyl)-ethylenediamine p.a. and make up to 50 ml with water in a graduated flask. Prepare immediately before use.


52.3.11 anhydrous sodium chloride, p.a.


52.3.12 n-butanol p.a.


52.3.13 chemical pattern. Pure Etopabato.


52.3.14 standard solution.


52.3.14.1 solution of 0.040 mg of etopabato by ml. weigh with precision of 0.1 mg, 40 g of substance pattern (52.3.13). Dissolve with methanol diluted (52.3.2) into graduated 100 ml. complete flask to volume with the same solvent and mix. Pipette 10.0 ml, make up to 100 ml with methanol (52.3.2) in a volumetric flask and mix. This solution is stable for one month.


52.3.14.2 solution of 0.016 mg of etopabato in 20 ml. take 5,0 ml of the solution (52.3.14.1) make up to 250 ml with methanol diluted (52.3.2) in a volumetric flask and mix. Prepare before use.


52.4 procedure.


52.4.1 extraction. Weigh, with precision of 1 mg, finely divided and homogenized sample that contains approximately 80 micrograms of etopabato. Place the test sample in a 250 ml Erlenmeyer flask (52.2.1) and add 100,0 ml of diluted methanol (52.3.2). Mix, close the Erlenmeyer flask and shake for one hour by the agitator (52.3.4). Stop decant, filter out and delete the first ml of the filtrate.


52.4.2 purification. All the operations described in this point should be made quickly.


Enter a separating funnel of 100 ml (52.2.2) 20.0 ml of clear extract, add 5.0 ml of HCL diluted 1/10 (52.3.4) and 20.0 ml of chloroform (52.3.6). First shake cautiously and then vigorously for three minutes. Let stand until the separation of the phases and collect the cloroformica phase in a second separating funnel of 100 ml (52.2.2).


Remove the acid phase two successive times by 30.0 ml of chloroform (52.3.5) each time. Meet the cloroformicos extracts in the second funnel and eliminate the acidic phase. Add 10 ml of solution of soda ash (52.3.7) to the cloroformica solution, shake for three minutes and let stand until the separation of the phases. Collect the cloroformica phase in a third funnel of 100 ml (52.2.2) and remove the aqueous phase. Add 10 ml of solution of soda ash (52.3.7) to the cloroformica solution, shake for three minutes and let stand until the separation of the phases.


Collect the cloroformica phase in a fourth funnel of 100 ml (52.2.2), wash twice successively with 25 ml of water each time, separate the aqueous phases and collect quantitatively cloroformico extract in a flask of 250 ml (52.2.4). Meet the aqueous phases, wash the gaps separating funnels by some ml of chloroform (52.3.6) then wash the aqueous phase through the chloroform. Separate the cloroformica phase and add it to the extract collected in a flask.


52.4.3 hydrolysis. Evaporate cloroformico extract up to 2 ml approximately on a water bath at 50 ° C in the Rotary evaporator to the vacuum (52.2.4). Dissolve the residue with 2 or 3 ml of methanol (52.3.1). Transfer quantitatively the solution to a centrifuge tube 50 ml (52.2.6) with two portions of 10 ml and a portion of 5 ml of hydrochloric acid 0, 3N (52.3.5). Add a few granules of pumice stone, homogenize and attached to the tube a refrigerant of air (52.2.7). Immerse the tube in a boiling water bath and keep it for forty five minutes. Leave to cool then under a stream of cold water.


52.4.4 development of coloration and measurement of absorbance. Add 1.0 ml of nitrite of sodium (52.3.8), stir and leave to sit for two minutes. Add 1.0 ml of solution of sulfamate ammonium (52.3.9), stir and let stand 2 minutes. Add 1.0 ml of solution of N-(1-naphthyl)-ethylenediamine (52.3.10), stir and leave to stand for 10 minutes. Add 5.0 g of sodium chloride (52.3.11) and 10,0 ml of n-butanol (52.3.12), shake vigorously until complete dissolution of sodium chloride.


Transfer layer supernatant butanolica with help of a pipette, a centrifuge for 15 ml (52.2.6) and centrifuge tube. Then measure absorbance AA in spectrophotometer at 555 nm by comparison with the n-butanol (52.3.12).


52.4.5. blank test. Carry out a blank test applying the same operating method, starting from the point 52.4.2 20.0 ml of diluted methanol (52.3.2). Measure the absorbence AB a 555 nm by comparison with the n-butanol (52.3.12).


52.4.6 benchmark. Carry out a test by applying the same operating method, starting from the point 52.4.2 20.0 ml of solution pattern (52.3.14.2). Measure the AC absorbance at 555 nm by comparison with the n-butanol (52.3.12).


52.5 calculations. Content in mg of etopabato per kg of sample is given by the formula: A - W x 80 H - W x 80 H - P as X: = absorbance of the sample solution.


To = absorbance of the resulting solution of the blank test.


To = absorbance of the resulting solution of the test pattern.


P = the weight in grams of the sample.


The difference between the results of two parallel determinations carried out on the same sample must not exceed:-20 per 100, in relative value, for contents in etopabato less than 7.5 mg/kg.


-1.5 mg/kg, in absolute value, for contents of between 7.5 and 10 mg/kg.


-15 per 100, in relative value, for contents greater than 10 mg/kg.


52.6 references. Fifth Directive of the Commission of March 25, 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 you to determine the dinitolmida (DOT) in feedingstuffs, concentrates and premixes. The 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 aluminum oxide and filtered. An aliquot of the filtrate is evaporated to dryness. The residue is collected by dimethylformamide and treated with ethylenediamine. A purple color develops. The dinitolmida is determined by spectrophotometry at 560 nm.


53.2 material and devices.


53.2.1 Erlenmeyer of 250 ml, standard grinding.


53.2.2 reflux, standardized grinding coolant.


53.2.3. filter Crucible, porosity G 3, diameter 60 mm.


53.2.4 vacuum filtration apparatus (e.g. Witt apparatus).


53.2.5 water bath, regulated at 50 ° C.


53.2.6 spectrophotometer with cuvettes of 10 mm thick.


53.3 reagents.


53.3.1 acetonitrile to 85 per 100 (v/v). Mix 850 ml of pure acetonitrile and 150 ml of water, distill the mixture before use; collect the fraction that distilled from 75 to 77 ° C.



53.3.2 oxide of aluminium for column chromatography. Burnt for two hours at least at 750 ºc, cool in a desiccator and keep in Glass stoppered Topaz. Before use, moisten as follows: insert into a Topaz 10 g glass jar of aluminium oxide and 0.7 ml water, seal and heat for five minutes in a boiling water bath, stirring all the time with energy. Cool by shaking it. Monitor activity by submitting to the analysis, from the point (53.4.1), a certain amount of solution (53.3.6). The recovery of the dinitolmida must be 100 per 100 ± 2 per 100.


53.3.3 n, N-dimethylformamide to 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 2.0 per 100 water content.


53.3.5 standard substance: 3.5 dinitro-o-Toluamide pure that meets the following characteristics: melting point: 177 ° C.


Coefficient of molecular extinction at 248 nm in acetonitrile: 13.1 / 103.


Coefficient of molecular extinction at 266 nm in N, N-dimethylformamide: 10.1 / 103.


53.3.6 standard solution. Weigh, with precision of 0.1 mg, 40 mg of substance pattern (53.3.5). Dissolve with acetonitrile (53.3.1) in a volumetric flask of 200 ml. complete volume with the same solvent and mix. Take 20.0 ml and make up to 100 ml with acetonitrile (53.3.1) in a volumetric flask and mix. 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 premixes, weigh 1 mg, 1 g. to introduce the sample in a 250 ml (53.2.1) Erlenmeyer flask and add 65 ml of Acetonitrile (53.3.1). Mix, attach the reflux condenser to the Erlenmeyer flask (53.2.2) and heat in the water bath (53.2.5) for 30 minutes, stirring it continuously. Cool with cold water. Add 20 g of aluminum (53.3.2) oxide, stir for three minutes and leave to decant.


Put a graduated flask of 100 ml on filtration (53.2.4) apparatus, adjust the filter Crucible (53.2.3) and filter the liquid vacuuming. Then transfer the pellet to the crucible with a few ml of Acetonitrile (53.3.1) and vacuum. Cut the vacuum, replace the sediment in suspension with a few ml of Acetonitrile (53.3.1) and vacuuming again. Repeat these operations until the filtrate volume reaches 95 ml approximately. Make up the volume to 100 ml with acetonitrile (53.3.1) and mix.


If necessary, take an aliquot and dilute by acetonitrile (53.3.1) to obtain a solution containing 5 to 15 micrograms of dinitolmida per ml.


