Feedingstuffs Rules, 2005

L.N. 99 of 2005

VETERINARY SERVICES ACT, 2001

(ACT XXIII OF 2001)

Methods of Analysis for the Official Control of

Feedingstuffs Rules, 2005

IN exercise of the powers conferred by article 22 of the

Veterinary Services Act, 2001 the Minister for Rural Affairs and the

Environment has made the following regulations>-

The title of these regulations is Methods of Analysis for Official Title, Scope and

Applicability.

Control of Feedingstuffs Regulations, 2004.

(2) The scope of these regulations is to implement the rules

found under EU Commission Directive 71#393#EEC establishing EC

methods for the official control of feedingstuffs.

(3) The Member States shall require that analyses for official

controls of feedingstuffs as regards their contents of moisture, volatile

nitrogenous bases, total phosphorus and crude oils and fats be carried

out according to the methods described in the Schedule to these

Regulations.

I. DETERMINATION OF MOISTURE

1. Purpose and Scope

This method makes it possible to determine the moisture content of

feedingstuffs. It does not cover the analysis of milk products as straight

feedingstuffs, the analysis of mineral substances and mixtures composed

predominantly of mineral substances, the analysis of animal and vegetable fats

and oils or the analysis of the oil seeds and olaginous fruit defined in Council

Regulation No 136/66/EEC (1) of 22 September 1966 on the establishment of a

common organization of the market in oils and fats.

The determination of the moisture content of oil seeds and oleaginous fruit is

described in Annex III to Commission Regulation (EEC) No 1470/68 (2) of 23

September 1968 on the drawing and reduction of samples and the determination

of the oil content, impurities

and Schedule

moisture in oil seeds.

17 pages

Principle

The sample is desiccated under specified conditions which vary according to the

nature of the feedingstuff. The loss in mass is determined by weighing. It is

necessary to carry out preliminary drying when dealing with solid feedingstuffs

which have a high moisture content.

Apparatus

3.1. Crusher of non moisture-absorbing material which is easy to clean, allows

rapid, even crushing without producing any appreciable heating, prevents

contact with the outside air as far as possible and meets the requirements

laid down in 4.1.1. and 4.1.2. (e.g. hammer or watercooled micro-crushers,

collapsible cone mills, slow motion or cogwheeled crushers).

3.2. Analytical balance, accurate to 0·5 mg.

3.3. Dry containers of non-corrodible metal or of glass with lids ensuring

airtight closure; working surface allowing the test sample to be spread at

about 0·3 g/cm2.

3.4. Electrically heated isothermal oven (± 1 ºC) properly ventilated and

ensuring rapid temperature regulation 3 .

(3) For the drying of cereals, flour, groats and meal, the oven must have a thermal capacity such that, when pre-

set at 131 ºC, it will return to that temperature in less than 45 minutes after the maximum number of test samples

have been placed inside to dry simultaneously. Ventilation must be such that, when as many samples of common

wheat as it can contain are dried for two hours, the results differ from those obtained after four hours of drying by

less than 0·15%.

3.5. Adjustable electrically heated vacuum oven fitted with an oil pump and

either a mechanism for introducing hot dried air or a drying agent (e.g.

calcium oxide).

3.6. Desiccator with a thick perforated metal or porcelain plate, containing an

efficient drying agent.

Procedure

N.B.: The operations described in this section must be carried out immediately

after opening the packages of samples. Analysis must be carried out at least in

duplicate.

4.1. Preparation

4.1.1. Feedingstuffs other than those coming under 4.1.2 and 4.1.3

Take at least 50 g of the sample. If necessary, crush or divide in such a

way as to avoid any variation in moisture content (see 6).

4.1.2. Cereals and groats

Take at least 50 g of the sample. Grind into particles of which at least

50% will pass through a 0·5 mm mesh sieve and will leave no more than

10% reject on a 1 mm round- meshed sieve.

4.1.3. Feedingstuffs in liquid or paste form, feedingstuffs predominantly

composed of oils and fats

Take about 25 g of the sample, weigh to the nearest 10 mg, add an

appropriate quantity of anhydrous sand weighed to the nearest 10 mg and

mix until a homogeneous product is obtained.

4.2. Drying

4.2.1. Feedingstuffs other than those coming under 4.2.2 and 4.2.3

Weigh a container (3.3) with its lid to the nearest 0·5 mg. Weigh into the

weighed container, to the nearest 1 mg, about 5 g of the sample and

spread evenly. Place the container, without its lid, in the oven preheated

to 103 ºC. To prevent the oven temperature from falling unduly,

introduce the container as rapidly as possible. Leave to dry for four

hours reckoned from the time when the oven temperature returns to 103

ºC. Replace the lid on the container, remove the latter from the oven,

leave to cool for 30 to 45 minutes in the desiccator (3.6) and weigh to

the nearest 1 mg.

For feedingstuffs composed predominantly of oils and fats, dry in the

oven for an additional 30 minutes at 130 ºC. The difference between the

two weighings must not exceed 0·1% of moisture.

4.2.2. Cereals, flour, groats and meal

Weigh a container (3.3) with its lid to the nearest 0·5 mg. Weigh into the

weighed container, to the nearest 1 mg, about 5 g of the crushed sample

and spread evenly. Place the container, without its lid, in the oven

preheated to 130 ºC. To prevent the oven temperature from falling

unduly, introduce the container as rapidly as possible. Leave to dry for

two hours reckoned from the time when the oven temperature returns to

130 ºC. Replace the lid on the container, remove the latter from the

oven, leave to cool for 30 to 45 minutes in the desiccator (3.6) and

weigh to the nearest 1 mg.

4.2.3. Compound feedingstuffs containing more than 4% of sucrose or lactose

straight feedingstuffs such as locust beans, hydrolyzed cereal products,

malt seeds, dried beet chips, fish and sugar solubles; compound

feedingstuffs containing more than 25% of mineral salts including water

of crystallization.

Weigh a container (3.3) with its lid to the nearest 0·5 mg. Weigh into the

weighed container, to the nearest 1 mg, about 5 g of the sample and

spread evenly. Place the container, without its lid, in the vacuum oven

(3.5) preheated to between 80 ºC and 85 ºC. To prevent the oven

temperature from falling unduly, introduce the

container as rapidly as possible.

