The Feeding Stuffs (Sampling and Analysis) Regulations 1999

• Citation: S.I. 1999/1663
• Sections: 39

Section 1 Title, commencement and interpretation

(1) These Regulations may be cited as the Feeding Stuffs (Sampling and Analysis) Regulations 1999, and shall come into force on 6th July 1999.

(2) In these Regulations “ the Act ” means the Agriculture Act 1970, and “the sampling Directive” means First Commission Directive 76/371/ EEC establishing the methods of sampling for the official control of feeding stuffs .

(3) Any reference in these Regulations to a numbered regulation or Schedule shall be construed as a reference to the regulation or Schedule bearing that number in these Regulations.

Section 2 Prescribed amount for the purposes of the definition of sampled portion

(1) The prescribed amount of material for the purposes of the definition of sampled portion in section 6691) of the Act, so far as it relates to feeding stuffs, shall be determined in accordance with the provisions of this regulation.

(2) In relation to a solid feeding stuff in packages the prescribed amount shall be the quantity of material present or 5 tonnes, whichever is the less.

(3) In relation to a solid feeding stuff in bulk containers, the prescribed amount shall be–

(a) the contents of the lowest number of containers which together hold not less than 5 tonnes; or

(b) if all the containers together hold less than 5 tonnes, or if all the feeding stuff is in one container, the quantity of material present; or

(c) if any container holds not less than 5 tonnes, the content of any such container.

(4) In relation to a solid feeding stuff which is loose in heaps or bays, the prescribed amount shall be–

(a) the contents of the lowest number of heaps or bays which together contain not less than 5 tonnes; or

(b) if all the heaps or bays together contain less than 5 tonnes, or if all the feeding stuff is in one heap or bay, the quantity of material present; or

(c) if any heap or bay contains not less than 5 tonnes, the content of any such heap or bay.

(5) In relation to a liquid or semi-liquid feeding stuff in containers, the prescribed amount shall be–

(a) the contents of the lowest number of containers which together hold not less than 5,000 litres; or

(b) if all the containers together hold less than 5,000 litres, or if all the feeding stuff is in one container, the quantity of material present; or

(c) if any container holds not less than 5,000 litres, the content of any such container.

Section 3 Manner of taking, preparing, marking, sealing and fastening of samples

The manner in which samples of feeding stuffs are to be taken, prepared, marked, sealed and fastened shall be as prescribed in paragraphs 1 to 9 of Schedule 1 and paragraph 10 of that Schedule shall have effect for the purposes of the certificate referred to in regulation 7.

Section 4 Methods of sending part of a sample

Any part of a sample of a feeding stuff required to be sent to any person in pursuance of subsection (1)(b) or (2) of section 77 of the Act shall be sent by registered post or by recorded delivery, or delivered or given by hand.

Section 5 Qualifications of agricultural analysts and deputy agricultural analysts

The prescribed qualifications for an agricultural analyst or a deputy agricultural analyst for the purposes of section 67(5) of the Act, insofar as it relates to feeding stuffs, are that–

(a) he shall be a Chartered Chemist or shall possess a Mastership in Chemical Analysis awarded by the Royal Society of Chemistry,

(b) he shall be a Fellow or a Member of the Royal Society of Chemistry, and

(c) his practical experience of the analysis and examination of feeding stuffs shall be attested by another agricultural analyst or deputy agricultural analyst appointed under section 67(3) of the Act.

Section 6 Application of methods of analysis

(1) Subject to paragraph (2) below, for the purpose of determining whether a substance of a class or description–

(a) listed in column 1 of Annex I to Part II of Schedule 2, or

(b) to which the method of analysis specified in Annex II , or the method specified in Annex III , to that Part relates–

is present or active in a sample of a feeding stuff to be analysed pursuant to the Act, or what quantity or proportion of such a substance is present or active as aforesaid, the provisions specified in Part I of that Schedule under the heading “GENERAL PROVISIONS” shall apply, and–

(i) in relation to a substance of a class or description listed (whether by itself or by reference to its activity) in column 1 of Annex I to Part II of that Schedule, the relevant method of analysis set out in the European Community provision in force specified in the corresponding entry in columns 2 and 3 of that Annex shall be used, and

(ii) in relation to a substance to which the method of analysis specified in Annex II, or the method specified in Annex III, to Part II of that Schedule relates, the method applicable to that substance shall be used

and, where more than one method is set out in columns 2 and 3 of Annex I to Part II of that Schedule in relation to the same substance, the notes to that Annex shall have effect to specify which is the relevant method.

