1978L0633 — EN — 19.08.1981 — 001.001 — 1
Thisdocumentismeantpurelyasadocumentationtoolandtheinstitutionsdonotassumeanyliabilityforitscontents
►B EIGHTCOMMISSIONDIRECTIVE
of15June1978
establishingCommunitymethodsofanalysisfortheofficialcontroloffeedingstuffs
(78/633/EEC)
(OJ L 206, 29.7.1978, p. 43)
Amended by:
Official Journal
No page date
►M1Commission Directive 81/680/EEC of 30 July 1981 L 246 32 29.8.1981
▼B
EIGHTCOMMISSIONDIRECTIVE
of15June1978
establishingCommunitymethodsofanalysisfortheofficialcontrol
offeedingstuffs
(78/633/EEC)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community,
Having regard to Council Directive 70/373/EEC of 20 July 1970 on the
introduction of Community methods of sampling and analysis for the
official control of feedingstuffs(1), as last amended by the Act of
Accession, and in particular Article 2 thereof,
Whereas that Directive requires that official control of feedingstuffs be
carried out using Community methods of sampling and analysis for thepurpose of checking compliance with requirements arising under theprovisions laid down by law, regulation or administrative action
concerning the quality and composition of feedingstuffs;
Whereas Commission Directives 71/250/EEC of 15 June 1971(
2), 71/
393/EEC of 18 November 1971(3), 72/199/EEC of 27 April 1972(4),
73/46/EEC of 5 December 1972(5), 74/203/EEC of 25 March1974(6),
75/84/EEC of 20 December 1974(7) and 76/372/EEC of 1 March
1976(8) have already established a number of Community methods of
analysis; whereas the progress of work since then makes it advisable to
adopt an eighth set of methods;
Whereas the measures provided for in this Directive are in accordance
with the opinion of the Standing Committee for Feedingstuffs,
HAS ADOPTED THIS DIRECTIVE:
Article 1
1. Member States shall require that analyses for official controls of
feedingstuffs, as regards their content of zinc bacitracin, flavophospho-lipol, iron, copper, manganese and zinc, be carried out in accordancewith the methods described in the Annex to this Directive.
Article 2
Member States shall, on 1 January 1979, bring into force the laws,
regulations or administrative provisions necessary to comply withth isDirective. They shall forthwith notify the Commission thereof.
Article 3
This Directive is addressed to the Member States.1978L0633 — EN — 19.08.1981 — 001.001 — 2
(1) OJ No L 170, 3. 8. 1970, p. 2.
(2) OJ No L 155, 12. 7. 1971, p. 13.
(3) OJ No L 279, 20. 12. 1971, p. 7.
(4) OJ No L 123, 29. 5. 1972, p. 6.
(5) OJ No L 83, 30. 3. 1973, p. 21.
(6) OJ No L 108, 22. 4. 1974, p. 7.
(7) OJ No L 32, 5. 2. 1975, p. 26.
(8) OJ No L 102, 15. 4. 1976, p. 8.▼M1
▼B
▼B
ANNEX
1.DETERMINATIONOF ZINCBACITRACINBYDIFFUSIONINAN
AGARMEDIUM
1. PURPOSE AND SCOPE
The method is for the determination of zinc bacitracin in feedingstuffs,
concentrates and premixes. The lower limit of determination is 5 mg/kg
(5 ppm)(*). The method is not valid in the presence of interfering
substances, particularly large amounts of copper and ascorbic acid.
2. PRINCIPLE
The sample is extracted at pH 2 with a mixture of methanol/water/
hydrochloric acid. The extract is brought to pH 6·5, concentrated
(where necessary) and diluted. Its antibiotic activity is determined by
measuring the diffusion of zinc bacitracin in an agar medium inoculated
with Micrococcus luteus (Syn. M. flavus). Diffusion is shown by the
formation of zones of inhibition of the micro-organism. The diameter
of these zones is taken to be in direct proportion to the logarithm of
the antibiotic concentration over the range of antibiotic concentrations
employed.