Bring the pressure up to 100 Torr and leave to dry for four hours at this

pressure, either in a current of hot, dry air or using a drying agent (about

300 g for 20 samples). In the latter instance, disconnect the vacuum

pump when the prescribed pressure has been reached. Reckon drying

time from the moment when the oven temperature returns to 80 ºC to 85

ºC. Carefully bring the oven back to atmospheric pressure. Open the

oven, place the lid on the container immediately, remove the container

from the oven, leave to cool for 30 to 45 minutes in the desiccator (3.6)

and weigh to the nearest 1 mg. Dry for an additional 30 minutes in the

vacuum oven at 80 ºC to 85 ºC and reweigh. The difference between the

two weighings must not exceed 0·1% of moisture.

4.3. Preliminary drying

4.3.1. Feedingstuffs other than those coming under 4.3.2

Solid feedingstuffs with a high moisture content which makes

crushing difficult must be subjected to preliminary drying as follows

Weigh 50 g of uncrushed sample to the nearest 10 mg (compressed

or agglomerated feedingstuffs may be roughly divided if necessary)

in a suitable container (e.g. a 20 × 12 cm aluminium plate with a 0·5

cm rim). Leave to dry in an oven from 60 ºC to 70 ºC until the

moisture content has been reduced to between 8% and 12%. Remove

from the oven, leave to cool uncovered in the laboratory for one hour

and weigh to the 10 mg. Crush immediately as indicated in 4.1.1 and

dry as indicated in 4.2.1 or 4.2.3 according to the nature of the

feedingstuff.

4.3.2. Cereals

Grain with a moisture content of over 17% must be subjected to

preliminary drying as follows

Weigh 50 g of unground grain to the nearest 10 mg in a suitable

container (e.g. a 20 × 12 cm aluminium plate with a 0·5 cm rim).

Leave to dry for 5 to 7 minutes in an oven at 130 ºC. Remove from

the oven, leave to cool uncovered in the laboratory for two hours and

weigh to the nearest 10 mg. Grind immediately as indicated in 4.1.2

and dry as indicated in 4.2.2.

Calculation of results

The moisture content, as a percentage of the sample, is calculated by using

the following formulae

5.1. Drying without preliminary drying

E

E = initial mass, in grammes, of the test sample,

m = mass, in grammes, of the dry test sample.

5.2. Drying with preliminary drying

 ( M ' − m ) M

 100

 Mm 

= 100  1 −



 E

 EM ' 

M '

E = initial mass, in grammes, of the test sample,

M = mass, in grammes, of the test sample after preliminary drying,

M' = mass, in grammes, of the test sample after crushing or grinding,

m = mass, in grammes, of the dry test sample.

5.3. Repeatibility

The difference between the results of two parallel determinations carried out on

the same sample should not exceed 0·2% of moisture.

Observation

If crushing proves necessary and if this is seen to alter the moisture content of

the product, the results of the analysis of the components of the feedingstuff

must be corrected on the basis of the moisture content of the sample in its initial

state.

II. DETERMINATION OF VOLATILE NITROGENOUS BASES

A. MICRODIFFUSION

1. Purpose and Scope

This method makes it possible to determine the content of volatile nitrogenous

bases, expressed as ammonia, in feedingstuffs.

2. Principle

The sample is extracted with water and the solution clarified and filtered. The

volatile nitrogenous bases are displaced by microdiffusion using a solution of

potassium carbonate, collected in a solution of boric acid and titrated with

sulphuric acid.

3. Reagents

3.1. 20% (w/v) solution of trichloroacetic acid.

3.2. Indicator: dissolve 33 mg of bromocresol green and 65 mg of methyl red in

100 ml of 95%—96% (v/v) of ethanol.

3.3. Boric acid solution: in a 1 litre graduated flask dissolve 10 g of boric acid

A.R. in 200 ml of 95%—96% (v/v) ethanol and 700 ml of water. Add 10

ml of indicator (3.2.). Mix and, if necessary, adjust the colour of the

solution to light red by adding a solution of sodium hydroxide. 1 ml of this

solution will fix a maximum of 300 µg of NH3.

3.4. Saturated potassium carbonate solution: dissolve 10 Og of potassium

carbonate A.R. in 100 ml of boiling water. Leave to cool, filter.

3.5. Sulphuric acid 0·02 N.

4. Apparatus

4.1. Mixer (tumbler): approximately 35 to 40 r.p.m.

4.2. Glass or plastic Conway cells (see diagram).

4.3. Microburettes graduated in 1/100 ml.

5. Procedure

Weigh 10 g of sample to the nearest 1 mg and place with 100 ml of water in a

200 ml graduated flask. Mix in the tumbler for 30 minutes. Add 50 ml of

trichloroacetic acid solution (3.1), make upto volume with water, shake

vigorously and filter through a pleated filter.

Using a pipette, introduce 1 ml of boric acid solution (3.3) into the central part

of the Conway cell and 1 ml of the sample filtrate into the crown of the cell.

Cover partially with the greased lid. Drop 1 ml of saturated potassium carbonate

solution (3.4) quickly into the crown and close the lid so that the cell is airtight.

Turn the cell carefully rotating it in a horizontal plane so that the two reagents

are mixed. Leave to incubate either for at least four hours at room temperature

or for one hour at 40 ºC.

Using a microburette (4.3), titrate the volatile bases in the boric acid solution

with sulphuric acid 0·02 N (3.5).

Carry out a blank test using the same procedure but without a sample to be

analysed.

6. Calculation of results

1 ml of H2SO4 0·02 N corresponds to 0·34 mg of ammonia. Express the result

as a precentage of the sample.

Repeatibility

The difference between the results of two parallel determinations carried out

on the same sample should not exceed

10%, in relative value, for ammonia contents of less than 1·0%;

0·1%, in absolute value, for ammonia contents of 1·0% or more.

Observation

If the ammonia content of the sample exceeds 0·6%, dilute the initial filtrate.

B. BY DISTILLATION

1. Purpose and Scope

This method makes it possible to determine the content of volatile nitrogenous

bases, expressed as ammonia, in fish- meal containing practically no urea. It is

applicable only to ammonia contents of less than 0·25%.

2. Principle

The sample is extracted with water and the solution clarified and filtered. The

volatile nitrogenous bases are displaced at boiling point by adding magnesium

oxide and collected in a specific quantity of sulphuric acid, the excess of which

is back-titrated with a solution of sodium hydroxide.

3. Reagents

3.1. 20% (w/v) solution of trichloroacetic acid.