53.4.2 development of coloration and measurement of absorbance. Enter respectively on three 50 ml glasses, A, B and C, 4.0 ml of the solution obtained in (53.4.1). Add only to the vessel C, 1.0 ml of standard solution (53.3.6). Take the three vessels on the water bath (53.2.5) located under a well ventilated gas hood and evaporate to dryness under air flow. Let cool to room temperature.


Add 10.0 ml of N, N-demetilformamida (53.3.3) to A vessel and 2.0 ml respectively to vessels B, C. leave in contact for a few minutes, stirring lightly until complete dissolution of the residue. Then add 8.0 ml of ethylenediamine (53.3.4) in vessels B and C and mix. Measure five minutes exactly after the addition of ethylenediamine, the absorbance of the three solutions using the spectrophotometer (53.2.6) at 560 nm, using the N, N-dimethylformamide (53.3.3) as a target.


53.5 calculations. Content in mg of dinitolmida per kg of sample is given by the formula: A - X F A - F A - B 3 P as X: = absorbance of the solution (witness).


To = absorbance of the solution B (sample).


To = absorbance of the solution C (internal standard).


P = the weight in grams of the sample.


F = dilution factor (staged the event (53.4.1)).


The difference between the results of two parallel determinations carried out on the same sample must not exceed:-10 mg/kg, in absolute value, for contents in dinitolmida of less than 100 mg/kg.


-10 per 100, in relative value, for contents of between 100 and 5,000 mg/kg.


-From 500 mg/kg, in absolute value, for contents of between 5,000 and 10,000 mg/kg.


-5 by 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 Nicarbazina (mix 4 equimolecular, 4-dinitrocarbanilide and 2-hydroxy-4, 6-dimethyl-pyrimidine) 54.1 principle. The method enables you to determine nicarbazina in feedingstuffs, concentrates and premixes containing not more than 5 per 100 forrejeras flour. The derivatives of the nitrofuran, the acetilenheptina and carbadox interfere. The detection limit is 20 mg/kg.


The sample is subjected to extraction with N, N-dimethylformamide. The extract is purified in column chromatography of aluminium oxide: the nicarbazina is eluting with ethanol. The eluate is treated with a solution of sodium hydroxide in ethanol: a yellow colour develops. The nicarbazina is determined spectrophotometrically at 430 nm.


54.2 material and devices.


54.2.1 mouth standard 250-ml Erlenmeyer flask.


54.2.2 reflux, mouth standard condenser.


54.2.3. boiling water bath.


54.2.4 centrifuge with 120 ml tubes.


54.2.5 column chromatography of glass (inside diameter: 25 mm, length 300 mm).


54.2.6 spectrophotometer with cuvettes of 10 mm thick.


54.2.7 burettes graduated to 1/10 ml.


54.3 reagents.


54.3.1 n, N-dimethylformamide p.a.


54.3.2 oxide of aluminium for column chromatography. Burnt for two hours at least at 750 ºc, cool in a desiccator and keep in Topaz with Cap glass bottle. Before using it, to monitor their activities by subjecting to analysis from the point (54.4.2), a certain amount of pattern (54.3.8.3). The recovery of nicerbazina must be 100 per 100 ± 2 per 100.


54.3.3 95 per 100 ethanol (v/v).


54.3.4 ethyl alcohol of 80 per 100 (v/v).


54.3.5 50 per 100 solution (w/v) sodium p.a. hydroxide


54.3.6 1 per 100 solution in ethanol (w/v) of sodium hydroxide. Take 1 ml of the solution (54.3.5) in a 50 ml graduated flask and make up to volume with ethanol 80 per 100 (54.3.4). Prepared at the time of employment.


54.3.7 chemical pattern. Pure Nicarbazina, coefficient of molecular extinction at 362 nm in N, N-dimethylformamide is 37.8 x 10 3.


54.3.8 standard solution.


54.3.8.1 solution 1.25 mg of nicarbazina by ml. weigh to the nearest 0,1 mg, 125 mg of substance pattern (54.3.7). Dissolved in 75 ml of N, N-dimethylformamide (54.3.4), in a 100 ml graduated flask heating slightly. Let cool, make up to volume with the same solvent and mix. Store away from light.


54.3.8.2 solution of 0.125 mg of nicarbazina by ml. take 10 ml of the solution (54.3.8.1) and make up to 100 ml with N, N-dimethylformamide (54.3.1) in a volumetric flask and mix thoroughly.


54.3.8.3 solution, 0.025 mg of nicarbazina by ml. take 20 ml of the solution (54.3.8.2), make up to 100 ml with N, N-dimethylformamide (54.3.1) in a volumetric flask and mix thoroughly.


54.4 procedure.


54.4.1 extraction. Weigh, with precision of 1 mg, 10 g of finely divided and homogeneous sample. For concentrates and premixes weigh 1 g with the accuracy of 1 mg, insert the sample in an Erlenmeyer flask of 250 ml (54.2.1) and add 100 ml of N, N-dimethylformamide (54.3.1) exactly. Mix, attach to a (54.2.2) reflux condenser and heat in a water bath (54.2.3) for 15 minutes shaking from time to time. Cool under running cold water.


Then decant the supernatant liquid to a centrifuge tube (54.2.4) and centrifuge for 3 minutes approximately.


If necessary pipette 25 ml of the supernatant and diluted with N, N-dimethylformamide (54.3.1) to obtain a solution containing from 2 to 10 micrograms of nicarbazina per ml.


54.4.2 chromatography. Insert into a column chromatographic (54.2.5) 30 g of aluminium oxide (54.3.2) in suspension in N, N-dimethylformamide (54.3.1). Let down the liquid up to 1 cm above the column of oxide aluminum and then enter in the column, 25 ml of the extract obtained in (53.4.1). Let the fluid avoiding leaving the column dries and wash the column with 10 ml of N, N-dimethylformamide (54.3.1) each time three times. Elute immediately with 70 ml of ethanol to 95 per 100 (54.3.3). Remove the 10 first ml of eluate and collect the rest into fractions as follows:-a fraction to) 5 ml.


(-A fraction b) 50 ml in a volumetric flask.


(-A fraction c) 5 ml.


Check that the fractions to)) and (c) present no yellowing by addition of solution in ethanol (54.3.6) sodium hydroxide. Continue operations on the fraction b) as shown in (54.4.3).


54.4.3 development of coloration and measurement of absorbance. Place 20 ml of fraction b respectively) in two volumetric flasks A and B of 25 ml. Add to the flask to 5.0 ml of solution in ethanol (54.3.6) sodium hydroxide and the flask B 5.0 ml of ethanol 95 per 100 (54.3.3). Homogenize.


Measure the absorbance of the solutions at 430 nm using white as a mixture of 20.0 ml of ethanol 95 per 100 (54.3.3) and 5 ml of solution in ethanol hidrnoxido of sodium (54.3.6) in the first five minutes.



The solution B the difference between absorbance and is interpolated curve calibrated (54.4.4) to obtain the amount of nicarbazina.


54.4.4 curve of calibration. Treating 25 ml of the solution pattern (54.3.8.3) by chromatography as described in (54.4.2). Transfer quantitatively to a graduated burette (54.2.7) the fraction b) of eluate and pipette in dry 25 ml flasks 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 nicarbazina). Add to each flask 5.0 ml of solution in ethanol (54.3.6) sodium hydroxide make up to volume with ethanol 95 per 100 (54.3.3) and mix thoroughly.


Measure the absorbance of the solutions at 430 nm, in the first five minutes as target using a mixture of 20 ml of ethanol 95 per 100 (54.3.3) and 5.0 ml of solution of sodium hydroxide in ethanol. Plot the curve pattern representing along the abscissa and the absorbance corresponding quantities of nicarbazina mg.


54.5 calculations. The amount of nicarbazina in mg per kg of sample is given by the formula: X F x 10,000 H x F x 10,000 P x F x 10,000 being: A = quantity, in mg, of nicarbazina determined by Spectrophotometric measurement.


P = weight, in g, of the sample.


F = dilution factor (eventually performed in 54.4.1).


The difference between the results of two parallel determinations carried out on the same sample must not exceed the following values:-10 mg/kg, in absolute value, for contents in nicarbazina of less than 100 mg/kg.


-10 per 100, in relative value, for contents ranging from 100 to 5,000 mg/kg.


-500 mg/kg, in absolute value, for contents between 5,000 and 10,000 mg/kg.


-5 by 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 (carboxylate ethyl-4-hydroxy-6, 7-diiso-butoxy-3-quinoline) 55.1 principle. The method allows the determination of buquinolate in feedingstuffs, concentrates and premixes. The lower limit of detection is 10 mg/kg. Decoquinate interferes in the dosage.