3. MICRO-ORGANISM: MICROCOCCUS LUTEUS ATCC 10240
3.1.Maintenanceofstockculture
Inoculate Micrococcus luteus on to agar slopes of culture medium (4.1).
Incubate for 24 hours at 30 to 37 ºC, store in a refrigerator at about 4 ºC
and reinoculate every two weeks onto agar slopes.
3.2.Preparationofthebacterialsuspension (a)
Harvest the growth from a recently prepared agar slope (3.1) with 2 to
3 ml of sodium chloride solution (4.3). Use this suspension to inoculate
250 ml of culture medium (4.1) contained in a Roux flask and incubate
for 18 to 20 hours at 30 to 37 ºC. Harvest the growth in 25 ml of
sodium chloride solution (4.3) and mix. Dilute the suspension to one-
tenth with sodium chloride solution (4.3). The light transmission of the
suspension must be about 75 %, measured at 650 nm in a 1 cm cell
against sodium chloride solution (4.3).
4. CULTURE MEDIA AND REAGENTS
4.1.Culturemedium (b)
Meat peptone 6·0 g
Tryptone 4·0 gYeast extract 3·0 g
Meat extract 1·5 g
Glucose 1·0 gAgar 15·0 g
Water 1 000 ml
pH 6·5 to 6·6 (after sterilization)
4.2.Assaymedium (
b)
Tryptone 10·0 g
Yeast extract 3·0 g
Meat extract 1·5 g
Glucose 1·0 gAgar 20·0 gTween 80 1 ml
Water 1 000 ml
pH 6·5 (after sterilization)
4.3. Sodium chloride solution 0·8 % (w/v): dissolve 8 g of sodium chloride
a.p. in water and dilute to 1 000 ml; sterilize.
4.4. Mixture of methanol, pure/water/hydrochloric acid a.p. (d: 1·18 to
1·19): 80/17·5/2·5 (v/v/v).1978L0633 — EN — 19.08.1981 — 001.001 — 3
(*) 1 mg feedingstuff grade zinc bacitracin is equivalent to 42 international units (i.u.).
(a) Other methods may be used provided that it has been established that they give similar
bacterial suspensions.
(b) Any commercial culture medium of similar composition and giving the same results
may be used.
▼B
4.5.Phosphatebuffer,pH6·5
Potassium hydrogen phosphate K2HPO4a.p. (22·15 g).
Potassium dihydrogen phosphate KH2PO4a.p. (27·85 g).
Water to 1 000 ml.
4.6. Hydrochloric acid a.p. (d: 1·18 to 1·19).
4.7. Hydrochloric acid a.p. (0·1 N).4.8. Sodium hydroxide 1 N solution a.p.
4.9. Bromocresol purple solution 0·04 % (w/v): dissolve 0·1 g of bromo-
cresol purple in 18·5 ml of 0·01 N sodium hydroxide solution. Make
up the volume to 250 ml with water and mix.
4.10. Standard substance: zinc bacitracin of known activity (in i.u.).
5. STANDARD SOLUTIONS
Weighout a quantity of standard zinc bacitracin (4.10) corresponding to
1 050 i.u. (according to the activity indicated). Add 5 ml of 0·1 N
hydrochloric acid (4.7) and leave to stand for 15 minutes. Add 30 ml
of water, adjust the pH to 4·5 with phosphate buffer pH 6·5 (4.5) (about
4 ml), make up to a volume of 50 ml withwater and mix well (1 ml
= 21 i.u.).
From this solution prepare by successive dilution with phosphate buffer
pH 6·5 (4.5) the following solutions:
S
80·42 i.u./ml
S40·21 i.u./ml
S20·105 i.u./ml
S10·0 525 i.u./ml
6. PREPARATION OF THE EXTRACT
6.1.Extraction
6.1.1.Concentrates, premixes and mineral feeds
Weighout a quantity of sample of 2·0 to 5·0 g, add 30 ml of th e
mixture (4.4) and shake briefly. Check that the pH is about 2. Shake
for 10 minutes, add 30 ml of phosphate buffer pH 6·5 (4.5) and shake
for 15 minutes. Dilute with phosphate buffer (4.5) to obtain an expected
zinc bacitracin content of 0·42 i.u./ml) (= U8).