3.2. Magnesium oxide A.R.

3.3. Anti- foaming emulsion (e.g. silicone).

3.4. Sulphuric acid 0·1 N.

3.5. Sodium hydroxide solution 0·1 N.

3.6. 0·3% (w/v) solution of methyl red in 95%—96% (v/v) ethanol.

4. Apparatus

4.1. Mixer (tumbler): approximately 35 to 40 r.p.m.

4.2. Distilling apparatus of the Kjeldahl type.

5. Procedure

Weigh 10 g of the sample to the nearest 1 mg and place with 100 ml of water in

a 200 ml graduated flask. Mix in the tumbler for 30 minutes. Add 50 ml of

trichloroacetic acid solution (3.1), make upto volume with water, shake

vigorously and filter through a pleated filter.

Take a quantity of clear filtrate appropriate for the presumed content of volatile

nitrogenous bases (100 ml is usually suitable). Dilute to 200 ml and add 2 g of

magnesium oxide (3.2) and a few drops of anti- foaming emulsion (3.3).The

solution should be alkaline to litmus paper; otherwise add some magnesium

oxide (3.2). Distil about 150 ml of the solution in the Kjeldahl apparatus and

collect the distillate in an Erlenmeyer flask containing an accurately measured

volume (25 to 50 ml) of sulphuric acid 0·1 N (3.4). While distilling, avoid

overheating of the sides. Boil the sulphuric acid solution for two minutes, cool it

and back-titrate the excess sulphuric acid with the sodium hydroxide solution

0·1 N (3.5) in the presence of the methyl red indicator (3.6).

Carry out a blank test using the same procedure but without a sample to

be analysed.

6. Calculation of results

1 ml of H2SO4 0·1 N corresponds to 1·7 mg of ammonia. Express the result as a

percentage of the sample.

Repeatibility

The difference between the results of two parallel determinations carried out on

the same sample should not exceed, in relative value, 10% of ammonia.

III. DETERMINATION OF TOTAL PHOSPHORUS

PHOTOMETRIC METHOD

1. Purpose and Scope

This method makes it possible to determine the content of total phosphorus in

feedingstuffs. It is particularly appropriate for the analysis of products low in

phosphorus. In certain cases (product rich in phosphorus), a gravimetric method

may be used.

2. Principle

The sample is mineralised, either by dry combustion (in the case of organic

feedingstuffs) or by acid digestion (in the case of mineral compounds and

liquid feedingstuffs), and placed in an acid solution. The solution is treated with

molybdovanadate reagent. The optical density of the yellow solution thus

formed is measured in a spectrophotometer at 430 nm.

3. Reagents

3.1. Calcium carbonate A.R.

3.2. Hydrochloric acid A.R., d: 1·1 (approx 6 N).

3.3. Nitric acid A.R., d: 1·045.

3.4. Nitric acid A.R., d: 1·38 to 1·42.

3.5. Sulphuric acid A.R., d: 1·84.

3.6.Molybdovanadate reagent: mix 200 ml of ammonium heptamolybdate

solution (3.6.1), 200 ml of ammonium monovanadate solution (3.6.2) and 134

ml of nitric acid (3.4) in a 1 litre graduated flask. Make up to volume with

water.

3.6.1. Ammonium heptamolybdate solution: dissolve in hot water 100 g of

ammonium heptamolybdate A.R. (NH4) 6Mo7O24.4H2O. Add 10

ml of ammonia (d: 0·91) and make up to 1 litre with water.

3.6.2. Ammonium monovanadate solution: 2·35 g of ammonium

monovanadate A.R. NH4VO3 dissolve in 400 ml of hot water. Stirring

constantly, slowly add 20 ml of dilute nitric acid (7 ml of HNO3 (3.4) +

13 ml of H2O and make upto 1 litre with water.

3.7. Standard solution of 1 mg phosphorus per ml: dissolve 4·387 g of

potassium dihydrogen phosphate A.R. KG2PO4 in water. Make up to 1

litre with water.

4. Apparatus

4.1. Silica or porcelain ashing crucibles.

4.2. Electric muffle- furnace with thermostat set at 550 ºC.

4.3. 250 ml Kjeldahl flask.

4.4. Graduated flasks and precision pipettes.

4.5. Spectrophotometer.

4.6. Test tubes about 16 mm in diameter, with stoppers graded to a diameter of

14·5 mm; capacity: 25 to 30 ml.

5. Procedure

5.1. Preparation of the solution

According to the nature of the sample, prepare a solution as indicated in

5.1.1 or 5.1.2.

5.1.1. Usual procedure

Weigh 1 g or more of the sample to the nearest 1 mg. Place the test

sample in a Kjeldahl flask, add 20 ml of sulphuric acid (3.5), shake to

impregnate the substancecompletely with acid and to prevent it from

sticking to the sides of the flask, heat and keep at boiling point for 10

minutes. Leave to cool slightly, add 2 ml of nitric acid (3.4), heat

gently, leave to cool slightly, add a little more nitric acid (3.4) and

bring back to boiling point. Repeat this procedure until a colourless

solution is obtained. Cool, add a little water, decant the liquid into a

500 ml graduated flask, rinsing the Kjeldahl flask with hot water.

Leave to cool, make upto volume with water, homogenise and filter.

5.1.2. Samples containing organic substances and free from calcium and

magnesium dihydrogen phosphates

Weigh about 2·5 g of the sample to the nearest 1 mg in an ashing

crucible. Mix the test sample until completely merged with 1 g of

calcium carbonate (3.1). Ash in the oven at 550 ºC ± 5 ºC until white

or grey ash is obtained (a little charcoal does not matter).

Transfer the ash into a 250 ml beaker. Add 20 ml of water and

hydrochloric acid (3.2) until effervescence ceases. Add a further 10 ml

of hydrochloric acid (3.2). Place the beaker on a sand bath and

evaporate until dry to make the silica insoluble. Redissolve the residue

in 10 ml of nitric acid (3.3) and boil on the sand bath for 5 minutes

without evaporating until dry. Decant the liquid into a 500 ml

graduated flask, rinsing the beaker several times with hot water. Leave

to cool, make up to volume with water, homogenise and filter.

5.2. Development of coloration and measurement of optical density

Dilute an aliquot part of the filtrate obtained by 5.1.1 or 5.1.2 to obtain a

phosphorus concentration of not more than 40 µg/ml. Place 10 ml of this

solution in a test tube (4.6) and add 10 ml of molybdovanadate reagent (3.6).