(2) After 31st October 1999, paragraph (1) above shall cease to apply to the following substances listed in column 1 of Annex I to Part II of Schedule 2–

(a) menadione (vitamin K3);

(b) theobromine;

(c) vitamin A; and

(d) volatile mustard oil,

and shall cease to apply to starch insofar as it falls to by analysed by the pancreatic method as mentioned in the notes to that Annex.

(3) Where microscopic examination is carried out in order to–

(a) determine whether any constituent of animal origin is present in a sample of a feeding stuff to by analysed pursuant to the Act; or

(b) estimate the quantity of any such constituent present in any such sample,

then–

(i) the general provisions specified in Part I of Schedule 2 shall apply, and

(ii) the procedure set out in Commission Directive 98/88/ EC establishing guidelines for the microscopic identification and estimation of constituents of animal origin for the official control of feeding stuffs shall be used.

(4) Where a sample of a feeding stuff is to be analysed pursuant to the Act and neither paragraph (1) above nor paragraph (3) above applies–

(a) if there is an applicable standard of the kind referred to in the first indent of Article 18.3 of Council Directive 95/53/EC fixing the principles governing the organisation of official inspections in the field of animal nutrition (as amended), analysis shall be carried out in accordance with that standard.

(b) if there is no such standard, it shall be carried out in accordance with any scientifically valid method the application of which does not contravene any general principle of the Treaty establishing the European Community.

Section 7 Form of certificate of analysis

The certificate of analysis of any feeding stuff to be sent pursuant to section 7794) of the Act shall be in the form set out in Part I of Schedule 3, and shall be completed in accordance with the notes setout in Part II of that Schedule.

Section 8 Period within which analysis of the oil content of a feeding stuff must be carried out

Where a sample of a feeding stuff has been taken by an inspector in the prescribed manner and sent to an agricultural analyst for analysis, any analysis of the oil content of that feeding stuff shall be disregarded unless it is carried out before the expiry of the period of three weeks commencing with the date of sampling.

Section 9 Modification of the Agriculture Act 1970 as respects metrication

For the purposes of its application to feeding stuffs, the Act shall continue to be modified as follows–

(a) in the definition of sampled portion in section 66(1), the words “five tonnes or 5,000 litres” shall be substituted for the words “five tons or 1,000 gallons or the prescribed metric substitution”;

(b) in section 68(2)(b), the words “the sales in quantities of not more than 25 kilograms” shall be substituted for the words “to ales of small quantities (that is to day, sales in quantities of not more than fifty six pounds or the prescribed metre substitution)”; and

(c) in section 76(5), the words “six kilograms” shall be substituted for the words “fourteen pounds or the prescribed metric substitution”.

Section 10 Revocation

The Feeding Stuffs (Sampling and Analysis) Regulations 1982 , the Feeding Stuffs (Sampling and Analysis) (Amendment) Regulation 1984 , the Feeding Stuffs (Sampling and Analysis) (Amendment) Regulations 1985 and the Feeding Stuffs (Sampling and Analysis) (Amendment) Regulations 1994 are hereby revoked.

Section 1

In the case of feeding stuffs in packages or containers, except where section 68(2)(b) of the Act applies, only unopened packages or containers, which appear to the inspector proposing to take the sample to be the original packages or containers of the feeding stuff, shall be selected for the purpose of the sample.

Section 1

Each container of a final sample shall be so secured and sealed by the person taking the sample that the container cannot be opened without breaking the seal; alternatively the container may be place in a stout envelope or in a linen, cotton or plastic bag, and this further receptacle then secured and sealed in such a manner that the contents cannot be removed without breaking the seal or the receptacle.

Section 2

The sample shall be taken and prepared as quickly as possible, having regard to the precautions necessary to ensure that it remains representative of the sampled portion. Instruments, surfaces and containers used in sampling shall be clean and dry.

Section 2

A label shall be attached to the container or receptacle containing the final sample and sealed in such a manner that it cannot be removed without the seal being broken. The label shall be marked with the following particulars, which shall be visible without the seal being broken:

(a) name of the inspector and the authority by which he was authorised to take the sample;

(b) identification mark given by the inspector to the sample;

(c) place of sampling;

(d) date of sampling;

(e) name of the material; and

(f) identification code, batch reference number or consignment identification of the material sampled, where readily available.

Section 3

No sample shall be drawn from any part of the sampled portion which appears to be damaged.