6.1.2.Protein concentrates
Weighout a quantity of sample of 10·0 g, add 50 ml of th e mixture
(4.4) and shake briefly. Check that the pH is about 2. Shake for 10
minutes, add 50 ml of phosphate buffer pH 6·5 (4.5) and shake for 15
minutes. Dilute with phosphate buffer (4.5) to obtain an expected zinc
bacitracin content of 0·42 i.u./ml (= U8).
6.1.3.Other feeds
Weighout a quantity of sample of 10·0 g (20·0 g for an expected zinc
bacitracin content of 5 mg/kg), add 25 ml of the mixture (4.4) and
homogenize for about 10 minutes. Add 25 ml of phosphate buffer pH
6·5 (4.5), shake for 15 minutes and centrifuge. Take 20 ml of the super-
natant solution and adjust the pH to 6·5 by means of 1 N sodium
hydroxide solution (4.8) with the bromocresol purple solution (4.9) as
indicator. Evaporate to about 4 ml in a rotary evaporator at a tempera-
ture not exceeding 35 ºC. Dilute the residue with water to obtain an
expected zinc bacitracin content of 0·42 i.u./ml (= U8).
6.2.Assaysolutions
From solution U8prepare solutions U4(expected content: 0·21 i.u./ml),
U2(expected content: 0·105 i.u./ml) and U1(expected content:
0·0525 i.u./ml) by means of successive dilution (1 + 1) with phosphate
buffer (4.5).
7. ASSAY PROCEDURE
7.1.Inoculationoftheassaymedium
Inoculate the assay medium (4.2) with the bacterial suspension (3.2) at
about 50 ºC. By preliminary trials on plates withassay medium (4.2)
determine the quantity of bacterial suspension required to give the1978L0633 — EN — 19.08.1981 — 001.001 — 4
▼B
largest and clearest zones of inhibition with the various concentrations
of zinc bacitracin.
7.2.Preparationoftheplates
Diffusion through agar is carried out in plates with the four concentra-
tions of the standard solution (S8,S4,S2,S1) and the four concentrations
of the assay solution (U8,U4,U2,U1). These four concentrations of
extract and standard must necessarily be placed in eachplate. To th is
effect, select plates big enough to allow at least eight holes with a
diameter of 10 to 13 mm and not less than 30 mm between centres to
be made in the agar medium. The test may be carried out on plates
consisting of a sheet of glass with a faced aluminium or plastic ring
placed on top, 200 mm in diameter and 20 mm high.
Pour into the plates a quantity of the medium (4.2), inoculated as in
point 7.1, to give a layer about 2 mm thick (50 ml for a plate of
200 mm diameter). Allow to set in a level position, bore the holes and
place in them exactly measured volumes of assay and standard solu-
tions (between 0·10 and 0·15 ml per hole, according to the diameter).
Apply eachconcentration at least four times so th at eachdetermination
is subject to an evaluation of 32 zones of inhibition.
7.3.Incubation
Incubate the plates for 16 to 18 hours at 28 to 30 ºC.
8. EVALUATION
Measure the diameter of the zones of inhibition to the nearest 0·1 mm.
Record the mean measurements for each concentration on semi-loga-
rithmic graph paper showing the logarithm of the concentrations in
relation to the diameters of the zones of inhibition. Plot the best fit lines
of both the standard solution and the extract, for example as below.
Determine the ‘best fit’ point for the standard lowest level (SL) using
the formula:
(a) SL ¼7S1þ4S2þS4/C02S8
10
Determine the ‘best fit’ point for the standard highest level (SH) using
the formula:
(b) SH ¼7S8þ4S4þS2/C02S1
10
Similarly calculate the ‘best fit’ points for the extract lowest level (UL)
and the extract highest level (UH) by substituting U1,U2,U4and U8for
S1,S4and S8in the above formulae.