Homogenise and leave to stand for at least 10 minutes at 20 ºC. Measure the

optical density in a spectrophotometer at 430 nm against a solution obtained

by adding 10 ml of the molybdovanadate reagent (3.6) to 10 ml of water.

5.3. Calibration curve

From the standard solution (3.7) prepare solutions containing respectively

5, 10, 20, 30 and 40 µg of phosphorus per ml. Take 10 ml of each of these

solutions and add thereto 10 ml of molybdovanadate reagent (3.6).

Homogenise and leave to stand for at least 10 minutes at 20 ºC. Measure

the optical density as indicated in 5.2. Trace the calibration curve by

plotting the optical densities against the corresponding quantities of

phosphorus. For concentrations between 0 and 40 µg/ml, the curve will be

linear.

6. Calculation of results

Determine the amount of phosphorus in the test sample by using the calibration

curve.

Express the result as a percentage of the sample.

Repeatibility

The difference between the results of two parallel determinations carried out on

the same sample should not exceed

3%, in relative value, for phosphorus contents of less than 5%;

0·15%, in absolute value, for phosphorus contents of 5% or more.

IV. DETERMINATION OF CRUDE OILS AND FATS

1. Purpose and scope

This method is for the determination of crude oils and fats in feedingstuffs. It

does not cover the analysis of oil seeds and oleaginous fruit defined in Council

Regulation 136/66/EEC of 22 September 1966.

The use of the two procedures described below depends on the nature and

composition of the feedingstuff and the reason for carrying out the analysis.

1.1. Procedure A — Directly extractable crude oils and fats

This method is applicable to feed materials of plant origin, except those

included within the scope of Procedure B.

1.2. Procedure B — Total crude oils and fats

This method is applicable to feed materials of animal origin and to all

compound feeds. It is to be used for all materials from which the oils and fats

cannot be completely extracted without prior hydrolysis (eg glutens, yeast,

potato proteins and products subject to processes such as extrusion, flaking and

heating).

1.3. Interpretation of results

In all cases where a higher result is obtained by using Procedure B than by

Procedure A, the result obtained by Procedure B shall be accepted as the true

value.

2. Principle

2.1. Procedure A

The sample is extracted with light petroleum. The solvent is distilled off and

the residue dried and weighed.

2.2. Procedure B

The sample is treated under heating with hydrochloric acid. The mixture is

cooled and filtered. The residue is washed and dried and submitted to the

determination according to Procedure A.

3. Reagents

3.1. Light petroleum, boiling range: 40 to 60 ºC. The bromine value must be

less than 1 and the residue on evaporation less than 2 mg/100 ml.

3.2. Sodium sulfate, anhydrous.

3.3. Hydrochloric acid, c = 3 mol HCl/l

3.4. Filtration aid, e.g. Kieselguhr, Hyflo-supercel.

4. Apparatus

4.1. Extraction apparatus. If fitted with a siphon (Soxhlet apparatus), the reflux rate

should be such as to produce about 10 cycles per hour; if of the non-siphoning

type, the reflux rate should be about 10 ml per minute.

4.2. Extraction thimbles, free of matter soluble in light petroleum and having

a

porosity consistent with the requirements of point 4.1.

4.3. Drying oven, either a vacuum oven set at 75 ºC ± 3 ºC or an air-oven set at 100

ºC ± 3 ºC.

5. Procedure

5.1. Procedure A (see point 8.1)

Weigh 5 g of the sample to the nearest 1 mg, transfer it to an extraction thimble

(4.2) and cover with a fat- free wad of cotton wool. Place the thimble in an

extractor (4.1) and extract for six hours with light petroleum (3.1). Collect the

light petroleum extract in a dry, weighed flask containing fragments of pumice

stone 1 .

Distil off the solvent. Dry the residue maintaining the flask for one and a

half hours in the drying oven (4.3). Leave to cool in a desiccator and

weigh. Dry again for 30 minutes to ensure that the weight of the oils and

fats remains constant (loss in weight between two successive weighings

must be less than 1 mg).

5.2. Procedure B

Weigh 2,5 g of the sample to the nearest 1 mg (see point 8.2), place in a

400 ml beaker or a 300 ml conical flask and add 100 ml of hydrochloric

acid (3.3) and fragments of pumice stone. Cover the beaker with a watch

glass or fit the conical flask with a reflux condenser. Bring the mixture to

a gentle boil over a low flame or a hot-plate and keep it there for one hour.

Do not allow the product to stick to the sides of the container.

Cool and add a quantity of filtration aid (3.4) sufficient to prevent any loss

of oil and fat during filtration. Filter through a moistened, fat-free, double

filter paper. Wash the residue in cold water until a neutral filtrate is

obtained. Check that the filtrate does not contain any oil or fats. Their

presence indicates that the sample must be extracted with light petroleum,

using Procedure A, before hydrolysis.

Place the double filter paper containing the residue on a watch glass and

dry for one and a half hours in the air oven (4.3) 100 ºC ± 3 ºC.

Place the double filter paper containing the dry residue in an extraction

thimble (4.2) and cover with a fat- free wad of cotton wool. Place the

thimble in an extractor (4.1) and proceed as indicated in the second and

third paragraphs of point 5.1.

6. Expression of result

Express the weight of the residue as a percentage of the sample.

7. Repeatability

The difference between the results of two parallel determinations carried out on

the same sample by the same analyst should not exceed

Where the oil or fat has to undergo subsequent quality tests, replace the fragments of pumice stone by glass beads.

— 0,2 % in absolute value, for contents of crude oils and fats lower than 5 %,

— 4,0 % relative to the highest result for contents of 5 to 10 %,

— 0,4 % in absolute value, for contents above 10 %.

Observations

8.1. For products with a high content of oils and fats, which are difficult to

crush or unsuitable for drawing a homogeneous reduced test sample, proceed as

follows.

Weigh 20 g of the sample to the nearest 1 mg and mix with 10 g or more of

anhydrous sodium sulfate (3.2). Extract with light petroleum (3.1) as indicated

in point 5.1. Make up the extract obtained to 500 ml with light petroleum (3.1)

and mix. Take 50 ml of the solution and place in a small, dry, weighed flask

containing fragments of pumice stone 1 .

Distil off the solvent, dry and proceed as indicated in the last paragraph of point

5.1.

