1973L0046 — EN — 13.08.1998 — 003.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B FOURTHCOMMISSIONDIRECTIVE
of5December 1972
establishingCommunitymethodsofanalysisfortheofficial controloffeedingstuffs
(73/46/EEC)
(OJ L 83, 30.3.1973, p. 21)
Amended by:
Official Journal
No page date
►M1Commission Directive 81/680/EEC of 30 July 1981 L 246 32 29.8.1981
►M2Commission Directive 92/89/EEC of 3 November 1992 L 344 35 26.11.1992
►M3Commission Directive 98/54/EC of 16 July 1998 L 208 49 24.7.1998
▼B
FOURTHCOMMISSIONDIRECTIVE
of5December 1972
establishingCommunitymethodsofanalysisfortheofficialcontrol
offeedingstuffs
(73/46/EEC)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community;
Having regard to the Council Directive of 20 July 1970(1) on the intro-
duction of Community methods of sampling and analysis for theofficial control of feedingstuffs, as amended by the Directive No 72/
275/EEC of 20 July 1972(
2), and in particular Article 2 thereof;
Whereasthat Directive requiresthat official controlsof feedings tuffs
be carried out using Community methods of sampling and analysis for
the purpose of checking compliance with requirements arising underthe provisions laid down by law, Regulation or administrative actionconcerning the quality and composition of feedingstuffs;
WhereasDirectivesNo 71/250/EEC of 15 June 1971(
3), No 71/393/
EEC of 18 November 1971(4) and No 72/199/EEC of 27 April
1972(5) have already established a number of Community methods of
analysis; whereas the progress of work since then makes it advisable to
adopt a fourth set of methods;
Whereasthe meas uresprovided for in thisDirective are in accordance
with the Opinion of the Management Committee for Feedingstuffs;
HAS ADOPTED THIS DIRECTIVE:
Article 1
The Member States shall require that analyses for official controls of
feedingstuffs as regards moisture contents of animal and vegetable
fats and oils and magnesium and crude fibre contents of feedingstuffs
be carried out according to the methodsdes cribed in Annex I to thisDirective.
Article 2
The Member States shall require that analyses for official controls of
feedingstuffs as regards their contents of retinol (vitamin A),►
M3 ◄be carried out according to the methods
described in Annex II to this Directive.
Article 3
The Member Statess hall, not later than 1 January 1974, bring into
force the laws, regulations or administrative provisions necessary tocomply with this Directive. They shall forthwith notify the Commissionthereof.1973L0046 — EN — 13.08.1998 — 003.001 — 2
(1) OJ No L 170, 3. 8. 1970, p. 2.
(2) OJ No L 171, 29. 7. 1972, p. 39.
(3) OJ No L 155, 12. 7. 1971, p. 13.
(4) OJ No L 279, 20. 12. 1971, p. 7.
(5) OJ No L 123, 29. 5. 1972, p. 6.▼M1
▼B
▼M1
▼B
▼B
Article 4
This Directive is addressed to the Member States.1973L0046 — EN — 13.08.1998 — 003.001 — 3
▼B
ANNEX I
1.DETERMINATION OF MOISTURE IN ANIMAL AND VEGETABLE
FATSANDOILS
1.PurposeandScope
This method makes it possible to determine the water and volatile substances
content of animal and vegetable fatsand oils .
2.Principle
The sample is dried to constant weight at 103 ºC. The loss in mass is deter-
mined by weighing.
3.Apparatus
3.1. Flat-bottomed dish, of a corrosion-resistant material, 8 to 9 cm in
diameter and approximately 3 cm high.
3.2. Mercury thermometer with a strengthened bulb and expansion tube at
the top end, graduated from approximately 80 ºC to at least 110 ºC,
and approximately 10 cm in length.
3.3. Sand bath or electric hot-plate.3.4. Desiccator, containing an efficient drying agent.
3.5. Analytical balance.
4.Procedure
Weigh out to the nearest mg approximately 20 g of the homogenized sample
into the dry, weighed dish (3.1) containing the thermometer (3.2). Heat on
the sand bath or hot-plate (3.3), stirring continuously with the thermometer,
so that the temperature reaches 90 ºC in about 7 minutes.