Section 3

The container or receptacle referred to above may also be secured and sealed by the holder of the material sampled or person acting on his behalf.

Section 4

Where any appreciable portion of the feeding stuff appears to be mouldy, or is otherwise apparently unsuitable for feeding purposes, separate samples shall be drawn of the unsuitable portion and of the residue of the feeding stuff respectively. These shall be treated as separate sampled portions.

Section 4

The label referred to above shall be signed or initialled by the person taking the sample or by or on behalf of the holder of the material sampled.

Section 5

(1) An inspector who intends to take a sample in accordance with the provisions of section 76(1)(b) of the Act shall satisfy himself that the conditions in which the material concerned is stored are not such as might have caused undue deterioration thereof, and that it appears not to have been contaminated by any other material.

(2) The provisions of sub-paragraph (1) above shall not apply as respect any feeding stuff purchased for the purpose of resale in the course of trade.

Section 6

The sampling apparatus shall consist of materials which cannot contaminate the feeding stuff to be sampled.

Section 7

Subject to paragraph 8 below, in the absence of good reason to the contrary, the sampling apparatus for solid feeding stuffs shall be taken from among the following:

(a) a flat-bottomed shovel with vertical sides;

(b) a sampling spear with dimensions appropriate to the characteristics of the sampled portion in all respects, including dimensions of the container and particle size of the feeding stuff;

(c) mechanical apparatus which, if used for the purpose of sampling a feeding stuff being moved at the time the sample is taken, must be capable of taking samples right across the flow of the product;

(d) apparatus designed to divide the sample into approximately equal parts for taking incremental samples, and for the preparation of reduced and final samples.

Section 8

A sampling spear shall not be used if the material is in a package or container containing not more than 50 kg and, prior to the taking of a sample, the manufacturer objects to such use on the ground that the material is unsuitable.

Section 9

The sample shall be taken, prepared and packaged in accordance with the requirements specified, in the Annex to the Sampling Directive–

(a) in paragraphs 5A and 5B under the heading “QUANTITATIVE REQUIREMENTS” (as set out in section A of the Table to this Part); and

(b) in paragraphs 6.2 to 6.4 under the heading “INSTRUCTION FOR TAKING, PREPARING AND PACKAGING THE SAMPLES” (as set out in Section B of the Table to this Part).

Section 10

Any sample taken in accordance with the preceding paragraphs of this Schedule shall be considered as representative of the sampled portion.

Section 1 Introduction

(a) In general a single method of analysis applies for the determination of the presence or quantity of a substance in feeding stuffs. Where two or more methods are prescribed the choice between them shall, except where otherwise indicated, be left to the agricultural analyst concerned; the method used must however be indicated in the certificate of analysis.

(b) the result given in the analysis report shall be the average value obtained from at least two independent determinations, carried out on separate portions of the sample, and of satisfactory repeatability.

(c) The result shall be expressed, in the manner laid down in the method of analysis, to an appropriate number of significant figures and shall be corrected, if necessary, to the moisture content of the final sample prior to preparation (see paragraph 3(d) below).

Section 1 Scope and Field of Application

This method is for the determination of uric acid and its salts in dried poultry waste and in feeding stuffs containing dried poultry waste.

Section 1 Scope and Field of Application

This method is for the determination of isobutylidenediurea in feeding staffs.

Section 2 Reagents and Apparatus

(a) Unless otherwise specified in the method of analysis concerned, all reagents must be analytically pure. The purity of the reagents, especially when determining trace elements, must be checked by a blank test. Depending upon the results obtained, further purification of the reagents may be required.

(b) Where any operation involves preparation of solutions, dilution, rinsing or washing, as part of a method of analysis, water must be used unless the specification of the method indicates otherwise.

(c) Water should, in the absence of good reason to the contrary, be demineralized or distilled. Where indicated in the method of analysis concerned it must be subjected to special purification procedures.

(d) All instruments or apparatus used must be clean, especially when very small amounts of substances have to be determined.

Section 2 Principle

Uric acid is extracted with neutral ethanotic formaldehyde solution, precipitated as silver magnesium urate, redissolved in sodium thiosulphate solution and determined spectrophotometrically.

Section 2 Principle

The sample is hydrolysed, liberating isobutyraldehyde, the concentration of which is determined by gas chromatography.

Section 3 Preparation of the sample for analysis

(a) Samples must be prepared in such a way that the amounts weighed out, as provided for in the methods of analysis, are homogeneous and representative of the final sample.