Record the calculated SL and SH values on the same graph paper and
join them to give the ‘best fit’ line for the standard solution. Similarly
record UL and UH and join them to give the ‘best fit’ line for the
extract.
In the absence of any interference the lines should be parallel. For prac-
tical purposes the lines can be considered parallel if the values (SH-SL)
and (UH-UL) do not differ by more than 10 % from their mean value.
If the lines are found to be non-parallel, either U1and S1or U8and S8may be discarded and SL, SH, UL and UH calculated, using the alter-
native formulae, to give alternative ‘best fit’ lines:
(a′)S L¼5S1þ2S2/C0S4
6or5S2þ2S4/C0S8
6
(b′)S H¼5S4þ2S2/C0S1
6or5S8þ2S4/C0S2
6
and similarly for UL and UH. The alternative best fit lines should be
checked for parallelism as before. The fact that the result has been
calculated from three levels should be noted on the final report.
When the lines are considered as being parallel , calculate the logarithm
of the relative activity (log. A) by means of one of the following
formulae.1978L0633 — EN — 19.08.1981 — 001.001 — 5
▼B
For four levels
(c) log :A¼U1þU2þU4þU8/C0S1/C0S2/C0S4/C0S8 ðÞ /C2 0/C1602
U4þU8þS4þS8/C0U1/C0U2/C0S1/C0S2
For three levels
(d) log :A¼U1þU2þU4/C0S1/C0S2/C0S4 ðÞ /C2 0/C1401
U4þS4/C0U1/C0S1
or
(d′) log :A¼U2þU4þU8/C0S2/C0S4/C0S8 ðÞ /C2 0/C1401
U8þS8/C0U2/C0S2
Real activity = supposed activity × relative activity.
When the lines are considered as not being parallel, repeat the determi-
nation. If parallelism is still not achieved, calculate the logarithm of the
relative activity (log. A) by means of formula (c). The result obtained
must however be considered as approximate and this should be noted in
the final report.
9. REPEATABILITY
The difference between the results of two determinations carried out on
the same sample by the same analyst should not exceed:
20 % related to the highest value for contents of zinc bacitracin from 5
and up to 25 mg/kg;
5 mg/kg, in absolute value, for contents greater than 25 and up to
50 mg/kg;
10 % related to the highest value for contents above 50 mg/kg.
2.DETERMINATIONOFFLAVOPHOSPHOLIPOLBYDIFFUSIONIN
ANAGARMEDIUM
1. PURPOSE AND SCOPE
The method is for the determination of flavophospholipol in feeding-
stuffs, concentrates and premixes. The lower limit of determination is
1 mg/kg (1 ppm).
2. PRINCIPLE
The sample is extracted with diluted methanol by heating under reflux.
After centrifuging, the extract is purified (where necessary) by treat-
ment withion exch ange resins and diluted. Its antibiotic activity is
determined by measuring the diffusion of flavophospholipol in an agar
medium inoculated with Staphylococcus aureus. Diffusion is shown by
the formation of zones of inhibition of the micro-organism. The
diameter of these zones is taken to be in direct proportion to the loga-
rithm of the antibiotic concentration over the range of antibiotic
concentrations employed.
3. MICRO-ORGANISM: STAPHYLOCOCCUS AUREUS ATCC 6538 P
3.1.Maintenanceofstockculture
Inoculate Staphylococcus aureus onto agar slopes of culture medium
(4.1). Incubate for 24 hours at 37 ºC, store in a refrigerator at about
4 ºC and reinoculate every monthonto agar slopes.
3.2.Preparationofthebacterialsuspension (a)
Set aside two tubes containing the stock culture (3.1) and reinoculate
them weekly. Incubate for 24 hours at 37 ºC and store in a refrigerator
at about 4 ºC.
24 hours before the assay, inoculate with this growth two to four tubes
containing slopes of culture medium (4.1). Incubate for 16 to 18 hours1978L0633 — EN — 19.08.1981 — 001.001 — 6
(a) Other methods may be used provided that it has been established that they give similar
bacterial suspensions.