The buquinolate is extracted from the sample with chloroform. The extract is evaporated to dryness, the residue is collected with chloroform and the solution is thin layer chromatography. The buquinolate is eluted by ethanol and certain espectrofluorimetricamente by comparison with solutions pattern.


55.2 material and devices.


55.2.1 Erlenmeyer flask of 50 ml and 250 ml with screw cap.


55.2.2 agitator.


55.2.3. centrifuge tubes of 15 ml with screw cap.


55.2.4 water bath termorregulado at 50 ° C.


55.2.5 equipment for thin layer chromatography.


55.2.6 glass plates for chromatography, thin layer of 200 x 200 mm, prepared as follows: spread evenly over the plates a 0.5 mm thick layer gel from silica G (55.3.5) (55.6.1). Left to dry for 15 minutes in the air. Then are introduced for two hours at 110 ° C in the range (55.2.11). Cooling in a desiccator containing silica as dehydrating agent gel.


You can use ready-made equivalent plates.


55.2.7 0,50 ml pipetting devices.


55.2.8 collector of zones for thin layer chromatography.


55.2.9 short-wave UV lamp.


55.2.10 Spectrofluorimeter equipped with a xenon lamp two Monochromators.


55.2.11 stove forced air and adjustable temperature from 0 ° C to 150 ° C.


55.2.12 rotary device of vacuum flask of 250 ml with evaporation.


55.3 reagents.


55.3.1 chloroform p.a.


55.3.2 ethanol 96 per 100 (v/v) p.a.


55.3.3 mix cloroformo-etanol. Mix 10 volumes of chloroform (55.3.1) and a volume of ethanol (55.3.2).


55.3.4 80 100 ethanol (v/v) p.a.


55.3.5 gel G silica for thin layer chromatography.


55.3.6 Buquinolate quality pattern.


55.3.7 standard solutions: 55.3.7.1 standard solutions buquinolate 0.1 mg/ml. Weigh 50 mg of substance pattern (55.3.6) with the precision of 0,1 mg. dissolve in chloroform (55.3.1) and hot, in a water bath at 50 ° C. Transfer quantitatively into a volumetric flask of 250 ml. leave to cool to room temperature. Complete with chloroform (55.3.1) until the number of seats and mix thoroughly.


55.3.7.2 solutions work pattern. Pipette into 25 ml volumetric flasks; 5,0, 10,0, 15,0, 20.0 and 25.0 ml of the solution (55.3.7.1). Make up to the number of seats with chloroform (55.3.1) and mix thoroughly. Prepared at the time of your use. These solutions contain respectively 0.04, 0.08, 0.12, 0.16 and 0.20 mg/ml of buquinolate.


55.4 procedure.


55.4.1. preparation of the sample. Grind the sample in such a way that it passes through a sieve with 1 mm (conforming to ISO R 565 recommendations).


55.4.2 extraction. Weigh with 1 mg sample prepared according to (55.4.1) containing approximately 1.25 mg of buquinolate and insert quantitatively into a 250 ml (55.2.1) Erlenmeyer flask. Add 100,0 ml of chloroform (55.3.1). Mix, cover and shake for one hour on the shaker (55.2.2). Let decanted, filtered and despise the first ml of the filtrate.


Enter 80 ml of clear filtrate into the flask from the Rotary evaporator (55.2.12). Evaporate almost to dryness on a water bath (55.2.4). Transfer the oily residue quantitatively to a 10 ml flask with help of chloroform and a narrow stem funnel through. Make up to volume with chloroform (55.3.1) and if the solution is not clear, centrifuged for three minutes at 3000 rpm in a closed tube.


55.4.3 thin layer chromatography. Deposit promptly on a plate of chromatography (55.2.6) with a micropipette (55.2.7) and a respective distance of 2 cm, the volumes of 0.25 ml of the extract obtained in (55.4.2) and five pattern (55.3.7.2) solutions.


Elute the chromatogram in the camera's development until the solvent front reaches the upper edge of the plate, using chloroform (55.3.1) as eluent. Dry with the help of a stream of air. Back to the plate in the camera's development, using chloroform/ethanol mixture as eluent (55.3.3) until the solvent front has advanced about 12 cm. remove the camera plate. Let evaporate the solvent. Examine the plate under UV light of short wavelength (55.2.9) and delimit the stains of buquinolate (0.4 to 0.6 Rf value) with the help of a needle.


55.4.4 Elution. Pick up silica from each area defined with the help of a collector of zones (55.2.8) in a tube from centrifuge (55.2.3). Add to each 10 ml of ethanol (55.3.4) tube, shake for twenty minutes and then centrifuge for five minutes at 3,000 rpm. Decant the clear solutions in the Erlenmeyer flask of 50 ml (55.2.1).


55.4.5 measurement of fluorescence. Scale to 100 the spectrofluorimeter (55.2.10) with the help of the eluate (55.4.4) obtained from the solution pattern more concentrated. Use for the excitation wavelength of between 300 and 289 nm which give more intense, emission fluorescence wavelength of 375.


Measure the intensity of fluorescence of other eluates (55.4.4) under these conditions. From the found values determine the amount (C) of buquinolate mg contained in 10 ml of the sample aluato.


55.5 calculation. Content in mg of buquinolate per kg of sample is given by the formula: C x C 50,000 x 50,000 x 50,000 being P: C = quantity in mg of buquinolate determined by the measured espectrofluorimetrica.


P = weight, in grams, of the sample taken for analysis.


The difference between the results of two parallel determinations carried out on the same sample should not differ from:-50 by 100 of the higher result for buquinolate values between 10 and 20 mg/kg.


-10 mg/kg, in absolute value for values between 20 and 100 mg/kg.


-10 per 100 of the higher result for values between 100 and 5,000 mg/kg.


-500 mg/kg, in absolute value for values between 5,000 and 10,000 mg/kg.


-5 per 100 of the higher result for values greater than 10,000 mg/kg.


55.6 observations.


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 conical flask of 250 ml with CAP, by shaking vigorously for thirty to forty-five seconds. The suspension well homogenized once, quickly turns on the applicator and spreads evenly over the plates, 0.5 mm thick layer. Left to dry for 15 minutes in the air; then they introduce on the stove (55.2.11) for two hours at 110 ° C. Cooling in a desiccator containing silica as dehydrating agent gel.


55.7 references. Sixth Commission directive of 20 December 1974 (75/84/EEC). «Official Journal of the European communities» number L, 32/26, February 5, 1975.


56 sulphaquinoxaline (2 sulfanilamidoquinoxalina) 56.1 principle. The method allows the determination of sulphaquinoxaline in compound feedingstuffs, hub and premixes. The lower limit of the determination is 20 mg/kg. They interfere with other sulfonamides and the arsanilic acid.


The sulphaquinoxaline is extracted from the sample with dimethylformamide and chloroform. It hydrolyzes in an alkaline medium. After neutralizing the amino derivative is diazota and copulate with N-(1-naphthyl)-ethylenediamine. The absorbance of the solution is measured at 545 nm.


56.2 material and devices.


56.2.1 Erlenmeyer of 250 ml with standard mouth and glass stopper.


56.2.2 agitator.


56.2.3 crucibles filter porosity 3, diameter 80 mm, with kitasatos.



56.2.4 separating funnels of 250 ml.


56.2.5 graduated flasks of 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 cuvettes of 20 mm optical pass-thru.


56.3 reagents.


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 p.a. sodium hydroxide and 25 g of sodium chloride p.a. make up to 500 ml with distilled water and mix.


56.3.5 p.a. concentrated hydrochloric acid (d = 1,18).


56.3.6 solution 0.1 per 100 (w/v) of sodium nitrate. Dissolve in distilled water 100 mg of sodium nitrite p.a., fill with distilled water to 100 ml and mix. Prepare immediately before use.


56.3.7 solution 0.5 per 100 (w/v) of ammonium sulfamate. Dissolve in water distilled 500 mg of ammonium sulfamate p.a. make up to 100 ml with distilled water and mix thoroughly. Prepare immediately before use.


56.3.8 solution 0.1 per 100 (w/v) N-(1-naphthyl)-ethylenediamine dihydrochloride. Dissolve in hydrochloric acid for 0.1 per 100 (v/v) 100 mg of the dihydrochloride of N-(1-naphthyl)-ethylenediamine p.a. make up to 100 ml with the same acid and mix. Prepare immediately before use.


56.3.9 chemical pattern. Pure sulphaquinoxaline.


56.3.10 standard solution. Weigh to 0,1 mg, 250 mg of substance pattern (56.3.9). Dissolve in about 50 ml of sodium hydroxide solution (25 ml of sodium hydroxide solution 0,1 N ° 25 ml of water), make 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 sulphaquinoxaline.