Reduce the heat, watching the frequency with which bubblesris e from the
bottom of the dish. The temperature must not exceed 105 ºC. Continue to
stir, scraping the bottom of the dish, until bubbles stop forming.
In order to ensure complete elimination of moisture, reheat several times to
103 ºC ± 2 ºC, cooling to 93 ºC between successive heatings. Then leave to
cool to room temperature in the desiccator (3.4) and weigh. Repeat this
operation until the loss in mass between two successive weighings no longer
exceeds2 mg.
N.B.An increase in the mass of the sample after repeated heating indicates
an oxidation of the fat, in which case calculate the result from the
weighing carried out immediately before the mass began to increase.
5.Calculationofresults
The moisture content, as a percentage of the sample, is given by the
following formula:
M
1/C0M2 ðÞ /C1100
Mo
where:
Mo= mass, in grammes, of the test sample;
M1= mass, in grammes, of the dish with its contents before heating;
M2= mass, in grammes, of the dish with its contents after heating.
Results lower than 0·05 % must be recorded as ‘lower than 0·05 %’.
Repeatability
The difference in moisture between the results of two parallel determinations
carried out on the same sample must not exceed 0·05 %, in absolute value.1973L0046 — EN — 13.08.1998 — 003.001 — 4
▼B
2.DETERMINATIONOFMAGNESIUM
— by atomic absorption spectrophotometry —
1.PurposeandScope
This method makes it possible to determine the quantity of magnesium in
feeding stuffs. It is particularly appropriate for determining magnesium
contentslower than 5 %.
2.Principle
The sample is ashed and dissolved in dilute hydrochloric acid. If it contains
no organic substances, it is dissolved directly in dilute hydrochloric acid. The
solution is diluted and the magnesium content determined by atomic absorp-
tion spectrophotometry at 285·2 nm, by comparison with standard solutions.
3.Reagents
3.1. Hydrochloric acid a.p. d: 1·16.
3.2. Concentrated hydrochloric acid a.p. d: 1·19.3.3. Magnesium ribbon or wire, or magnesium sulphate heptahydrate, dried
at room temperature.
3.4. Strontium salt solution (chloride or nitrate) at 2·5 % (w/v) strontium
(= 76·08 g SrC1
2·6 H2O a.p., or 60·38 g Sr (NO3)2a.p.).
3.5. Standard magnesium solution: weigh out to the nearest mg 1 g magne-
sium (3.3) which has previously had its oxide coating carefully
removed, or the corresponding quantity (10·143 g) of magnesium
sulphate heptahydrate (3.3). Place in a 1 000 ml graduated flask, add
80 ml hydrochloric acid (3.1), leave to dissolve and make up to
1 000 ml with water. 1 ml of thiss olution contains1·000 mg magne-
sium.
4.Apparatus
4.1. Platinum, silica or porcelain ashing crucibles.
4.2. Thermostatically controlled electric muffle furnace.
4.3. Atomic absorption spectrophotometer.
5.Procedure
5.1.Preparation of the sample solution
5.1.1.Feeding stuffs composed exclusively of mineral substances
Weigh out to the nearest mg 5 g of the sample into a 500 ml
graduated flask with 250 to 300 ml water. Add 40 ml hydro-
chloric acid (3.1), bring to the boil and keep the liquid gently
boiling for 30 minutes. Leave to cool, make up to volume with
water, mix and filter into a dry beaker through a dry pleated
filter. Discard the first 30 ml of the filtrate. In the presence of
silica, treat 5 g of sample with a sufficient quantity (15—30 ml)
of hydrochloric acid (3.2), evaporate to dryness on a water bath
and transfer to an oven at 105 ºC for one hour. Proceed as from
the third sentence of 5.1.2.