(b) All the necessary operations must be performed in such a way as to avoid, as far as possible, any change in, or contamination of, the sample. Grinding, mixing and sieving should be carried out as quickly as possible, with minimal exposure of the sample to air and light. Overgrinding is to be avoided. Mills and grinders likely to heat the sample appreciably should not be used. Nevertheless, where some loss or gain of moisture is unavoidable, allowance should be made for such changes (see sub-paragraph (d) below). Manual grinding is recommended for feeding stuffs which are particularly sensitive to heat. Care should also be taken to ensure that the apparatus itself is not a source of contamination by trace elements.

(c) If the final sample as received consists of unopened packages or containers then, immediately prior to the preparation of the sample for analysis, all the contents shall be thoroughly mixed together.

(d) If the sample is appreciably moist, or if for any reason the preparation cannot be carried out without significant changes in the moisture content of the sample, determine the moisture content before and after preparation, using the method specified in columns 2 and 3 of Annex I to Part II of this Schedule, appearing opposite to the reference to “Moisture” in column 1 of that Annex.

(e) When a microscopial examination for the presence of undesirable substances is required then, in the absence of good reason to the contrary–

(i) the sample should be crushed and ground only to such an extent as facilitates the examination, and

(ii) grinding to pass 1 mm should not be used where it could lead to difficulties in identifying the undesirable substances listed in Schedule 5 of the Feeding Stuffs Regulations 1995 .

Procedure

(f) Mix the sample thoroughly either mechanically or manually. Divide the sample into two equal portions (the quartering method should be used where applicable). Preliminary crushing and/or grinding may be necessary, if the sample is in a coarse condition, to facilitate division. Keep one of the portions in a suitable container, i.e. non-corrodible, clean and dry and fitted with an air-tight stopper, and prepare the other portion or a representative part of it, of at least 100 g, as indicated below.

(i) Feeding stuffs which can be ground as such

Unless otherwise specified in the method of analysis concerned, sieve the whole sample through a sieve having apertures of 1 mm square , in accordance with recommendation ISO R565, after grinding, if necessary.

Mix the sieved sample and collect it in a suitable container, i.e. non-corrodible, clean and dry and fitted with an air-tight stopper. Mix again, immediately before weighing out the amounts for analysis.

(ii) Feeding stuffs which can be ground only after drying

Unless otherwise specified in the method of analysis concerned, dry the sample to reduce its moisture content to a level of 8–12%, in accordance with the preliminary drying procedure specified in point 4.3 of the method referred to in sub-paragraph (d) above, until grinding enables the sample to be passed wholly through a sieve having apertures of 1 mm square . Then proceed as indicated in sub-paragraph (f)(i) above.

(iii) Liquid or semi-liquid feeding stuffs

Collect the sample in a suitable container, i.e. non-corrodible, clean and dry and fitted with an air-tight stopper. Mix thoroughly immediately before weighing out the amount for analysis.

(iv) Other feeding stuffs

A sample which cannot be prepared according to any of the above procedures should be treated by any other procedure which ensures that the amounts weighted out for analysis are homogeneous and representative of the final sample.

Storage of samples

(g) Samples must be stored at such a temperature as will cause no compositional changes. A sample intended for the analysis of vitamins, or substances which are particularly sensitive to light, should be placed in a brown glass container.

Section 3 Reagents

(3.1) Sodium hydroxide solution: dissolve 50 g sodium hydroxide in 50 ml water, mix well and store in a suitable plastic container.

(3.2) Formaldehyde solution: the strength of the commercially available solution should be checked as follows: mix 3 ml formaldehyde solution with 50 ml IN sodium hydroxide solution and 25 ml hydrogen peroxide solution (20 volumes). Heat on a steam bath until effervescence stops. Cool, and titrate with IN hydrochloric acid using phenolphthalein indicator. Carry out a blank titration using 3 ml water in place of the formaldehyde.

1 ml of 1N sodium hydroxide ≡ 0.0300 g formaldehyde

where B = blank titre; and

T = sample titre.

(3.3) Neutral ethanolic formaldehyde solution: mix an appropriate volume of formaldehyde solution (3.2) containing 17.5 g of formaldehyde with 250 ml water and 500 ml ethanol. Adjust the pH of the solution to 7.0 with 0.1N sodium bhydroxide solution. Dilute to 1,000 ml with water, mix and gain adjust the pH to 7.0 if necessary.