56.4 procedure.


56.4.1. preparation of the sample. Grind the sample in such a way that passes entirely through a mesh of 1 mm (in accordance with recommendation ISO r 565).


56.4.2 extraction. Weigh a sample that contains between 0.25 to 1.25 mg of sulphaquinoxaline with mg accuracy. Introduce quantitatively into a 250 ml Erlenmeyer flask (56.2.1) and add 20 ml of N, N, dimethylformamide (56.3.1). Mix and heat for thirty minutes on a water bath (56.2.7). Cool under running cold water. Add 60 ml of chloroform (56.3.2) stopper the Erlenmeyer flask and shake for 30 minutes with the help of the agitator. (56.2.2). filter the liquid over the filter Crucible (56.2.3) settled in kitasatos and sucking lightly. Wash the Erlenmeyer flask with four servings of 5 ml of chloroform (56.3.2) and on transfer liquids to the crucible. Quickly then transfer the filtrate to a separating funnel (56.2.4), wash the kitasato with the help of approximately 15 ml of chloroform (56.3.2) and transfer the liquid to the separating funnel.


56.4.3 hydrolysis. Add to the separating funnel 50 ml of alkaline solution (56.3.4) and 5 ml of ethanol (56.3.3). Mix well, avoiding the formation of emulsion, either inverting the funnel about 20 times either shaking horizontally. Let stand until the separation of the two phases (the separation is usually completed around fifteen minutes).


Transfer the upper phase (aqueous phase) to a volumetric flask of 250 ml (56.2.5). Extract of the cloroformica phase with three lots alkaline solution (56.3.4) and quantitatively transfer the aqueous extract after each extraction, to the graduated flask (56.2.5). Make up to the number of seats with distilled water and mix thoroughly.


Introduce 25.0 ml of the solution into a volumetric flask of 50 ml (56.2.5), add 5.0 ml of hydrochloric acid (56.3.5). Make up to the number of seats with distilled water and mix thoroughly. Filter, if necessary and delete the first 15 ml of the filtrate. Introduce 10 ml of the solution, two tubes (56.2.6) A and B, respectively.


56.4.4 development of color and the absorbance measurement. Add to each tube 2.0 ml of nitrite of sodium (56.3.6) stir and allow to stand for three minutes. Add 2.0 ml of sulfamate ammonium (56.3.7), stir and leave to sit for two minutes. Add below 1.0 ml of N dihydrochloride solution (1-naphthyl) ethylenediamine (56.3.8) in A tube and 1.0 ml of distilled water tube B. mix well the contents of each tube. Connect the tubes to a tube of water with the help of rubber seals and apply a small vacuum to remove the dissolved nitrogen.


Measure the absorbance Aa and Ab after ten minutes, solutions in the spectrophotometer (56.2.8) to 545 nm taking the distilled water as white. Determine the amount from the value Aa-Ab (C) of sulphaquinoxaline present in the solution of the sample referring to the curve pattern (56.4.5) previously made.


56.4.5 curve pattern. Enter in the graduated flasks of 100 ml (56.2.5), volumes of 2.0; 4.0; 6.0; 8.0 and 10.0 ml of the standard solution (56.3.10) for 50, 100, 150, 200 and 250 micrograms of sulphaquinoxaline. Add 8 ml of hydrochloric acid (56.3.5) to each flask filling volume with distilled water and homogenizing.


Take 10 ml of each solution, corresponding respectively to 5, 10, 15, 20, and 25 micrograms of sulphaquinoxaline, and insert it into the tubes (56.2.6). Develop color as shown in 56.4.4, first paragraph. Then measure the absorbance at 545 nm using distilled water as white. Plot the curve pattern by ordinate the abscissa and the absorbance value the corresponding quantity of sulphaquinoxaline expressed in micrograms.


56.5 calculations. The amount in mg of sulphaquinoxaline per kg of sample is given by the following formula: sulphaquinoxaline mg/kg = C x 50 sulphaquinoxaline mg/kg = C x 50 sulphaquinoxaline mg/kg = P x 50 to be: C = number of sulphaquinoxaline in micrograms determined by measured spectrophotometrically.


P = weight, in grams, of the sample taken for testing.


The difference between the results of two parallel determinations carried out on the same sample should not be greater of:-10 mg/kg, in absolute value, for contents of sulphaquinoxaline between 20 and 100 mg/kg.


-10 per 100 of the higher result for contents ranging from 100 to 5,000 mg/kg.


-500 mg/kg, in absolute value, for contents of between 5,000 and 10,000 mg/kg.


-5 per 100 of the highest result for contents of 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 February 5, 1975.


57 furazolidone (n-(5-nitro-2-furfuriliden)-3-amino-2-oxazolidona) 57.1 principle. The method allows the determination of furazolidone in feedingstuffs, concentrates and premixes. The lower limit of determination is 10 mg/kg.


Furazolidone is extracted with acetone, once the sample has been previously defatted with light petroleum. The extract is purified in a column chromatography of aluminium oxide and furazolidone is eluting with acetone. The eluate is evaporated to dryness and the residue is collected in Amyl alcohol. Furazolidone is removed with a solution of urea and the absorbance of the extract is measured at 375 nm.


57.2 material and devices.


57.2.1 graduated flasks of 100 and 250 ml Topaz color glass.


57.2.2 separating funnels of glass 100 ml 57.2.3 Topaz Twisselman or Soxhlet extraction apparatus.


57.2.4 25 x 80 extraction thimbles or 28 x 100 mm.


57.2.5 column of glass for chromatography, internal diameter of 10 mm and 300 mm length.


57.2.6 water bath termorregulado up to 150 ° C.


57.2.7 spectrophotometer with cuvettes of 10 mm thick.


57.3 reagents.


57.3.1 acetone p.a.


57.3.2 chromatography neutral aluminum oxide. Particle size 100 to 240 mesh 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. Drying the oxide of aluminum at 105 ° C until constant weight.


57.3.3 acetate Amyl p.a.


57.3.4 Amyl alcohol. Mixture of isomers should be a.


57.3.5 light petroleum, p. eb 40-60 ° C.


57.3.6 urea solution. Mix 90 g urea p.a. with 100 ml of distilled water, heat gently to facilitate dissolution.


57.3.7 chemical pattern. Furazolidone pure.


57.3.8 standard solutions. Weigh to the nearest 0,1 mg about 25 mg of substance pattern (57.3.7). Place it in a flask quantitatively graduated 250 ml (57.2.1) to make up to capacity with acetone (57.3.1) and mix (1 ml of this solution contains 100 micrograms of furazolidone).


57.4 procedure. All manipulations must be carried out avoiding direct: 57.4.1 sample preparation. Grind the sample in such a way that pass through a 1 mm mesh sieve, in accordance with recommendation ISO R 565.


57.4.2 extraction. Weighing with accuracy of 1 mg, 5-20 g well homogenised sample (containing as maximum 1 mg of furazolidone) in a cartridge removal (57.2.4). Put it into the stripping apparatus (57.2.3) and extract with light petroleum (57.3.5). To the Soxhlet apparatus are needed 13 to 17 cycles of solvent; for other appliances the duration of extraction should not be less than thirty minutes. Then remove the cartridge from the appliance, remove the residual solvent drying cartridge and its content with the help of a stream of hot air.



For extraction, itself, replace the cartridge into the stripping apparatus and remove with acetone (57.3.1). For the appliance Soxhlet are at least required 25 cycles of solvent; for other devices, the conditions necessary for obtaining a complete extraction must be determined previously. Evaporate the extract acetone over the water bath (57.2.6) to a volume of 5 to 10 ml and allow to cool to room temperature.


57.4.3 chromatography. Introduce a small plug of glass wool in the lower end of the column chromatographic (57.2.5) and tighten the CAP with the help of a rod, to a thickness of 2-3 mm. Prepare a suspension of oxide aluminium (57.3.2) acetone (57.3.1). Insert the suspension in the column chromatographic (57.2.5) and leave. The column thus obtained must have a height of 200 mm approximately. Let descend acetone (57.3.1) up to the upper surface of the column.


Decant the extract obtained in (57.4.2) on the column and wash the flask several times with acetone (57.3.1) transfer liquids on the column. Place an appropriate flask under the column and elute the furazolidone with acetone (57.3.1).


The total volume of acetone used, including that used for washing, must not exceed 150 ml.