5.1.2.Feeding stuffs composed predominantly of mineral substances
Weigh out to the nearest mg 5 g of the sample into a crucible and
ash at 550 ºC in the muffle furnace until an ash which is free
from carbonaceousparticlesisobtained, and leave to cool. In
order to eliminate silica, add to the ash a sufficient quantity
(15—30 ml) of hydrochloric acid (3.2), evaporate to dryness on
a water bath and transfer to an oven at 105 ºC for one hour. Treat
the residue with 10 ml hydrochloric acid (3.1) and transfer to a
500 ml graduated flask using warm water. Leave to cool and
make to volume with water. Mix and filter into a dry beaker
through a dry pleated filter. Discard the first 30 ml of the filtrate.
5.1.3.Feedingstuffs composed predominantly of organic substances
Weigh out to the nearest mg 5 g of the sample into a crucible and
ash at 550 ºC in the muffle furnace until an ash which is free
from carbonaceousparticlesisobtained. Treat the as h with 5 ml
hydrochloric acid (3.2), evaporate to dryness on a water bath and
then dry for one hour in the oven at 105 ºC in order to render the1973L0046 — EN — 13.08.1998 — 003.001 — 5
▼B
silica insoluble. Treat the ash with 5 ml hydrochloric acid (3.1),
transfer to a 250 ml graduated flask using warm water, bring to
the boil, leave to cool and make up to volume with water. Mix
and filter into a dry beaker through a dry pleated filter. Discard
the first 30 ml of the filtrate.
5.2.Measurement by atomic absorption
By diluting the standard solution (3.5) with water, prepare at least 5
reference solutions of increasing concentration, corresponding to the
optimal measuring range of the spectrophotometer. Add to each solution
10 ml strontium salt solution (3.4) and then make up the volume to
100 ml with water. Dilute with water one aliquot part of the filtrate
obtained from 5.1.1, 5.1.2 or 5.1.3, so as to obtain a magnesium concen-
tration which iswithin the limitsof concentration of the reference
solutions. The hydrochloric acid concentration of this solution must not
exceed 0·4 N. Add 10 ml strontium salt solution (3.4) and then make up
the volume to 100 ml with water. Measure the absorption of the solution
to be determined and of the reference solutions at 285·2 nm.
6.Calculationofresults
Calculate the quantity of magnesium in the sample by relation to the refer-
ence solutions. Express the result as a percentage of the sample.
Repeatability
The difference between the results of two parallel determinations carried out
on the same sample must not exceed 5 %, in relative value.
3.DETERMINATIONOFCRUDEFIBRE
1.Purposeandscope
This method makes it possible to determine fat-free organic substances in
feedingstuffs which are insoluble in acid and alkaline media and are conven-
tionally described as crude fibre.
2.Principle
The sample, defatted where necessary, is treated successively with boiling
solutions of sulphuric acid and potassium hydroxide of specified concentra-
tions. The residue is separated by filtration on a sintered-glass filter washed,dried, weighed and ashed within a range of 475 to 500 ºC. The loss of weight
resulting from ashing corresponds to the crude fibre present in the test
sample.
3.Reagents
3.1. Sulphuric acid, c = 0,13 mol/l.
3.2. Anti-foaming agent (e.g. n-octanol).
3.3. Filter aid (Celite 545 or equivalent), heated at 500 ºC for four hours
(8.6).
3.4. Acetone.
3.5. Light petroleum boiling-range 40 to 60 ºC.
3.6. Hydrochloric acid, c = 0,5 mol/l.
3.7. Potassium hydroxide solution, c = 0,23 mol/l.
4.Apparatus
4.1. Heating unit for digestion with sulphuric acid or potassium hydroxide
solution, equipped with a support for the filter crucible (4.2) and
provided with an outlet tube with a tap to the liquid outlet and vacuum,
possibly with compressed air. Before use each day preheat the unit with
boiling water for five minutes.
4.2. Glass filter crucible with fused sintered glass filter plate pore size 40-
90 µm. Before first use, heat to 500 ºC for a few minutes and cool (8.6).