(3.4) Scucinate buffer solution: dissolve by heating, 29.5 g of succinic acid in 750 ml water and 20 ml sodium hydroxide solution (3.1). Cool, add an appropriate volume of formaldehyde solution (3.2) containing 17.5 g of formaldehyde, mix well and adjust the pH to 6.0 with sodium hydorxide solution (3.1). Dilute to 1,000 ml with water, mix and gain adjust the pH to 6.0 if necessary.

(3.5) sodium thiosulphate solution: 25 g sodium thiosulphate (Na 2 S 2 O 3 .5H 2 O) per 1,000 ml.

(3.6) Silver lactate solution: dissolve, by heating, 3 g silver lactate in 50 ml water and 1 ml lactic acid. Dilute to 100 ml with water, filter, and store in dark glassware. Do not expose to strong light.

(3.7) Ammoniacal magnesium solution: dissolve 8.75 g magnesium sulphate (MgSO 4 .7H 2 O) and 17.5 g ammonium chloride in 50 ml water. Add 30 ml ammonia solution (d = 0.88 g/ml) mix well and dilute to 100 ml with water.

(3.8) Benedict and Hitchcock reagent: mix 35 ml silver lactate solution (3.6) with 15 ml ammoniacal magnesium solution (3.7). Add 50 ml ammonia solution (d = 0.88 g /ml). Mix well, Prepare immediately before use.

(3.9) Standard uric acid solution: weigh to the nearest 0.1 mg, 250 mg of uric acid and transfer to a 150 ml round-bottomed flask fitted with a reflux condenser. Add 100 ml ethanolic formaldehyde solution (3.3) and boil under reflux on a steam bath for 30 minutes, shaking frequently. Cool, transfer the solution to a 250 ml graduated flask, wash the round-bottomed flask with ethanolic formaldehyde solution (3.3) and combine the washings with the uric acid solution. Dilute to the mark with ethanolic formaldehyde solution (3.3) and mix. 1 ml contains 1 mg of uric acid.

(3.10) Light petroleum, boiling range 40–60°C.

Section 3 Reagents

(3.1) Toluene.

(3.2) Sodium sulphate, anhydrous.

(3.3) Buffer solution pH1: dissolve 27.2 g sodium acetate trihydrate in 300 ml IM hydrochloric acid and add 700 ml water.

(3.4) Buffer solution pH 0.65: dissolve 27.2 g sodium acetate trihydrate in 400 ml IM hydrochloric acid and add 600 ml water.

(3.5) Isobutylidenediurea.

(3.6) internal standard solution: dilute 5 ml isopropyl acetate to 100 ml with toluene (3.1).

Section 4 Apparatus

(4.1) Spectrophotometer, with 10 mm silica cells.

(4.2) Percolation tubes, glass. Upper part: approximately 240 mm long, 18 mm internal diameter, lower part: approximately 120 mm long, 8 mm internal diameter.

Section 4 apparatus

(4.1) 250 ml conical flasks with ground glass or PTFE stoppers.

(4.2) Stoppered centrifuge tubes.

(4.3) Gas chromatograph with flame ionisation detector.

(4.4) Column:

either (i) 1.5 m glass column (4 mm internal diameter) packed with 5% QV17 on Gas Chrom Q, 80–100 mesh,

or (ii) 1.5 m glass column (4 mm internal diameter) packed with 5% Carbowax 20M-TPA on Diatomite C-AAW, 80–100 mesh.

(4.5) Water bath: hotplate stirrer on which is placed a 2,000 ml beaker (or suitable vessel) containing water maintained at 40–50°C.

Section 5 Procedure

Extraction of Uric Acid

(5.1) From dried poultry waste:

(5.1.1) Weigh to the nearest 0.001 g, about 0.4 g dried poultry waste and place in a 150 ml round-bottomed flask. Add 60 ml ethanolic formaldehyde solution (3.3), fit a reflux condenser onto the flask and heat on a steam bath for 1 hour. Cool and filter by suction through a sintered glass crucible (porosity 4) into a 100 ml graduated flask. Wash out the round-bottomed flask with 3 × 10 ml portions of ethanolic formaldehyde solution (3.3) passing each portion through the crucible into the graduated flask. Dilute to 100 ml with ethanolic formaldehyde solution and mix.

From feeding stuffs:

(5.1.2) Weigh to the nearest 0.001 g, between 4 g and 5 g of prepared sample. Transfer to a glass percolation tube (4.2) fitted with a small paper cup to retain the feed. Remove the fat from the feed by extraction with light petroleum (3.10). Transfer quantitatively the defatted sample to a 150 ml round-bottomed flask and remove the residual solvent with a slow current of air. Continue as in 5.1.1, second sentence “… Add 60 ml ethanolic formaldehyde solution (3.3) …”.