57.4.4 extraction and measurement of absorbance. Evaporate the eluate obtained in 57.4.3 to dryness on a water bath (57.2.6) (see 57.5.1). Dissolve the residue in 10 ml of Amyl alcohol (57.3.4) and quantitatively transfer the solution into a separating funnel (57.2.2). Rinse the flask with successive portions of 10 ml of amyl acetate (57.3.3) and 10 ml of the solution of urea (57.3.6). Transfer these solutions to a separating funnel (57.2.2) and shake vigorously for two minutes. Let stand for three to four minutes and collect the aqueous phase into a volumetric flask of 100 ml (57.2.1). Repeat the washing and extraction with the help of four portions of 10 ml of solution of urea (57.3.6) and pick up each time the aqueous phase in the flask. Make the contents of flask 100 ml solution of urea (57.3.6) and mix. Measure the absorbance of the solution in the spectrophotometer (57.2.7) at 375 nm taking the solution of urea (57.3.6) as a target. Determine the amount of furazolidone referring to the standard curve (57.4.5).


57.4.5 curve pattern. Prepare four-column chromatographic (57.2.5) according to the indicated operating model (57.4.3 first paragraph). Enter in column volumes of 2.5, 5.0, 7.5, and 10 ml of solution (57.3.8).


Eluting each column with 150 ml of acetone (57.3.1) and continue the operating mode as shown in 57.4.4. Plot the curve pattern putting in absorbance ordinates and abscissas the corresponding amounts from furazolidone in micrograms.


57.5 calculations. The value in milligrams of furazolidone per kg of sample is given by the following formula: C - P is: C = quantity in micrograms of furazolidone determined by photometric measurement.


P = the weight in grams of the sample taken for analysis.


The difference between the results of two determinations performed in parallel and carried out on the same sample must not differ from:-50 by 100 of the higher result for furazolidone values between 10 and 20 mg/kg.


-10 mg/kg, in absolute value for values between 20 and 100 mg/kg.


-10 per 100 of the higher result for values between 100 and 5,000 mg/kg.


-500 mg/kg, in absolute value for values between 5,000 and 10,000 mg/kg.


-5 per 100 of the higher result for values greater than 10,000 mg/kg.


57.6 observations.


57.6.1 occasionally gets a small amount of diacetone-alcohol, produced by condensation of acetone on aluminum oxide, not obstructing the 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 February 5, 1975.


58 determination of halofuginone DL-trans-7-bromo-6-cloro-3(3-(3-hidroxi-2 Hydrobromide - piperidil) acetonil) - quinazolin-4-(3 H) - ona 58.1 principle. Determination of halofuginone in feedingstuffs. The limit of determination is 1 mg/kg.


After discussing it with hot water, the halofuginone is extracted as free base with ethyl acetate, and subsequently passed as hydrochloride to an aqueous acidic solution. The extract is purified by ion-exchange chromatography. The content of halofuginone is determined by high performance liquid chromatography of reversed-phase (HPLC) using a UV detector.


58.2 material and devices.


58.2.1 ultrasonic bath.


58.2.2. Rotary evaporator.


58.2.3. centrifuge.


58.2.4. HPLC equipment with variable wavelength or photodiode array detector UV detector.


58.2.4.1 liquid chromatography column (300 mm x 4 mm) with c, 10 lm or equivalent column filling.


58.2.5. equipped with a porous glass filter and a key column of glass (300 nm x 10 mm).


58.2.6 filters of fiberglass, with a diameter of 150 mm.


58.2.7 membrane, 0.45 lm filters.


58.2.8 membrane, 0.22 lm filters.


58.3 reagents.


58.3.1 acetonitrile, HPLC grade.


58.3.2 amberlita XAD-2 resin.


58.3.3 ammonium acetate.


58.3.4 ethyl acetate.


58.3.5 glacial acetic acid.


((58.3.6 Halofuginone standard pattern, DL-trans-7-bromo-6-cloro-3-(3-(3-hidroxi-2-piperidil hydrobromide) acetonil) - quinazolin-4-(3 H) - one, and 7.64.


58.3.6.1. booking of halofuginone (100 lg/ml) standard solution. Weigh 50 mg of halofuginone (58.3.6) with an accuracy of 0.1 mg in a 500 ml volumetric flask; dissolve the product in ammonium (58.3.18) acetate buffer solution, fill the container up to the mark with the buffer solution and mix. Stored in the dark and at 5 ºc, this solution is stable for three weeks.


58.3.6.2 solutions for calibration. Pour 1,0, 2,0, 3,0, 4,0 and 6,0 ml of the standard solution of reserve (58.3.6.1) in a series of graduated flasks of 100 ml. fill up to the mark with mobile phase (58.3.21) and mix the liquids. These solutions have concentrations of halofuginone of 1,0, 2,0, 3,0, 4,0 and 6,0 lg/ml respectively. These solutions should be prepared each time that they will use.


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 (ethylenediaminetetraacetic acid, disodium salt).


58.3.14. water, HPLC grade.


58.3.15 solution carbonate sodium 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 to complete a litre and add chloride sodium (58.3.12) until the solution is saturated.


58.3.17 hydrochloric acid, approximately 0,1 mol/l. dilute 10 ml of hydrochloric acid (58.3.7) in water, up to 1 litre.


58.3.18 buffer of ammonium acetate, 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 to 1 litre.


58.3.19 preparation of resin XAD-2 amberlita. Wash the right quantity of resin (58.3.2) with water until it removed all the ions chloride, which is checked by the test with nitrate of silver (58.3.20) in the discarded aqueous phase. Subsequently wash the resin with 50 ml of methanol (58.3.8), discard the methanol and store under new methanol resin.


58.3.20 solution of nitrate of silver, approx. 0.1 mol/l. dissolve 0,17 g of silver nitrate (58.3.9) in 10 ml of water.


58.3.21. HPLC mobile phase. Mix 500 ml of Acetonitrile (58.3.1) with 300 ml of ammonium acetate buffer solution (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 through a filter of 0,22 um (58.2.8) and Degas the solution (e.g. by ultrasound for ten minutes). Stored in the dark and in a closed container, this solution is stable for one month.


58.4 procedure. Note: Halofuginone as the free base is unstable in alkaline solutions and ethyl acetate. You must not remain in ethyl acetate for more than thirty minutes.


58.4.1 general.


58.4.1.1 a blank feed should be analysed to ensure that it does not contain halofuginone nor interfering substances are present.


58.4.1.2 should be to perform a recovery test, by analysing the blank feed after enrich it by addition of a quantity of halofuginone, similar to that present in the sample problem. Enriched to a level of 3 mg/kg, add 300 ll of the standard stock solution (58.3.6.1) to 10 g of the blank feed, mix and wait for ten minutes before proceeding to the extraction (58.4.2).


Note: For the purposes of this method, the blank feed should be similar in type to that of the sample problem and its analysis halofuginone should not be detected.



58.4.2 extraction. Weigh 10 g of the prepared sample to an accuracy of 0,01 g into a 200-ml centrifuge tube, add 0,5 g of ascorbate sodium (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 temperature environment, 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 fifteen seconds. Place the tube in the ultrasonic bath (58.4.1) for three minutes and unscrew the CAP. Centrifuge for 2 minutes and decant the phase of ethyl acetate in a 500 ml separating funnel through a filter of fiberglass (58.2.6). Repeat the extraction of the sample with a second portion of 100 ml of ethyl acetate. Wash extracts combined for one minute with 50 ml of sodium chloride-saturated sodium carbonate solution (58.3.16) and discard the aqueous layer.


Remove the organic layer for a minute with 50 ml of hydrochloric acid (58.3.17). Pass the acid layer less than a separator funnel of 250 ml. again remove the organic layer for a minute and half with other 50 ml of hydrochloric acid and combine with the first extract. Wash the two combined acid extracts by shaking them for approximately ten seconds with 10 ml of ethyl (58.3.4).


Quantitatively transfer the aqueous layer into a 250 ml round-bottomed flask and discard the organic phase. Evaporate all the ethyl acetate that is in acid solution using a rotary evaporator (58.4.2). The temperature of the water bath must not exceed 40 ° C. A vacuum of approximately 25 mbar all residual ethyl acetate is eliminated in five minutes at 38 ° C.


58.4.3 cleaning.


58.4.3.1 preparation of the resin column amberlita.


Prepare a XAD-2 column for each sample extract. Pour 10 g of resin prepared (58.3.19) in a glass column (58.2.5) using methanol (58.3.8). Place a small piece of glass wool in the top of the resin bed. Let the methanol from the column and wash the resin with 100 ml of water, cutting off the flow when the liquid reaches the top of the resin bed. Before using it, leave the column at rest for ten minutes to balance it. You must never allow column to dry.