4.3. Cylinder of at least 270 ml with a reflux condenser, suitable for boiling.
4.4. Drying oven with thermostat.
4.5. Muffle furnace with thermostat.1973L0046 — EN — 13.08.1998 — 003.001 — 6
▼M2
▼M2
4.6. Extraction unit consisting of a support plate for the filter crucible (4.2.)
and with a discharge pipe with a tap to the vacuum and liquid outlet.
4.7. Connecting rings to assemble the heating unit (4.1), crucible (4.2) and
cylinder (4.3) and to connect the cold extraction unit (4.6) and crucible.
5. Procedure
Weigh out to the nearest 0,001 g, 1 g of the prepared sample and place it in
the crucible (4.2), (see observations 8.1, 8.2 and 8.3) and add 1 g of filter aid
(3.3).
Assemble the heating unit (4.1) and the filter crucible (4.2), then attach the
cylinder (4.3) to the crucible. Pour 150 ml of boiling sulphuric acid (3.1) into
the assembled cylinder and crucible and if necessary add a few drops of anti-
foaming agent (3.2).
Bring the liquid to the boil within 5 ± 2 minutesand boil vigorous ly for
exactly 30 minutes.
Open the tap to the discharge pipe (4.1) and, under vacuum, filter the
sulphuric acid through the filter crucible and wash the residue with three
consecutive 30 ml portions of boiling water, ensuring that the residue is
filtered dry after each washing.
Close the outlet tap and pour 150 ml boiling potassium hydroxide solution
(3.7) to the assembled cylinder and crucible and add a few drops of anti-
foaming agent (3.2). Bring the liquid to boiling point within 5 ± 2 minutes
and boil vigorously for exactly 30 minutes. Filter and repeat the washing
procedure used for the sulphuric acid step.
After the final washing and drying, disconnect the crucible and its contents
and reconnect it to the cold extraction unit (4.6). Apply the vacuum and
wash the residue in the crucible with three consecutive 25 ml portions of
acetone (3.4) ensuring that the residue is filtered dry after each washing.
Dry the crucible to constant weight in the oven at 130 ºC. After each drying
cool in the desiccator and weigh rapidly. Place the crucible in a muffle
furnace and ash to constant weight at 475 ºC to 500 ºC for at least 30
minutes.
After each heating cool first in the furnace and then in the desiccator before
weighing.
Carry out a blank test without the sample. Loss of weight resulting from
ashing must not exceed 4 mg.
6.Calculationofresults
The crude fibre content as a percentage of the sample is given by the expres-
sion:
b/C0c ðÞ /C2 100
a
where
a = mass of sample in g;
b = loss of mass after ashing during the determination, in g;c = loss of mass after ashing during the blank test, in g.
7.Repeatability
The difference between two parallel determinationscarried out on the s ame
sample must not exceed:
— 0,3 in absolute value for crude fibre contents lower than 10 %,
— 3 % relative to the higher result, for crude fibre contents equal to or
greater than 10 %.
8.Observations
8.1. Feedingstuffs containing more than 10 % crude fat must be defatted
prior to analysis with light petroleum (3.5). Connect the filter crucible
(4.2) and itscontentsto the cold extraction unit (4.6) and apply vacuum
and wash the residue with three consecutive 30 ml portions of light
petroleum, ensuring that the residue is dry. Connect the crucible and its
contentsto the heating unit (4.1) and continue asdes cribed under 5.1973L0046 — EN — 13.08.1998 — 003.001 — 7
▼M2
8.2. Feedingstuffs containing fats which cannot be extracted directly with
light petroleum (3.5) must be defatted as shown in 8.1 and defatted
once more after boiling with acid.
After boiling with acid and the subsequent washing connect the crucible
and itscontentsto the cold extraction unit (4.6) and was h three times
with 30 ml acetone followed by three further washings with 30 ml
portionsof light petroleum. Filter under vacuum until dry and continue
the analysis as described under 5, beginning with potassium hydroxide
treatment.