Eliminate the solvent from the extraction residue left in the thimble, crush the

residue to a fineness of 1 mm, return it to the extraction thimble (do not add

sodium sulfate) and proceed as indicated in the second and third paragraphs of

point 5.1.

Calculate the content of oils and fats as a percentage of the sample by using the

following formula

(10 a + b) × 5

where

a = mass in grams of the residue after the first extraction (aliquot part of

the extract),

b = mass in grams of the residue after the second extraction.

8.2. For products low in oils and fats the test sample may be increased to 5 g.

8.3. Pet foods containing a high content of water may need to be mixed with

anhydrous sodium sulfate prior to hydrolysis and extraction as per

Procedure B.

Where the oil or fat has to undergo subsequent quality tests, replace the fragments of pumice stone by glass

beads.

8.4. In paragraph 5.2 it may be more effective to use hot water in place of cold

water to wash the residue after filtration.

8.5. The drying time of 1,5 h may need to be extended for some feedingstuffs.

Excessive drying should be avoided as this can lead to low results. A

microwave oven can also be used.

8.6. Pre-extraction by Procedure A prior to hydrolysis and re-extraction by

Procedure B is recommended if the crude oil/fat content is greater than 15

%. To some extent this depends on the nature of the feedingstuff and the

nature of the oil- fat in the feedingstuff.

Mitbug[ fl-Istamperija tal-Gvern — Printed at the Government Printing Press

0g potassium carbonate A.R. f’ 100 ml ilma

jaghli. Hallih jiksah, u iffiltrah.

3.5. Sulphuric acid 0·02 N.

4. Apparat

4.1. Mixer (tumbler): approssimament

bejn 35 u 40

r.p.m.

4.2. Conway cells tal-hgieg jew tal-plastik (ara d-

diagram).

4.3. Microburettes iggradwati f’ 1/100 ml.

5. Procedura

Izen 10g mil-kampjun sa l-eqreb 1mg u poggih f’ 100ml

ilma f’ graduated flask tal-200ml. Hallat fit-tumbler ghal

30 minuta. Zid 50ml soluzzjoni ta’ trichloroacetic acid

(3.1), zid sal-marka bl-ilma, ixxekja bil-qawwa u iffiltra

b’pleated filter.

Permezz ta’ pipetta, qattar 1ml soluzzjoni ta’ boric acid

(3.3) fil-parti centrali tal-Conway cell u 1ml mil-filtrat

tal-kampjun fil-crown tac-cellula. Aghtti parti minnha bl-

ghatu iggrisjat. Qattar 1ml soluzzjoni issaturata ta’

potassium carbonate(3.4) malajr fil-crown u aghlaq l-

ghatu halli c-cellula tkun izolata ghal kollox mill-arja.

Dawwar ic -cellula attentament u poggiha orizzontalmet

biex iz-zewg reagenti jithalltu. Halliha inkubata ghal mill-

inqas erba’ sighat f’temperatura ambjentali jew ghal

siegha f’40 grad Celsius.

Permezz ta’ microburette (4.3), ittitratja il-bazijiet volatili

fis -soluzzjoni ta’ boric acid bis -sulphuric acid 0.02 N

(3.5).

Aghmel blank test bl-istess procedura

izda minghajr

kampjun biex jigi analaizzat.

6. Kalkolu tar-Rizultati

1ml H2SO4 0.02N jikkorrispondu ghal 0.34mg ammonia.

Esprimi r-riziltat bhala persentagg tal-kampjun.

Repetizzjoni

Id-differenza bejn ir-rizultati ta’ zewg determinazzjonijiet paralleli

mill-istess kampjun ma jistghux jeccedu

10%, valur relattiv, ghal kontenut ta’ammonia ta’ inqas minn 1%;

0.1%, valur assolut, ghal kontenut ta’ ammonia ta’ 1.0% jew iktar.

7. Osservazzjoni

Jekk il-kontenut ta’ l- ammonia jeccedi 0.6%, hallat il-filtrat

inizzjali.

Cellola ta’ Conway

Skala 1/1

Cross Section ta’ l -ghatu S 2 S 3

Cross Section tac-cellola S S 1

Veduta tal- pjan tal-ghatu maghmul min h gieg migruf

Veduta tal- pjan tac- cellola

B.

BID-DISTILLAZZJONI

1. Ghan u skop

Dan il-metodu jippermetti d-determinazzjoni tal-kontenut

ta’ bazijiet nitrogeni volatili, espress bhala ammonia, fil-fish

meal li ma fih prattikalment ebda urea. Dan il-metodu

japplika biss fil-kaz ta’ kontenut ta’ ammonia ta’ inqas minn

0.25%.

2. Principju

Il-kampjun jigi estratt bl-ilma u s-soluzzjoni iccarata u

iffiltrata . Il-bazijiet nitrogeni volatili jigu spustati fil-boiling

point billi zzid il-magnesium oxide u tigbru f’ ammont

specifiku ta’ sulphuric acid, u l-eccess tieghu jigi titrati lura

f’ soluzzjoni ta’ sodium hydroxide.

Reagenti

3.1. 20% (w/v) soluzzjoni trichloroacetic acid.

3.2. Magnesium oxide A.R.

3.3. Anti-foaming emulsion (ez. silicone).

3.4. Sulphuric acid 0·1 N.

3.5. Soluzzjoni ta’ sodium hydroxide 0·1 N.

3.6. 0·3% (w/v) soluzzjoni ta’ methyl red f’ 95%—96%

(v/v) ethanol.

4. Apparatus

4.1. Mixer (tumbler): approssimament 35 sa 40 r.p.m.

4.2. Apparat ghad-distillazzjoni tat-tip Kjeldahl.

5. Procedura

Izen 10g mil-kampjun sa l-eqreb 1mg u poggi ma’ 100ml

ilma f’graduated flask tal-200ml. Hallat fit-tumbler ghal

30 minuta. Zid 50ml soluzzjoni ta’ trichloroacetic acid

(3.1), zid sal-marka bl-ilma, ixxekja bil-qawwa u iffiltra

permezz ta’ pleated filter.