▼B
at 37 ºC. Make a suspension of the growth in the sodium chloride solu-
tion (4.3). The light transmission of the suspension must be about 40 %,
measured at 578 nm in a 1 cm cell against sodium chloride solution
(4.3).
4. CULTURE MEDIA AND REAGENTS
4.1.Culturemedium (a)
Meat peptone 6·0 g
Tryptone 4·0 g
Yeast extract 3·0 gMeat extract 1·5 g
Glucose 1·0 g
Agar 15·0 gWater 1 000 ml
pH 6·5 (after sterilization)
4.2.Assaymedium
4.2.1.Base layer (
b)
Meat peptone 6·0 g
Yeast extract 3·0 g
Meat extract 1·5 gAgar 10·0 g
Water 1 000 ml
pH 6·5 (after sterilization)
4.2.2.Seed layer
As for point 4.1, withth e addition of 2·0 g of silicone anti-foaming
emulsion(
c).
4.3. Sodium chloride solution 0·4 % (w/v): dissolve 4 g of sodium choride
a.p. in water and dilute to 1 000 ml; sterilize.
4.4. Methanol, pure.4.5. Methanol 50 % (v/v): dilute 500 ml of methanol (4.4) with 500 ml of
water.
4.6. Methanol 80 % (v/v): dilute 800 ml of methanol (4.4) with 200 ml of
water.
4.7. Tris (hydroxymethyl) aminomethane a.p.4.8. Potassium chloride methanolic solution 1·5 % (w/v): dissolve 1·5 g of
potassium chloride a.p. in 20 ml of water, make up the volume to
100 ml withmeth anol (4.4).
4.9. Cation exchanger: Dowex 50 W×8, 20 to 50 mesh, Na form (cat. Serva
No 41600) or equivalent.
4.10. Anion exchanger: Dowex 1×2, 50 to 100 mesh, Cl form (cat. Serva
No 41010) or equivalent. Before use, keep for 12 to 14 hours in 80 %
methanol (4.6).
4.11. Glass wool.
4.12. pH indicator paper (pH 6·6 to 8·1).
4.13. Ascorbic acid.4.14. Standard substance: flavophospholipol of known activity.
5. APPARATUS
5.1. Glass tube for chromatography, internal diameter: 9 mm, length: 150 to
200 mm, fitted with a stopcock at the narrowed part of the lower end
and a ground-glass joint (to connect withth e dropping funnel (5.2)) at
the upper end.
5.2. Dropping funnel 250 ml, fitted witha stopcock and a ground-glass
joint.1978L0633 — EN — 19.08.1981 — 001.001 — 7
(a) Any commercial culture medium of similar composition and giving the same results
may be used, e.g. oxoid antibiotic medium 1 (CM 327) withan addition of oxoid agar
No 3 (L 13).
(b) Any commercial culture medium of similar composition and giving the same results
may be used, e.g. oxoid antibiotic medium 2 (CM 335) withan addition of oxoid agarNo 3 (L 13).
(
c) e.g. SE 2 from Wacker Chemie GmbH, Munich.
▼B
5.3. 250 ml conical flask withground-glass joint.
5.4. Reflux condenser withground-glass joint.
6. STANDARD SOLUTIONS
Dissolve an accurately weighed quantity of the standard substance
(4.14) in 50 % methanol (4.5) and dilute to give a stock solution
containing 100 µ g flavophospholipol per millilitre. Stored in stoppered
flasks at 4 ºC this solution is stable for up to two months.
From this stock solution prepare by successive dilution with 50 %
methanol (4.5) the following solutions:
S80·2 µg/ml
S40·1 µg/ml
S20·05 µg/ml
S10·025µg/ml
7. PREPARATION OF THE EXTRACT
7.1.Extraction
7.1.1.Concentrates, premixes and mineral feeds
Weighout a quantity of sample of 2·0 to 5·0 g and add about 150 mg
of ascorbic acid (4.13). Homogenize with150 ml of 50 % meth anol
(4.5) in a conical flask (5.3) and adjust the pH to 8·1 to 8·2 with about
400 mg of tris(hydroxymethyl)aminomethane (4.7). Check the pH with
indicator paper (4.12). Allow to stand for 15 minutes, then readjust the
pH to 8·1 to 8·2 with tris(hydroxymethyl)aminomethane (4.7) and boil
for 10 minutes under reflux (5.4) withconstant stirring. Allow to cool,
centrifuge the mixture and decant the extract.