8.3. If the feedingstuffs contain over 5 % of carbonates, expressed as
calcium carbonate, connect the crucible (4.2) with the weighed sample
to the heating unit (4.1). Wash the sample three times with 30 ml hydro-
chloric acid (3.6). After each addition let the sample stand for about one
minute before filtering. Wash once with 30 ml water and then continue
asdes cribed under 5.
8.4. If an apparatus in the form of a stand is used (several crucibles attached
to the same heating unit) no two individual determinations on the same
sample for analysis may be carried out in the same series.
8.5. If after boiling it is difficult to filter the acidic and basic solutions, use
compressed air through the discharge pipe of the heating unit and then
continue filtering.
8.6. The temperature for ashing should not be higher than 500 ºC in order to
extend the lifetime of the glass filter crucibles. Care must be taken to
avoid excessive thermal shock during heating and cooling cycles.1973L0046 — EN — 13.08.1998 — 003.001 — 8
▼B
ANNEX II
1.DETERMINATIONOFRETINOL(VITAMINA)
1.PurposeandScope
This method makes it possible to determine the quantity of retinol (Vitamin
A) in feedingstuffs, concentrates and premixes. The lower limit of the deter-
mination is10 000 IU/kg for highly pigmented feedsand 4 000 IU/kg for
others(1). Products are classified in two groups, according to their presumed
retinol content:
Group A: contentslower than 200 000 IU/kg;
Group B: contentsequal to or greater than 200 000 IU/kg.
2.Principle
The sample is hydrolised when hot with a potassium hydroxide solution in an
ethanol medium and in the presence of an antioxidant or in a nitrogen atmo-
sphere. The mixture is extracted with 1,2-dichlorethane. The extract is
evaporated to dryness and treated with light petroleum. The solution is chro-
matographed on a column of aluminium oxide (forGroup B products,
chromatography is only required in certain cases). For Group A products
the retinol isdetermined by s pectrophotometry at 610 nm after development
of a coloured complex according to the Carr-Price reaction; for Group B
productsby s pectrophotometry in the UV at 325 nm.
3.Reagents
(a)used for analysing products of Groups A and B
3.1. 96 % (v/v) ethanol.3.2. 10 % (w/v) sodium ascorbate solution a.p., or
3.3. Purified nitrogen.
3.4. 50 % (w/v) potassium hydroxide solution a.p.3.5. Potassium hydroxide solution 1 N a.p.
3.6. Potassium hydroxide solution 0·5 N a.p.
3.7. 1,2-dichlorethane a.p.
3.8. Light petroleum, boiling range: 30—50 ºC. If necessary, purify as
follows: stir 1 000 ml light petroleum with 20 ml lots of concen-trated sulphuric acid until the acid remains colourless. Remove
the acid and wash the light petroleum successively with 500 ml
water, twice with 250 ml of 10 % (w/v) sodium hydroxide solution
and three timeswith 500 ml water. Remove the aqueouslayer, dry
the light petroleum for 1 hour over active carbon and anhydrous
sodium sulphate, filter and distil.
3.9. Aluminium oxide, standardized according to Brockmann: ash for 8
hours at 750 ºC, cool in a desiccator and keep in a brown glass
bottle fitted with a ground-glass stopper. Before use in chromato-
graphy moisten as follows: place in a brown glass bottle 10 g
aluminium oxide and 0·7 ml water, seal with a stopper, reheat for
5 minutesin a boiling water bath while s haking. Leave to cool.
Verify the activity of the aluminium thusprepared by s ubjecting a
known quantity of retinol (3.17) (ca. 500 IU) to the procedure of
5.3 and 5.4 and checking recovery.
3.10. Basic aluminium oxide, degree of activity 1 (Woelm, Merck or
equivalent).
3.11. Pure diethyl ether. Remove peroxidesand tracesof water by chro-
matography on a column of basic aluminium oxide (3.10). (25 g
aluminium oxide per 250 ml diethyl ether.)