Determination

(5.2) Transfer by pipette 20 ml of the sample extract prepared as in 5.1.1 or 5.1.2 to a 50 ml centrifuge tube. Add 10 ml of Benedict and Hitchcock reagent 93.8), mix well and allow to stand in the dark for 1 hour. Centrifuge at 2,000 rpm for 15 minutes, pour off the supernatant liquid and allow to drain for 10 minutes. Carefully wipe off any remaining liquid without disturbing the precipitate, and add 20 ml sodium thiosulphate solution (3.5) to each tube. Dissolve the precipitate by stirring with a thin glass rod. Transfer by pipette 5 ml of this solution into a 200 mg graduated flask containing 40 ml succinate buffer solution (3.4). Dilute to 200 ml with water and mix well. Measure the absorbance of the solution at 294 mm in 10 mm silica cells against a solution prepared by mixing 5 ml sodium thiosulphate solution (3.5) with 40 ml succinate buffer solution (3.4) and diluting to 200 ml with water. Determine the quantity of uric acid present by reference to the calibration curve (5.3).

Calibration Curve

(5.3) Into a series of 50 ml centrifuge tubes, transfer by pipette 2, 4, 6, 8, 10 and 12 ml standard uric acid solution (3.9) (corresponding to 2, 4, 6, 8, 10 and 12 mg of uric acid) and make up to 20 ml with ethanolic formaldehyde solution (3.3). Add to each tube 10 ml Benedict and Hitchcock reagent (3.8), mix well and stand in the dark for 1 hour. Continue as in 5.2 from “… Centrifuge at 2,000 rpm. …”, Measure the absorbances of the solutions and plot the calibration curve using absorbances as the ordinates and the corresponding quantities of uric acid, in mg (as shown above) as the abseissae.

Section 5 Procedure

Hydrolysis

(5.1) Weigh to the nearest 0.001 g, between 3 and 7 g of the prepared sample containing about 0.2 g of isobutylidenediurea into a concical flask (4.1). Add 100 ml buffer solution (3.4) and 20.0 ml toluene (3.1) to the sample and place in the flask a magnetic bar. Stopper firmly to ensure that the flask remains tightly closed during the hydrolysis.

Place the flask in the water bath (4.5) and stir vigorously for 20 minutes. Remove the flask and immerse in an ice-water bath for 5 minutes. Add 15 g sodium sulphate (3.2) and 5.0 ml internal standard solution (3.6) to the contents of the flask. Stopper the flask again, shake, return to the water bath (4.5) and warm for 3 minutes with stirring. Cool in the ice-water bath for 5 minutes. Transfer slowly between 15 and 25 ml of the mixture to the centrifuge tube (4.2), stopper, and centrifuge for 5 minutes to separate the layers. (Repeat the transfer if insufficient toluene is decanted). Transfer a portion of the upper (toluene) layer to a test tube with a pasteur pipette.

Determination

(5.2) Inject between 0.5 and 1.0 μl of the toluene solution (5.1) into the gas chromatograph (4.3).

Suggested conditions:

Approximate retention times:

Measure the peak heights of the isobutyraldehyde and internal standard. Calculate the peak height ratio, isobutyraldehyde/internal standard, and from this value determine the quantity of isobutylidenediurea present by reference to the calibration curve (5.3).

Calibration curve

(5.3) Weigh to the nearest mg, 100, 200 and 300 mg isobutylidenediurea (3.5) into three conical flasks (4.1). Add 100 ml buffer solution (3.3), 20.0 ml toluene (3.1) and a magnetic bar to each. Stopper the flasks firmly. Continue as in 5.1 from “… Place the flask in the water bath …”. Inject the toluene solutions into the gas chromatograph (4.3), and measure the peak heights. Calculate the peak height ratios, isobutyraldehyde/internal standard, and plot the calibration curve using peak height ratios as the ordinates and the corresponding weights or isobutylidenediurea as the abseissae.

Section 6 Expression of the Results

The uric acid nitrogen content per cent of the sample is given by the formula

where:

mg uric acid (in the aliquot volume of the sample extract) as determined by photometric measurement; and

weight of sample in grams.

Section 6 Expression of the Results

The per cent content of isobutylidenediurea in the sample is given by the formula:

where:

weight of isobutyldenediurea (mg) read from the calibration curve; and

weight of sample in grams.