7.1.2.Other feeds
Weighout a quantity of samle of 5·0 to 30·0 g containing at least 30 µ g
of flavophospholipol. Homegenize with 150 ml of 50 % methanol (4.5)
in a conical flask (5.3) and adjust the pH to 8·1 to 8·2 with about
400 mg of tris(hydroxymethyl)aminomethane (4.7). Check the pH with
indicator paper (4.12). Allow to stand for 15 minutes, then readjust the
pH to 8·1 to 8·2 with tris(hydroxymethyl)aminomethane (4.7) and boil
for 10 minutes under reflux (5.4) withconstant stirring. Allow to cool,
centrifuge the mixture and decant the extract.
7.2.Purification (this stepmay be omitted for concentrates, premixes
andmineralfeeds)
Mix 110 ml of the extract with 11 g of the cation exchanger (4.9), boil
for one minute under reflux (5.4) withconstant stirring. Separate th e
cation exchanger by centrifugation or filtration. Mix 100 ml of the
extract with 150 ml of methanol (4.4) and store the solution for 12 to
15 hours at 4 ºC. Filter off the flocculent mass while cold.
Insert a glass wool plug (4.11) at the bottom end of a glass tube (5.1),
pour into the tube 5 ml of the anion exchanger (4.10) and wash the
column with 100 ml of 80 % methanol (4.6). Using the funnel (5.2),
transfer to the column a volume of filtrate of at least 100 ml which is
expected to contain 16 µ g of flavophospholipol (200 ml for a 30 g
sample of feedingstuff at 1 ppm). Where necessary, before application
to the column, dilute the filtrate with 80 % methanol (4.6) to obtain an
expected flavophospholipol content of 16 µ g/100 ml. Adjust the flow
rate to about 2 ml/minute. Discard the effluent. Then wash the column
with 50 ml of 80% methanol (4.6) and discard the effluent.
Elute the flavophospholipol with the methanolic solution of potassium
chloride (4.8) keeping the flow rate to about 2 ml/minute. Collect
50 ml of the eluate in a graduated flask, add 30 ml of water and mix.
This solution should have a flavophospholipol content of 0·2 µg/ml (=
U8).
7.3.Assaysolutions
Where necessary (i.e. when the purification step has been omitted),
dilute the extract obtained in point 7.1.1 with 50 % methanol (4.5) to
obtain an expected flavophospholipol content of 0·2 µ g/ml (= U8).
From solution U8prepare solutions U4(expected content: 0·1 µg/ml), U2(expected content: 0·05 µ g/ml) and U1(expected content: 0·025 µ g/ml)
by means of successive dilution (1 + 1) with50 % meth anol (4.5).1978L0633 — EN — 19.08.1981 — 001.001 — 8
▼B
8. ASSAY PROCEDURE
8.1.Inoculationoftheassaymedium
Inoculate the assay medium (4.2.2) with the bacterial suspension (3.2)
at about 50 ºC. By preliminary trials on plates withassay medium
(4.2.2) determine the quantity of bacterial suspension required to give
the largest and clearest zones of inhibition with the various concentra-
tions of flavophospholipol (about 30 ml/litre).
8.2.Preparationoftheplates
Diffusion through agar is carried out in plates with the four concentra-
tions of the standard solution (S8,S4,S2,S1) and the four concentrations
of the assay solution (U8,U4,U2,U1). These four concentrations of
extract and standard must necessarily be placed in eachplate. To th isef-
fect, select plates big enough to allow at least eight holes with a
diameter of 10 to 13 mm and not less than 30 mm between centres to
be made in the agar medium. The test may be carried out on plates
consisting of a sheet of glass with a faced aluminium or plastic ring
placed on top, 200 mm in diameter and 20 mm high.