3.12. Light petroleum solutions (3.8) at 4, 8, 12, 16 and 20 % (v/v)
diethyl ether (3.11).
3.13. Sodium sulphide solution 0·5 molar in 70 % (v/v) glycerine,
prepared from sodium sulphide a.p.1973L0046 — EN — 13.08.1998 — 003.001 — 9
(1) 1 IU = 0·3 µg of retinol.
▼B
(b)used exclusively for analysing Group A products
3.14. Crystallizable benzene a.p.
3.15. Chloroform a.p. Remove the ethanol, phosgene and traces of water
by chromatography on a column of basic aluminium oxide (3.10)
(50 g aluminium oxide per 200 ml chloroform; it isadvis able to
chromatograph the first 50 ml of the eluate a second time).
3.16. Carr-Price reagent: stir approximately 25 g antimony trichloride
a.p. (kept in a desiccator) with 100 ml chloroform (3.15) until the
solution is saturated. A slight deposit of antimony trichloride
causes no problem. Add 2 ml acetic anhydride a.p. Keep in a
refrigerator in a brown glass bottle with ground-glass stopper. The
solution keeps for several weeks.
3.17. Retinol — standardized spectrophotometrically.
(c)used exclusively for analysing Group B products
3.18. Isopropanol, for chromatography.
4.Apparatus
4.1. Water bath.
4.2. Vacuum evaporation apparatus with round flasks of different capacities.4.3. Glass chromatography tubes (length: 300 mm; internal diameter: about
13 mm).
4.4. Spectrophotometer with 10 mm cells. Measurements in the UV require
silica cells.
4.5. UV lampss uitable for 365 nm.
5.Procedure
N.B.All operations must be carried out away from direct light, if necessary
in brown glass equipment.
5.1.Test Sample
From the finely divided sample, take a test sample proportional to the
presumed retinol content, thus:
0·1—1·0 g for concentrates(contentsgreater than 20 000 IU/g);
3·0—5·0 g for premixes(contentsof between 400 and 20 000 IU/g);
10—20 g for mineral mixtures;
30 g for Group A products.Immediately place the test sample in a 500 ml flask with a ground-glass
stopper.
5.2.Hydrolysis and extraction (
1)
Add successively to the test sample 40 ml ethanol (3.1), 2 ml sodium
ascorbate solution (3.2)(2), 10 ml potassium hydroxide solution (3.4)
and 2 ml sodium sulphide solution (3.13).
Heat for 30 minutesat 70—80 ºC under a reflux condens er and then
leave to cool under a stream of water. Add 50 ml ethanol (3.1) and
100 ml 1,2-dichlorethane (3.7) (taken with a pipette). Shake vigorously
and then decant the supernatant liquid into a decanting container. Add to
the container 150 ml potassium hydroxide solution (3.5), shake for 30
seconds and leave to stand until the layers are separated. Collect the
dichlorethane layer (lower layer) in a decanting container, add 40 ml
potassium hydroxide solution (3.6), shake for 10 seconds and leave to
stand until the layers are separated. Collect the dichlorethane layer in a
decanting container and wash 6—8 times with 40 ml lots of water until
free of alkali (phenolphthalein test). Collect the dichlorethane layer and
remove the last traces of water using strips of filter paper.1973L0046 — EN — 13.08.1998 — 003.001 — 10
(1) For milk feedsand productswith a tendency to agglomerate or s well, double the quan-
tity of the reagents shown in the first and second paragraphs of 5.2.
(2) Sodium ascorbate need not be added when hydrolysis is carried out in a nitrogen atmo-
sphere.
▼B
Evaporate to dryness an aliquot part of the solution under vacuum and
on the water bath at 40 ºC. Rapidly treat the residue with 5 ml light
petroleum (3.8).
For Group A products, chromatograph ass hown in 5.3.1.
For Group B products, transfer the solution to a 50 ml graduated flask,
make up to volume with light petroleum (3.8), mix and measure the
optical density as shown in 5.4.2.