Hu kemm hemm bzonn mil-filtrat car ghal-kontenut

mistenni tal-bazijiet nitrogeni volatili (100ml ikunu

bizzejjed is-soltu). Hallat sa 200ml u zid 2g magnesium

oxide (3.2) u ftit qtar ta’ anti-foaming emulsion (3.3). Is-

soluzzjoni trid tkun alkaline fuq il-litmus paper , jekk le

zid ftit magnesium oxide (3.2). Iddistilla xi 150ml mis-

soluzzjoni fil-Kjeldahl apparatus u igbor id-distillat

f’Erlenmeyer flask li fih volum preciz ta’ (25 sa 50ml)

sulphuric acid 0.1N (3.4). Waqti d-distillazzjoni, evita l-

overheating tal-gnub. Aghlli s-sulphuric acid ghal zewg

minuti, keshu u aghmel back -titration ta’ l-eccess ta’

sulphuric acid bis-soluzzjoni ta’ sodium hydroxide 0.1N

(3.5) fil-prezenza indikatur tal-methyl red (3.6).

Aghmel blank test bl-istess procedura izda minghajr

kampjun ghall-analizi.

6. Kalkulazzjoni tar-Rizultati

1ml H2SO4 0.1N jikkorrispondi ghal 1.7mg ammonia.

Esprimi r-rizultat bhala persentagg tal-kampjun.

Repeatiblitiy

Id-differenza bejn ir-rizultati ta’ zewg determinazzjonijiet

paralleli mill-istess kampjun ma ghandhomx jeccedu 10%,

valur relattiv ta’ ammonia.

III. DETERMINAZZJONI TAT-FOSFRU TOTALI

METODU FOTOMETRIKU

1. Ghan u sskop

Dan il-metodu jiddetermina l- kontenut ta’ fosfru totali fl-ghalf.

Huwa partikolarment approprjat ghall-analizi tal-prodotti

b’kontenut baxx ta’ fosfru. F’ certu kazijiet (prodotti li fihom hafna

fosfru), jista’ jintuza metodu gravimetriku.

2. Principju

Il-kampjun jigi immineralizzat, jew bid-kombustjoni niexfa (fil-

kaz ta’ ghalf organici) inkella bl-digestjoni acidika (fil-kaz ta’

kompostiminerali u ghalf likwidu), u mqieghdin f’soluzzjoni

aciduza. Is-soluzzjoni tigi trattata bil-molybdovanadate reagent. L-

densita` ottika tas-soluzzjoni safra li tifforma titkejjel f’

spektrofotometru f’ 430nm.

3. Reagents

3.1. Calcium carbonate A.R.

3.2. Hydrochloric acid A.R., d: 1·1 (xi 6 N).

3.3. Nitric acid A.R., d: 1·045.

3.4. Nitric acid A.R., d: 1·38 sa 1·42.

3.5. Sulphuric acid A.R., d: 1·84.

3.6.Molybdovanadate reagent: hallat 200 ml soluzzjoni

ta’ ammonium heptamolybdate

(3.6.1), 200 ml

soluzzjoni ta’ ammonium monovanadate (3.6.2) u

134 ml nitric acid (3.4) f’ graduated flask tal-I litru.

Imla sal-marka bl-ilma.

3.6.1. Soluzzzjoni ta’ a mmonium heptamolybdate

holl 100 g ammonium heptamolybdate A.R.

(NH4) 6Mo7O24.4H2O. Zid 10ml ammonia

(d: 0·91) u imla sal-marka ta’ 1litru bl-ilma.

3.6.2. Soluzzjoni ta’ ammonium monovanadate: Holl

2·35 g ammonium monovanadate A.R.

NH4VO3 f’ 400 ml ilma jahraq. Filwaqt li

thawwad bla waqfien, zid bil-mod 20 ml dilute

nitric acid dilwit (7 ml HNO3 (3.4) + 13 ml

H2O) u imla sal-marka ta’ 1 litru bl-ilma..

3.7. Standard solution ta’ 1 mg fosfru ghal kull ml: holl

4·387 g potassium dihydrogen phosphate A.R.

KG2PO4 fl-ilma. Imla sal-marka ta’ 1 litru bl-ilma.

4. Apparat

4.1. Silica jew porcelain ashing crucibles.

4.2 Muffle-furnace ta’ l-elettriku bit- termostat issetjat fuq

550 ºC.

4.3. 250 ml Kjeldahl flask.

4.4. Graduated flasks u pipette tal-precizjoni.

4.5. Spektrofotometru.

4.6. Test tubes b’diametru ta’ xi 16mm, b’ stoppers

iggradati ghal diametru

ta’ 14·5 mm; kapacita’

25 sa 30 ml.

5. Procedura

5.1 Preparazzjoni tas-soluzzjoni

Skond in-natura tal-kampjun , ipprepara s-soluzzjoni kif

indikat f’ 5.1.1 jew 5.1.2.

5.1.1 Procedura tas-soltu

Izen 1g jew iktar mil-kampjun sa l-eqreb img.

Poggi l-kampjun tat-test f’Kjeldahl flask, zid

20ml sulphuric acid(3.5), ixxekja biex timla s-

sustanza kompletament bl-acidu u biex tevita li

tehel mal-gnub tal-flask, sahhan u zomm fuq il-

boiling point ghal ghaxar minuti. Halliha tiksah

ftit, zid 2ml nitric acid (3.4), sahhan ftit, halliha

tiksah ftit, zid ftit iktar nitric acid (3.4) u erga’

gibha ghal-boiling point. Irrepeti l-procedura

sakemm ikollok soluzzjoni bla kulur. Kessah, zid

ftit ilma, iddekantja l-likwidu f’graduated flask

tal-500ml, u lahlah il-Kjeldahl flask bl-ilma

jahraq. Hallih jiksah, imla sal-marka

bl-ilma,

omogenizza u iffiltra.

5.1.2

Kampjuni li fihom sustanzi organici u minghajr

calcium u magnesium dihydrogen phosphates

Izen xi 2.5g mil-kampjun sa l-eqreb mg f’

ashing crucible. Hallat il-kampjun tat-test

sakemm ikun imhallat kompletament ma’ 1g

calcium carbonate (3.1). Axxjah fil-forn

f’temepratura ta’ 550 grad ceslsius +/- 5 gradi

Celsius sakemm ikollok irmied abjad jew griz

(ma jimpurtax jekk ikun hemm xi ftit charcoal)

Poggi l-irmied f’ beaker tal-250ml . Zid 20ml

ilma u hydrochloric acid (3.2) sakemm tieqaf l-

effervexxenza . Zid 10ml ohra hydrochloric acid

(3.2). Poggi l-beaker fuq sand bath u evapora

sakemm jinxef biex is -silica tkun insolubli.