Pour into the plates a quantity of the medium (4.2.1) to give a layer
about 1·5 mm thick (45 ml for a plate of 200 mm diameter). Allow to
set in a level position and then over-layer with a quantity of the
medium (4.2.2) inoculated as in point 8.1 to give a layer 1 mm thick
(30 ml for a plate of 200 mm diameter). Allow to set again in a level
position, bore the holes and place in them exactly measured volumes of
assay and standard solutions (between 0·10 and 0·15 ml per hole,
according to the diameter).
Apply eachconcentration at least four times so th at eachdetermination
is subject to an evaluation of 32 zones of inhibition.
8.3.Incubation
Icubate the plates for 16 to 18 hours at 28 to 30 ºC.
9. EVALUATION
Measure the diameter of the zones of inhibition to the nearest 0·1 mm.
Record the mean measurements for each concentration on semi-loga-
rithmic graph paper showing the logarithm of the concentrations in
relation to the diameters of the zones of inhibition. Plot the best fit lines
of both the standard solution and the extract, for example as below.
Determine the ‘best fit’ point for the standard lowest level (SL) using
the formula:
(a) SL ¼7S1þ4S2þS4/C02S8
10
(b) SH ¼7S8þ4S4þS2/C02S1
10
Similarly calculate the ‘best fit’ points for the extract lowest level (UL)
and the extract highest level (UH) by substituting U1,U2,U4and U8for
S1,S2,S4and S8in the above formulae.
Record the calculated SL and SH values on the same graph paper and
join them to give the ‘best fit’ line for the standard solution. Similarly
record UL and UH and join them to give the ‘best fit’ line for the
extract.
In the absence of any interference the lines should be parallel. For prac-
tical purposes the lines can be considered parallel if the values (SH-SL)
and (UH-UL) do not differ by more than 10 % from their mean value.
If the lines are found to be non-parallel, either U1and S1or U8and S8may be discarded and SL, SH, UL and UH calculated, using the alter-
native formulae, to give alternative ‘best fit’ lines:
(a′)S L¼5S1þ2S2/C0S4
6or5S2þ2S4/C0S8
6
(b′)S H¼5S4þ2S2/C0S1
6or5S8þ2S4/C0S2
61978L0633 — EN — 19.08.1981 — 001.001 — 9
▼B
and similarly for UL and UH. The alternative best fit lines should be
checked for parallelism as before. The fact that the result has been
calculated from three levels should be noted on the final report.
When the lines are considered as being parallel, calculate the logarithm
of the relative activity (log. A) by means of one of the following
formulae.
For four levels
(c) log :A¼U1þU2þU4þU8/C0S1/C0S2/C0S4/C0S8 ðÞ /C2 0/C1602
U4þU8þS4þS8/C0U1/C0U2/C0S1/C0S2
For three levels
(d) log :A¼U1þU2þU4/C0S1/C0S2/C0S4 ðÞ /C2 0/C1401
U4þS4/C0U1/C0S1
or
(d′) log :A¼U2þU4þU8/C0S2/C0S4/C0S8 ðÞ /C2 0/C1401
U8þS8/C0U2/C0S2
Real activity = supposed activity × relative activity.
When the lines are considered as not being parallel, repeat the determi-
nation. If parallelism is still not achieved, calculate the logarithm of the
relative activity (log. A) by means of formula (c). The result obtained
must however be considered as approximate and this should be noted in
the final report.
10. REPEATABILITY
The difference between the results of two determinations carried out on
the same sample by the same analyst should not exceed:
0·5 mg/kg, in absolute value, for contents of flavophospholipol from 1
and up to 2 mg/kg;
25 % related to the highest value for contents greater than 2 and up to
10 mg/kg;
20 % related to the highest value for contents greater than 10 and up to
25 mg/kg;
5 mg/kg, in absolute value, for contents greater than 25 and up to
50 mg/kg;
10 % related to the highest value for contents above 50 mg/kg.