58.4.3.2 cleaning of the sample. (58.4.2) quantitatively transfer the extract to the top of the column resin prepared (58.4.3.1) and elute, discarding the eluate. The rate of Elution should not exceed 20 ml/minute. Rinse the round flask with 20 ml of hydrochloric acid (58.3.17) and use this liquid to wash the resin column. Remove all acid solution with a jet of air. Dispose of wash water. Add 100 ml of methanol (58.3.8) to the column and elute 5-10 ml, collecting the eluate in a round flask of 250 ml. leave the remaining methanol for 10 minutes so it equilibrate with the resin and continue the elution at a rate that does not exceed 20 ml/minute, collecting the eluate in the same round-bottomed flask. Evaporate the methanol on the Rotary evaporator (58.2.2); the temperature of the water bath should not exceed 40º C. transfer the residue quantitatively to a 10 ml volumetric flask using the mobile phase (58.3.21). Fill up to the mark with mobile phase and mix. Filter an aliquot through a membrane (58.2.7) filter. Reserve this solution for the determination by HPLC (58.4.4).


58.4.4. determination by HPLC.


58.4.4.1 parameters. The following conditions are offered as a guide; other conditions may be used provided that they give the same results.


Column liquid chromatography (58.2.4.1).


Mobile phase HPLC (58.3.21).


Flow rate: 1,5 to 2 ml per minute.


Detection wavelength: 243 nm.


Injection volume: 40 to 100 LL.


Check the stability of the chromatographic system, injecting several times the calibration (58.3.6.2) solution with a content of 3.0 lg/ml, until they have reached peak heights (areas) and retention times consistent.


58.4.4.2 curve of calibration. Inject each calibration (58.3.6.2) solution several times and measure the heights (areas) for each concentration peak. Plot a graph of calibrated using the heights or middle of the peaks of the solutions areas of calibration as the ordinates and the corresponding concentrations in lg/ml as the abscissae.


58.4.4.3 sample solution. Inject the sample extract several times (58.4.3.2), using the same volume used for solutions of calibration and determine the height (area) of the halofuginone peaks average.


58.5 calculations. From height (area) middle of the peaks of the sample solution halofuginone, determine the concentration of the sample in lg/ml solution referring to curve calibrated (58.4.4.2) 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 in lg/ml sample solution.


m: mass of the sample used for testing in grams.


58.6. validation of the results.


58.6.1 identity. The identity of the product can be confirmed by a co-cromatografia, or using a network of diodes (diode array) detector, by which the spectra of the extract of the sample and calibration (58.3.6.2) solution are compared with 6.0 lg/ml content.


58.6.1.1 co-cromatografia. Reinforce a sample extract by adding an appropriate amount of calibration (58.3.6.2) solution. The quantity of added halofuginone 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 according to the amount added, taking into account the dilution of the extract. The width of peak at half of its height, must be equal to the original width at + 10 for 100.


58.6.1.2 detection network of diodes (diode array). The results are evaluated according to the following criteria: to) the length of the wavelength of maximum absorption of the sample and pattern, Spectra recorded in 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 detector of diodes network, the margin is located usually in + 2 nm.


(b) between 225 and 300 nm, the sample and standard spectra recorded at the apex of the chromatographic peak, must not be different for those parts of the spectrum ranging from 10 to 100 per 100 relative absorption. This criterion is met when the same Maxima are present and at no point the deviation observed between the two Spectra exceeds 15 per 100 of the absorption of the analyzed product pattern;


(c) between 225 and 300 nm, the spectra of up-slope, Apex and the down-slope of the peak of the chromatogram of the sample extract must not be different from one another in what refers to those parts of the spectrum within the range of 10-100 by 100 relative absorption. This criterion is met when the same Maxima are present and at no point the deviation between the Spectra exceeds 15 per 100 of the absorption spectrum at the apex of the peak.


If it does not meet any of these criteria, it is not confirmed the presence of the product.


58.6.2 repeatability. The difference between the results of two parallel determinations carried out on the same sample must not exceed 0.5 mg/kg for a content of halofuginone up to 3 mg/kg.


58.6.3 recovery. The recovery of the sample blank reinforced must 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 arranged * in which three samples at eight laboratories were analyzed.


* The Analyst 1983, 108: 1252-1256.


Results shows A (white) upon receipt / sample B (flour): upon receipt / after two months / displays C (granulate): upon receipt / after two months medium (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 it detected.


DR: the reproducibility standard deviation.


CVR: coefficient of variation (percentage).


REC: recovery (percentage).


59. determination of the benzocuato of methyl 7-benziloxi-6-butil-3-metoxicarbonil-4-quinolona 59.1 principle. Methyl benzocuato 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 another extraction with dichloromethane. The content of methyl benzocuato is determined by high performance liquid chromatography on reversed-phase (HPLC) using a UV detector.


This method is intended for the determination of benzocuato of methyl in food for animals. The lower limit of determination is 1 mg/kg.


59.2 material and devices.


59.2.1 laboratory stirrer.


59.2.2. Rotary evaporator.


59.2.3 glass column (250 mm x 15 mm) fitted with a wrench and a deposit of 200 ml capacity, approximately.


59.2.4 team of HPLC with variable wavelength ultraviolet detector or diode network detector.


59.2.4.1 column for chromatography 300 mm x 4 mm, 10 lm C filled with liquid, or equivalent.


59.2.5 0.22 lm membrane filters.


59.2.6 0.45 lm membrane filters.


59.3 reagents.



59.3.1 dichloromethane.


59.3.2 methanol, HPLC quality.


59.3.3. mobile phase for HPLC. Mixture of methanol (59.3.2) and water (HPLC grade) 75 + 25 (V + V).


Filter through a 0.22 lm (59.2.5) filter and Degas the solution (e.g. by treatment with ultrasound for ten minutes).


59.3.4 methanesulfonic acid solution, c = 2 per 100. Dilute 20.0 ml of methanesulfonic acid to 1000 ml with methanol (59.3.2).


59.3.5 solution of hydrochloric acid, c = 10 per 100. Dilute 100 ml hydrochloric acid (q c.a. 1,18 g/ml) to 1000 ml with water.


59.3.6 cation exchange resin Amberlita CG-120 (Na), 100-200 mesh. The resin should be treated before use: prepare a slurry 100 g resin, 500 ml of hydrochloric acid (59.3.5) and bring to boiling on a hot plate, solution stirring continuously. Leave to cool and decant the acid. Filter in vacuo through a filter paper. Rinse the resin twice with lots of 500 ml water and then with 250 ml of methanol (59.3.2). Rinse the resin with another portion of 250 ml of methanol and dry by passing air through the filter cake. Keep the resin dried in capped bottle.


59.3.7 standard substance: pure methyl (7-benziloxi-6-butil-3-metoxicarbonil-4-quinolona) benzocuato.


59.3.7.1 mother of benzocuato methyl, 500 lg/ml standard solution. Despite pattern (59.3.7) with 0,1 mg 50 mg of the substance, dissolved in solution of methanesulfonic acid (59.3.4) in a 100 ml graduated flask, make up to volume and mix.


59.3.7.2 intermediate solution pattern of benzocuato of methyl, 50 lg/ml. 5.0 ml of the solution pattern mother of methyl benzocuato (59.3.7.1) to a 50 ml volumetric flask, make up to the volume with methanol (59.3.2) and mix.


59.3.7.3 solutions for calibration. Transfer 1,0, 2,0, 3,0, 4,0 and 5,0 ml of the standard solution intermediate benzoate of methyl (59.3.7.2) to a series of graduated flasks of 25 ml. make up to volume with mobile phase (59.3.3) and mix. These solutions have concentrations of benzocuato methyl 2.0, 4.0, 6.0, 8.0 and 10.0 lg/ml, respectively. These solutions should be prepared each time that they will use.


59.4 procedure.


59.4.1 general.


59.4.1.1 should be analysed a blank feed to ensure that it does not contain methyl benzocuato nor interfering substances are present.


59.4.1.2 should be to conduct a trial of recovery, by analysing the blank feed after enrich it by addition of a quantity of benzocuato methyl similar to that present in the sample. To enrich at a level of 15 mg/kg, add 600 ll of the solution pattern mother (59.3.7.1) to 20 g of the blank feed, mix and wait for ten minutes before proceeding to the extraction 59.4.2.


Note: For the purposes of this method, the blank feed should be of a type similar to that of the sample and on analysis, not methyl benzocuato should be detected.


59.4.2 extraction. Weigh to the nearest 0,01 gram approximately 20 g of the prepared sample and transfer to a flask of 250 ml. Add 100,0 ml of methanesulfonic acid solution (59.3.4) and shake mechanically (59.2.1) for thirty minutes. Filter the solution through filter paper and retain the filtrate for the phase partition liquid-liquid (59.4.3).