Erga’ holl ir-residwu f’10ml nitric acid (3.3) u

aghllih fuq is -sand bath ghal 5 minuti minghajr

ma tevapora sakemm jinxef. Iddekantja l-likwidu

go graduated flask tal-500ml, u lahlah il-beaker

ghal numru ta’ darbiet bl-ilma jahraq. Hallih

jiksah, zid sal-marka bl-ilma, onogenizzah u

iffiltrah.

5.2 Zvilupp tal-kulur u tkejjil tad- densita` ottika

Hallat parti alikwota tal-filtrat minn 5.1.1 jew 5.1.2 biex

ikollok koncentrazzjoni ta’ phosphorus ta’ mhux iktar

minn 40 ug/ml. Poggi 10ml minn din is -soluzzjoni f’ test

tube (4.6) u zid 10ml molybdovanadate reagent (3.6).

Omo genizzah u hallih joqghod ghal mill-inqas 10 minuti

f’ temepratura ta’ 20 grad Celsius. Kejjel id-densita`

ottika f’spektrofotometru fuq 430nm kontra soluzzjoni

billi zzid 10ml molybdovanadate reagent (3.6) ma’ 10ml

ilma.

5.3 Kurva ta’ Kalibrazzjoni

Mis -standard solution (3.7) ipprepara soluzzjonijiet b’ 5, 10,

20, 30, 40 u 50 ug phosphorus ghal kull ml rispettivament. Hu

10ml minn kull soluzzjoni u zid 10ml molybdovanadate

reagent (3.6). Omo genizzah u hallih joqghod ghal mill-inqas

10 minuti f’ temperatura ta’ 20 grad Celsius. Kejjel id-densita`

ottika kif indikat f’5.2.

Ittrejsja l- calibration curve billi tipplottja id-densitajiet ottici

kontra l-kwantitajiet korrispondenti ta’ fosfru. Ghal

koncentrazzjonijiet bejn 0 u 40ug/ml, il-kurva ghandha tkun

lineari.

6. Kalkolu tar-rizultati

Iddetermina l-ammont ta’ fosfru fil-kampjun tat-test permezz

tal-kurva ta’ kalibrazzjoni.

Esprimi r-riziltat bhala persentagg tal-kampjun.

Repetizzjoni

Id-differenza bejn ir-rizultati ta’ zewg determinazzjonijiet

paralleli mill-istess kampjun ma jistghux jeccedu

3%, valur relattiv, ghal kontenut ta’fosfru ta’ inqas minn 5%;

0.15%, valur assolut, ghal kontenut ta’ fosfru ta’ 5% jew iktar.

IV. DETERMINAZZJONI TA’ ZJUT MHUX MAHDUMIN U

XAHMIJIET

1. Ghan u skop

Dan il-metodu jiddetermina l-zjut mhux mahdumin u l-

xahmijiet fl-ghalf. Ma jkoprix l-analizi taz-zerriegha taz-zejt u

frott zejtni iddefinit fir-Regolament tal-Kunsill tal-Unjoni

Ewropea 136/66/KEE tat-22 ta’ Settembru 1966.

L-uzu taz-zewg proceduri deskritti hawn taht jiddependi mil-

kompozizzjoni u u n-natura ta’ l-ghalf u r-raguni ghall-analizi.

1.1 Procedura A – zjut mhux mahdumin u xahmijiet li jistghu

jigu estratti direttament

Dan il-metodu japplika ghal hwejjeg ta’ l-ikel li gejjin

mil-pjanti, hlief dawk li jaqghu taht Procedura B.

1.2 Procedura B – zjut mhux mahdumin u xahmijiet totali

Dan il-metodu japplika ghal hwejjeg ta’ l-ikel li gejjin

mill-annimali u ghal- ike l kompost. Ghandu jintuza ghal

kull materjal ta’ l-ikel li minnu ma jistghux jigu

kompletamant estratti zjut u xaham minghajr hydrolysis

(ez glutens, hmira, proteina tal-patata u prodotti soggetti

ghal processi bhal extrusion, flaking u tis hin).

1.3 Interpretazzjoni tar-rizultati

F’kull kaz fejn ikollok rizultat permezz ta’ Procedura B li

jkun oghla minn dak li ta’ Procedura A, ir-Rizultat ta’

Procedura B ghangu jkun accettat bhala l-valur reali.

2. Principju

2.1 Procedura A

Il-kampjun jigi estratt bil-light petroleum. Is-solvent

jitnehha bid-distillazzjoni

u r-residwu mnixxef u

mwiezen.

2.2 Procedura B

Il-kampjun jigi trattat bit-tis hin bil-hydrochloric acid. It-

tahlita trid titkessah u tigi filtrata. Ir-residwu jrid jinhasel

u jitnixxef u iddeterminat skond il-Procedura A.

3. Reagents

3.1. Light petroleum, boiling range: bejn 40 u 60 ºC. Il-

valur tal- bromine irid ikun inqas minn 1 u r-

residwu ta’ l-evaporazzjoni inqas minn 2 mg/100

ml.

3.2. Sodium sulfate, anhydrous.

3.3. Hydrochloric acid, c = 3 mol HCl/l

3.4. Ghajnuna ghall-filtrazzjoni ez. Kieselguhr, Hyflo-

supercel.

4. Apparat

4.1. Apparat ta’ l-estrazzjoni . Jekk ikun armat b’ siphon

(Soxhlet apparatus),ir- reflux rate ghandha tkun tali li

tipproduci madwar 10 cikli kull minuta; jekk mhux tat-

tip tas-siphon, ir-reflux rate ghandha tkun madwar

10 ml kull minuta.

4.2. Extraction thimbles, minghajr sustanzi li jinhallu fil-

light petroleum u li ghandhom porozita’ li hija

konsistenti mar-rekwiziti f’punt 4.1.

4.3. Forn ghat-tnixxif, jew forn bil-vakum issetjat fuq 75

ºC ± 3 ºC jewforn ta’ l-arja issetjat fuq 100 ºC ± 3 ºC.

5. Procedura

5.1 Procedura A (ara punt 8.1)

Izen 5g mil-kampjun sa l-eqreb 1mg, poggih f’extraction

thimble (4.2) u aghttih b’bicca tajjara li ma tkunx

xahmija.