5.3.Chromatography
5.3.1.Group A products
Fill a chromatography tube (4.3) to a height of 200 mm with 10 g
aluminium oxide (3.9) previously slurried with light petroleum
(3.8). Place in the tube the solution obtained in 5.2 and immedi-
ately add 20 ml light petroleum (3.8). Elute successively with
10 ml lotsof the light petroleum s olutionsat 4, 8, 12, 16 and
20 % diethyl ether (3.12) under pressure or partial vacuum, the
rate of flow being 2 to 3 dropsper s econd.
The carotene iseluted firs t(1). The retinol isgenerally eluted with
the light petroleum solution at 20 % diethyl ether (3.12). The
elution isfollowed under UV light (brief irradiation of the
column with the mercury lamp). The fluorescent zone of the
retinol isclearly s eparated from the yellow xanthophyll zones
following it. Collect the eluate fraction containing the retinol in
an Erlenmeyer flask.
5.3.2.Group B products
Chromatography must only be carried out if the optical density
measurements obtained in 5.4.2 do not conform to the require-
mentsgiven in 5.4.2.
If chromatography proves necessary, place in the chromatography
column an aliquot part of the solution in the light petroleum
obtained in 5.2, containing approximately 500 IU of retinol, and
chromatograph ass hown in 5.3.1.
5.4.Measurement of the optical density
5.4.1.Group A products
Evaporate to dryness under vacuum the eluate containing the
retinol obtained in 5.3.1. Treat the residue with 2 ml benzene
(3.14). Take 0·3 ml of thiss olution and add 3 ml of the Carr-
Price reagent (3.16). A blue colouring develops. Measure the
optical density with the spectrophotometer at 610 nm exactly 30
seconds after the reaction has begun. Determine the retinol
content by reference to a standard curve obtained from benzene
solutions of increasing retinol-standard concentrations treated
with Carr-Price reagent (2 to 16 IU retinol-standard (3.17) per
0·3 ml benzene (3.14) +3 ml Carr-Price reagent (3.16)). The stan-
dard curve must be checked regularly and frequently using the
standard and a freshly prepared Carr-Price reagent solution.
5.4.2.Group B products
Take an aliquot part of the solution in light petroleum obtained in
5.2 containing approximately 200 IU retinol. Evaporate to
dryness under vacuum and treat the residue with 25 ml isopro-
panol (3.18). Measure the optical density in the
spectrophotometer at 325, 310 and 334 nm. The absorption
maximum islocated at 325 nm. The retinol content of the s olu-
tion iscalculated asfollows :
E325· 18·30 = IU of retinol/ml
However, the ratio of the optical densities
E310:E325and E334:E325
must be 6 : 7 = 0·857.If one of these ratios differs appreciably from this value (< 0·830
or > 0·880), the measurement of the optical densities must be
preceded by chromatography in accordance with the method1973L0046 — EN — 13.08.1998 — 003.001 — 11
(1) Carotene content may be determined by optical density measurement at 450 nm;
E1%
1c m¼2600
▼B
given in 5.3.2. If the measurement of the optical densities carried
out after chromatography shows that the abovementioned ratios
still differ appreciably from the value of 0·857 (< 0·830 or
> 0·880), the determination must be carried out in accordance
with the method given for Group A products.
6.Calculationofresults
Calculate the retinol content of the sample taking into account the weight of
the test sample and the dilutions carried out in the course of analysis. Express
the results in IU of retinol per kg of feedingstuff or per kg of concentrate or
premix.
Repeatability
The difference between the results of two parallel determinations carried out
on the same sample must not exceed:
— 20 %, in relative value, for retinol contentslower than 75 000 IU/kg;
— 15 000 IU for contentsbetween 75 000 and 150 000 IU/kg;— 10 %, in relative value, for contentsbetween 150 000 and 250 000 IU/kg;
— 25 000 IU for contentsbetween 25 000and 500 000 IU/kg;
— 5 %, in relative value, for contentsgreater than 500 000 IU/kg.1973L0046 — EN — 13.08.1998 — 003.001 — 12
▼M3