59.4.3 partition liquid-liquid. Transfer 25.0 ml of the filtrate obtained in item 59.4.2 to a blister of decanting of 500 ml, containing 100 ml of hydrochloric acid (59.3.5). Add 100 ml of Dichloromethane (59.3.1) to blister and shake for one minute. Expected to occur the separation of layers and pour the lower (dichloromethane) layer into a round flask of 500 ml. repeat the extraction of the aqueous phase with other two portions of 40 ml of dichloromethane and combine with the first extract in the round flask. Evaporate the dichloromethane extract to dryness on the Rotary (59.2.2) to 40 ° C evaporator under reduced pressure. Dissolve the residue in 20-25 ml of methanol (59.3.2), plug the flask and store all the extract for ion-exchange (59.4.4) chromatography.


59.4.4. ion exchange chromatography.


59.4.4.1 preparation of the cation-exchange column. Plug the lower end of the glass (59.2.3) column with glass wool. Prepare a slurry with 5.0 g of the treated cation-exchange resin (59.3.6) and 50 ml of hydrochloric acid (59.3.5), pour into the glass column and allow to stand. Remove the acid excess, until its level is just above the resin surface and wash the column with water until the effluent neutral to litmus. Transfer 50 ml of methanol (59.3.2) column and elute until the resin surface.


59.4.4.2 column chromatography. Using a pipette, carefully transfer the extract obtained in 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 summary to the surface of resin and wash the column with 50 ml methanol, ensuring that the flow is not greater than 5 ml per minute. Discard the effluent. Elute the methyl column benzocuato using 150 ml of methanesulfonic acid (59.3.4) and collect the eluate from the column in a 250-ml erlenmeyer flask.


59.4.5 partition liquid-liquid. Transfer the eluate obtained in item 59.4.4.2 to a blister of settling of a liter. Rinse the erlenmeyer flask with 5 to 10 ml of methanol (59.3.2) and mix liquids of washing with the contents of the ampoule of decanting. Add 300 ml of a solution of HCL (59.3.5) and 130 ml of Dichloromethane (59.3.1). Stir for a minute and allow the phases to separate. Transfer the lower (dichloromethane) layer into a round flask of 500 ml. repeat these two extracts the first extraction of the aqueous phase with two new portions of 70 ml of dichloromethane and mix into the round flask. Evaporate to dryness extract of dichloromethane in the evaporator Rotary (59.2.2) to 40 ° C and reduced pressure. Dissolve the residue in the flask with approximately 5 ml of methanol (59.3.2) and quantitatively transfer the solution into a graduated flask 10 ml. Rinse the round flask with two new portions of 1 to 2 ml of methanol to the mark and mix. Filter an aliquot portion through a filter membrane (59.2.6). Reserve this solution for the determination by HPLC (59.4.6).


59.4.6 determination by HPLC.


59.4.6.1 parameters. The following conditions are offered as a guide: other conditions may be used provided that they will deliver the same results.


Liquid chromatographic column: (59.2.4.1).


HPLC mobile phase: mixture of methanol and water (59.3.3).


Flow: from 1 to 1,5 ml/minute.


Detection wavelength: 265 nm.


Injection volume: 20 to 25 ll.


Check the stability of the chromatographic system, injecting several times the calibration (59.3.7.3) solution containing 4 lg/ml, until heights (or areas) have reached peak and constant retention times.


59.4.6.2 curve of calibration. Inject each calibration (59.3.7.3) solution several times and measure the heights of the peaks (areas) for each concentration. Plot a graph of calibrated using stockings heights or areas of the peaks of the solutions of calibrated as the ordinates and the corresponding concentrations in lg/ml as the abscissae.


59.4.6.3 sample solution. Inject the sample extract several times (59.4.5), using the same volume used for solutions of calibration. Determine the height (area) middle of the peaks for methyl benzocuato.


59.5 calculations. From height (area) middle of the peaks of the sample solution methyl benzocuato, determine the concentration of the sample in lg/ml solution by reference to the calibration (59.4.6.2) curve content of methyl benzocuato w (mg/kg) is given by the following formula: w = / c x 40 m being: c = concentration of benzocuato methyl in lg/ml sample solution.


m = mass of the sample used for the test, in grams.


59.6. verification of the results.


59.6.1 identity. The identity of the analyte can be confirmed by a co-cromatografia or using a detector network of diodes (diode array), which compares the spectra of the extract of the sample and calibration (59.3.7.3) solution with a content of 10 lg/ml.


59.6.1.1 co-cromatografia. Reinforce a sample extract by adding a proper amount of the solution pattern intermediate (59.3.7.2). The quantity of added methyl benzocuato should be similar to the calculated amount of benzocuato of methyl found in the sample extract. Only the height of the peak of methyl benzocuato should be increased taking into account both the amount added and the dilution of the extract. The width of the peak, half of its maximum height, must not vary by ± 10 per 100 with respect to the original width.


59.6.1.2 detection network of diodes. The results are evaluated according to the following criteria: to) the length of the wavelength of maximum absorption of the sample and pattern, Spectra recorded in 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 detector of diodes network, the margin is usually located in ± 2 nm.



(b) between 220 and 350 nm, the sample and standard spectra recorded at the apex of the chromatographic peak, must not be different for those parts of the spectrum ranging from 10 to 100 per 100 relative absorption. This criterion is met when the same Maxima are present and at no observed point 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 up-slope, the apex and the sample extract chromatographic peak down-slope, must not be different from one another in what refers to those parts of the spectrum within the range of 10-100 by 100 relative absorption. This criterion is met when the same Maxima are present and at no observed point the deviation between the Spectra exceeds 15 per 100 of the absorption spectrum at the apex of the peak.


If it does not meet any of these criteria, it is not confirmed the presence of the analyte.


59.6.2 repeatability. The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 per 100 relative to the highest result for contents of between 4 and 20 mg/kg methyl benzocuato


59.6.3 recovery. The recovery of the sample blank reinforced must be at least 90 per 100.


59.7. results of a collaborative trial. Five samples in 10 laboratories were analyzed. The analyses were carried out in duplicate for each sample.


Results sample blank / flour 1 / granules / flour 2 / granules (mg/kg) / n.d. / 4.50 / 4.50 / 8.90 / 8,70 mg/kg S /-/ 0.30 / 0.20 / 0.60 / 0,50 CV (%) /-/ 6.70 / 4.40 / 6.70 / 5.70 S (mg/kg) /-/ 0.40 / 0.50 / 0.90 / 1.00 HP (percentage) /-/ 8.90 / 11.10 / 10.10 / 11.50 recovery (percentage) /-/ 92,00 / 93,00 / 92,00 / 89,00 S = standard deviation of repeatability.


CV = coefficient of variation of repeatability.


S = standard deviation of reproducibility.


CV = coefficient of variation of reproducibility.


59.8 references. Directive 93/117/EEC. «Official Journal of the European Communities» L 329, 30 December 1993.


60 determination of robenidine hydrochloride 1, 3-bis ((4-clorobenciliden) amino) Guanidine 60.1 principle. The sample is extracted with acidified methanol. The extract is dried out and an aliquot is purified on a column of aluminium oxide. Robenidine is eluting from the column with methanol, concentrates and complete to a volume with mobile phase. The content of robenidine is determined by liquid chromatography of high performance reversed-phase (HPLC) using a UV detector.


This method is intended for the determination of robenidine in feedingstuffs. The limit of determination is 5 mg/kg.


60.2 material and devices.


60.2.1. glass column. Amber Glass column key and a deposit of 150 ml capacity, with an inside diameter of 10.15 mm and a length of 250 mm.


60.2.2. oscillating agitator's laboratory.


60.2.3. Rotary evaporator.


60.2.4 team of HPLC with variable wavelength ultraviolet detector or diode network detector operating in the range 250-400 nm.


60.2.4.1 liquid chromatographic column: 300 mm x 4 mm with filling 10 lm C or equivalent.


60.2.5 (Whatman GF/A or equivalent) glass fiber filter paper.


60.2.6 0.22 lm membrane filters.


60.2.7 0.45 lm membrane filters.


60.3 reagents.


60.3.1 methanol.


60.3.2 acidified methanol. Transfer 4,0 ml hydrochloric acid (P ca, 1,18 g/ml) into a flask graduated 500 ml, make up to the volume with methanol (58.3.1.) and mix. This solution must be prepared before each use.


60.3.3 quality acetonitrile for HPLC.


60.3.4 molecular sieve. Mesh type 3A, 8-12 pearls (pearls of 1, 6-2, 5 mm, crystalline aluminosilicate, 0.3 mm of pore diameter).


60.3.5 aluminium oxide: acid, degree of activity I for column chromatography. Transfer 100 g of aluminium oxide into a suitable container and add 2.0 ml of water. Cover and shake for 20 minutes approximately. Store in a tightly closed container.