Poggi t-thimble f’ extractor (4.1) u estratta ghal 6 sighat

bil-light petroleum (3.1). Igbor l-estratt tal-light petroleum

fi flask niexef u mwiezen li fih frammenti tal-gebla

haffiefa. 1

Nehhi s-solvent bid-distillazzjoni . Nixxef ir-residwu billi

zzomm il-flask fil-forn ghat-tnixxif ghal siegha u nofs

(4.3). Hallih jiksah f’dessikatur u iznu. Erga nixfu ghal 30

minuta biex tkun cert li l-piz taz-zejt u x-xaham jibqa’

kostanti (telf fil-piz bejn id-darbtejn li tizen m’ghandux

ikun ta’ iktar minn 1mg).

Fejn iz-zejt jew ix-xaham irid jghaddi minn certu testijiet ta’

kwalita’, uza bococ tal-hgieg minflok fra mmenti tal-gebla haffiefa.

5.4 Procedura B

Izen 2.5g mil-kampjun sa l-eqreb 1mg (ara punt 8.2),

poggih f’ibeaker tal-400ml jew conical flask tat-300ml u

zid 100ml hydrochloric acid (3.3) u frammenti tal-gegla

haffiefa. Aghtti l- beaker b’watch glass jew qabbad il-

conical flask ma’ reflux condenser. Halli t-tahlita tghalli

bil-mod fuq fjamma baxxa jew hot-plate u zommha

hemm ghal siegha. Thallix il-prodott jehel mal-gnub tal-

kontenitur.

Kessah u zid kwantita’ ta’ filtration aid (3.4) bizzejjed

biex jigi evitat telf ta’ zejt u xaham waqt il-filtrazzjoni.

Iffiltraha minn go filter paper doppja li tkun imxarrba u li

ma tkunx xahmija. Ahsel ir-residwu b’ilma kiesah

sakemm tottjeni filtrat newtrali. Ara li l- filtrat ma jkunx

fih zejt jew xaham. Il-prezenza ta’ dawn hija indikazzjoni

li l-kampjun irid jigi estratt bil-light petroleum, permezz

ta’ Procedura A, qabel l-idrolisi.

Poggi d-filter paper doppja bir-residwu fuq watch glass u

nixxifha ghal siegha u nofs fil-forn ta’ l-arja (4.3) 100 ºC

± 3 ºC.

Poggi l-filter paper doppja bir-residwu niexef ) u

aghttih b’bicca tajjara li ma tkunx xahmija . Poggi t-

thimble f’ extractor (4.1) u kompli kif indikat fit-tieni u t-

tielet paragrafi ta’ punt 5.1.

6. Espressjoni tar-rizultat

Esprimi l-piz tar-residwu bhala persentagg tal-kamjun.

7. Repe tizzjoni

Id-differenza bejn ir-rizultati ta’ zewg determinazzjonijiet paralleli

fuq l-istess kampjun mill-istess analista ma jistghux jeccedu

0.2%, valur assolut, ghal kontenut ta’ zjut mhux mahdumin u

xahmijiet ta’ inqas minn 5%;

4% irrelatati mal-oghla rizultat ghal kontenut ta’ 5 sa 10%,

0.4%, valur assolut, ghal kontenut ta’ iktar minn 10%

8. Osservazzjonijiet

8.1 Fil-kaz ta’ prodotti b’kontenut gholi ta’ zejt u xaham, li ma

jistghux jitfarku facilment jew mhux adattati biex tottjeni

kampjun ta’ test ridott omo geniku, agmel dan li gej.

Izen 20g mil-kampjun sa l-eqreb 1mg u hallat ma’ 10g

jew iktar soluzzjoni ta’ anhydrous sodium sulfate (3.2).

Estratta bil-light petroleum (3.1) kif indikat f’ punt 5.1.

Imla l-estratt sal-marka ta’ 500ml bil-ilight petroleum

(3.1) u hallat. Hu 50ml mis -soluzzjoni u poggih f’flask

zghir, niexef u mwiezen li fih frammenti ta’ gebla

haffiefa. 1 Iddistilla s-solvent, nixxef u ipprocedi kif

indikat fl-ahhar paragrafu taht punt 5.1.

Elimina s-solvent mir-residwu tal-estrazzjoni li jibqa’ fit-

thimble, farrak ir-residwu sakemm ikollu finezza ta’

1mm, poggih lura fl-extraction thimble (izzidx sodium

sulfate) u ipprocedi kif indikat fit-tieni u t-tielet paragrafu

ta’ punt 5.1.

Ikkalkula l-kontenut ta’ zejt u xaham bhala persentagg tal-

kampjun permezz tal-formula

(10 a + b) × 5

Fejn

a= massa fi grammi tar-residwu wara l-ewwel estrazzjoni (l-

parti alikwota mill-estratt)

b= massa fi grammi tar-residwu wara t-tieni estrazzjoni

8.2 Fil-kaz ta’ prodotti li fihom kontenut baxx ta’ zejt u xaham

il-kampjun tat-test jista’ jigi mizjud ghal 5g.

8.3 Fil-kaz ta’ ikel ghall-annimali li ghandhom kontenut gholi

ta’ ilma jista’ jkun hemm bzonn li jithaltu ma’ anhydrous

sodium sulfate qabel l-idrolisi u l-estrazzjoni bhal

Procedura B.

8.4 F’paragrafu 5.2 jista’ jkun iktar effettiv jekk jintuza ilma

jahraq minflok ilma kiesah biex jinhasel ir-residwu wara

l-filtrazzjoni.

8.5 Jista’ jkun li l-perjodu ta’ tnixxif ta’ siegha u nofs ikollu

jigi estiz fil-kaz ta’ cert ghalf. Ghandu jigi evitat tnixxif

eccessiv ghax dan jista’ jaghti rizultati baxxi. Jista’

jintuza forn microwave.

8.6 L-estrazzjoni minn qabel b’procedura A qabel l-idrolisi u

l-estrazzjoni mill-gdid b’procecura B ghandha tintuza

jekk il-kontenut ta’ zejt mhux mahdum jew xaham ikun

oghla minn

15%. Sa certu punt dan jiddependi min-

natura ta’ l-ghalf u n-natura taz-zejt jew xaham fl-ghalf.

1 1

Fejn iz-zejt jew ix-xaham irid jghaddi minn certu testijiet ta’ kwalita’, uza bococ

tal-hgieg minflok frammenti tal-pumice stone.