60.3.6 solution of potassium dihydrogen phosphate, c == 0,025 mol/l. In a 1000 ml volumetric flask dissolve 3,40 g of potassium dihydrogen phosphate in water (HPLC quality), make up to volume and mix.


60.3.7 solution of disodiohidrogeno phosphate, c == 0,025 mol/l. In a 1000 ml volumetric flask dissolve 3,55 g of dibasic anhydrous sodium or 4,45 g of dihydrate. phosphate (or 8,95 g of dodecahydrate), make up to volume and mix.


60.3.8. mobile phase for HPLC. Mix the following reagents: 650 ml of Acetonitrile (60.3.3).


250 ml water (HPLC quality).


50 ml of solution of potassium dihydrogen phosphate (60.3.6).


50 ml of solution of disodiohidrogeno phosphate (60.3.7).


Filter through a 0.22 lm (60.2.6) filter and Degas the solution (e.g., through the application of ultrasound for ten minutes).


60.3.9 chemical pattern. Pure Robenidine: 1, 3-bis ((4-clorobenciliden) amino hydrochloride) Guanidine (and 758).


60.3.9.1 solution of robenidine stock standard: 300 lg/ml. Although pattern of robenidine (60.3.9) with 0,1 mg 30 mg of the substance. In a 100-ml volumetric flask dissolve in acidified methanol (60.3.2), make up to volume with the same solvent and mix. Wrap the flask with aluminium foil and store away from light.


60.3.9.2 robenidine standard solution: 12 lg/ml. transfer pattern mother (60.3.9.1) 10.0 ml of the solution into a 250 ml graduated flask, make up to volume with the mobile phase (60.3.8) and mix. Wrap the flask with aluminium foil and store away from light.


60.3.9.3 solution of calibration. Transfer 5,0, 10,0, 15,0, 20,0 and 25.0 ml of dissolution pattern intermediate (60.3.9.2) to a series of graduated flasks of 50 ml. make up to volume 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. It must be used glassware amber in all operations.


60.4.1 general.


60.4.1.1 a blank feed must be tested to verify that it does not contain neither robenidine nor interfering substances are present.


60.4.1.2 should be a trial of recovery, which will consist in analysing the blank feed enriched with the addition of a quantity of robenidine, similar to that contains the sample. To enrich a level of 60 mg/kg, transfer 3,0 ml of the solution pattern mother (60.3.9.1) to an erlenmeyer flask of 250 ml. evaporate the solution in a current of nitrogen until they left approximately 0.5 ml. Add 15 g of feed on white, mix and wait ten minutes before performing the extraction (60.4.2).


Note: For the purpose of this method, the blank feed should be similar in type to that of the sample and on analysis robenidine should not be detected.


60.4.2 extraction. Weigh to the nearest 0,01 g, 15 g of the prepared sample. Transfer to a flask of 250 ml and add 100,0 ml of acidified methanol (60.3.2), stopper and shake for one hour on the shaker (60.2.2). Filter the solution by a glass fibre filter paper (60.2.5) and collect all the filtrate in a flask of 150 ml. Add 7,5 g molecular sieve, cover and shake for five minutes. Filter immediately by a glass-fibre filter paper. Keep this solution to the phase of purification (60.4.3).


60.4.3 purification.


60.4.3.1 preparation of the aluminium oxide column. Introduce a small plug of glass fiber in the lower end of the glass column. Weigh 11.0 g of prepared aluminium oxide (60.3.5) and transfer to column. During this phase, should seek to minimize exposure to the atmosphere. Tap the bottom of the loaded column to settle the aluminium oxide.


60.4.3.2 purification of the sample. Transfer with a precision pipette to column 5.0 ml of sample extract prepared at the point 60.4.2. Place the tip of the pipette near the wall of the column and make sure the solution is absorbed into aluminum oxide. Elute the robenidine from the column with 100 ml of methanol (60.3.1), maintaining a flow of 2 - 3 ml/min, and collect the eluate in a round flask of 250 ml. evaporate the methanol solution under reduced pressure until dryness at a temperature of 40 ° C, using a rotary evaporator (60.2.3). Dissolve the residue once again in 3-4 ml. (60.3.8) mobile phase and transfer quantitatively into a graduated flask 10 ml. Rinse the flask with several portions of 1-2 ml of mobile phase and transfer washings to the volumetric flask. Make up to volume with the same solvent and mix. Filter an aliquot through a 0.45 mL (60.2.7) filter. Keep this solution for the determination by HPLC (60.4.4).


60.4.4 determination by HPLC.


60.4.4.1 parameters. The following conditions are offered as a guide; other conditions may be used provided that they give the same results.


Column for HPLC (60.2.4.1).


Mobile phase HPLC (60.3.8).


Flow rate: 1,5 to 2 ml per minute.


Detector wavelength: 317 nm.


Injection volume: 20 to 50 LL.


Check the stability of the chromatographic system, injecting several times the calibration (60.3.9.3) solution containing 3.6 lg/ml, until heights or peak areas and constant retention times have been achieved.



60.4.4.2 curve of calibration. Inject each calibration (60.3.9.3) solution several times and measure the heights (areas) for each concentration peak. Plot a graph of calibrated using the heights or middle of the peaks of the solutions areas of calibration as the ordinates and the corresponding concentrations in lg/ml as the abscissae.


60.4.4.3 sample solution. Inject the sample extract several times (60.4.3.2), using the same volume used for solutions of calibration and determine the height (or area) average of the robenidine peaks.


60.5 calculations. From (area) height average of the peaks of the sample solution robenidine, determine the concentration of the sample solution in lg/ml by referencing the curve calibrated (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 = concentration of the sample solution robenidine lg/ml.


m = mass, in grams, of the sample used for the test.


60.6. verification of the results.


60.6.1 identity. The identity of the analyte can be confirmed by a co-cromatografia, or using a detector of diodes, using network which compare the spectra of the extract of the sample and calibration (60.3.9.3) solution with a content of 6.0 lg/ml.


60.6.1.1 co-cromatografia. Reinforce a sample extract by adding an appropriate amount of calibration (60.3.9.3) solution. The quantity of added robenidine should be similar to the calculated amount of robenidine found in the sample extract.


Only you need to increase the height of the robenidine peak, taking into account both the amount added and the dilution of the extract. The peak width, at half of its maximum height, must be within ± 10 per 100 of the original width.


60.6.1.2 detection network of diodes. The results are evaluated according to the following criteria: a) wavelength which gives the maximum absorption of the sample and standard spectra 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 detector of diodes network, the margin is usually located in ± 2 nm.


(b) between 250 and 400 nm, the sample and standard spectra recorded at the apex of the chromatographic peak must not be different for those parts of the spectrum ranging from 10 to 100 per 100 of relative absorbance. This criterion is met when the same Maxima are present and at no observed point the deviation between the two Spectra exceeds 15 per 100 of the absorbance of the standard analyte.


(c) between 250 and 400 nm up-slope spectra, Apex and the down-slope of the peak of the chromatogram of the sample extract must not be different from one another in what refers to those parts of the spectrum ranging from 10 to 100 per 100 of relative absorbance. This criterion is met when the same Maxima are present and at no observed point the deviation between the Spectra exceeds 15 per 100 of the absorbance of the spectrum of the apex of the peak.


If it does not meet any of these criteria, it is not confirmed the presence of the analyte.


60.6.2 repeatability. The difference between the results of two parallel terminations carried out on the same sample must not exceed 10 per 100 relative to the highest result for robenidine content higher than 15 mg/kg.


60.6.3 recovery. The recovery of the sample blank reinforced must be at least 85 per 100.


60.7. results of a collaborative trial. CEE organized a collaborative trial in which four samples of feed for poultry and rabbits, both in the form of meal and granules were examined in 12 laboratories. Duplicate analysis of each sample is carried out and the following results were obtained: birds: flour / granule / rabbits: flour / granule mean (mg/kg) / 27,00 / 27.99 / 43.6 / 40.1 S (mg/kg) / 1.46 / 1.26 / 1.44 / 1.66 HP (percentage) / 5.4 / 4.5 / 3.3 / 4.1 S (mg/kg) / 4.36 / 3.36 / 4.61 / 3.91 CV (%) / 16.1 / 12.0 / 10.6 / 9.7 recovery (percentage) / 90.0 / 93.3 / 87.2 / 80.2 S = standard deviation of repeatability.


CV = coefficient of variation of repeatability.


S = standard deviation of reproducibility.


CV = coefficient of variation of reproducibility.


60.8 references. Directive 93/117/EEC. «Official Journal of the European Communities» L 329, 30 December 1993.

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