3.DETERMINATION OF THE TRACE ELEMENTS IRON, COOPER,
MANGANESEANDZINC
1. PURPOSE AND SCOPE
The method makes it possible to determine the trace elements iron,
copper, manganese and zinc in feedingstuffs. The lower limits of deter-
mination are:
iron (Fe): 20 mg/kg
copper (Cu): 10 mg/kg
manganese (Mn): 20 mg/kg
zinc (Zn): 20 mg/kg
2. PRINCIPLE
The sample is brought into solution in hydrochloric acid after destruc-
tion of organic matter, if any. The elements iron, copper, manganese
and zinc are determined, after appropriate dilution, by atomic absorp-
tion spectrometry.
3. REAGENTS
IntroductorycommentsFor preparation of the reagents and analytical solutions use water free
from the cations to be determined, obtained either by double distilling1978L0633 — EN — 19.08.1981 — 001.001 — 10
▼B
water in a borosilicate glass or quartz still or by double treatment on
ion exchange resin.
The reagents must be of at least analytical grade (a.p.). Freedom from
the element to be determined must be checked in a blank experiment. If
necessary, the reagents must be further purified.
In place of the standard solutions decribed below, commercial standard
solutions may be used provided that they are guaranteed and have been
checked before use.
3.1. Hydrochloric acid a.p. (d:1·19).
3.2. Hydrochloric acid a.p. (6 N).3.3. Hydrochloric acid a.p. (0·5 N).3.4. Hydrofluoric acid 38 to 40 % (v/v) having an iron content of less than
1 mg Fe/litre and a residue after evaporation of less than 10 mg (as
sulphate)/litre.
3.5. Sulphuric acid a.p. (d: 1·84).
3.6. Hydrogen peroxide a.p. (approximately 100 volumes of oxygen (30 %
by weight) ).
3.7. Standard iron solution (1 000 µg Fe/ml) prepared as follows: dissolve
1 g of iron wire a.p. in 200 ml of 6 N hydrochloric acid (3.2), add
16 ml of hydrogen peroxide (3.6) and make up to one litre with water.
3.7.1. Working standard iron solution (100 µg Fe/ml) prepared by diluting the
standard solution (3.7) 1 + 9 withwater.
3.8. Standard copper solution (1000 µg Cu/ml) prepared as follows:
dissolve 1 g of copper in powder form (a.p.) in 25 ml of 6 N hydro-
chloric acid (3.2), add 5 ml of hydrogen peroxide (3.6) and make up
to one litre withwater.
3.8.1. Working standard copper solution (10 µg Cu/ml) prepared by diluting
the standard solution (3.8) 1 + 9 with water and then diluting the
resulting solution 1 + 9 withwater.
3.9. Standard manganese solution (1 000 µg Mn/ml) prepared as follows:
dissolve 1 g of manganese in powder form (a.p.) in 25 ml of 6 N hydro-
chloric acid (3.2) and make up to one litre with water.
3.9.1. Working standard manganese solution (10 µg Mn/ml) prepared by
diluting the standard solution (3.9) 1 + 9 with water and then diluting
the resulting solution 1 + 9 with water.
3.10. Standard zinc solution (1 000 µg Zn/ml) prepared as follows: dissolve
1 g of zinc in strip or leaf form (a.p.) in 25 ml of 6 N hydrochloric
acid (3.2) and make up to one litre withwater.
3.10.1. Working standard zinc solution (10 µg Zn/ml) prepared by diluting the
standard solution (3.10) 1 + 9 with water and then diluting the resulting
solution 1 + 9 withwater.
3.11. Lanthanum choride solution prepared as follows: dissolve 12 g of
lanthanum oxide in 150 ml of water, add 100 ml of 6 N hydrochloric
acid (3.2) and make up to one litre withwater.
4. APPARATUS
4.1. Muffle furnace withtemperature regulation and recorder.1978L0633 — EN — 19.08.1981 — 001.001 — 11
▼B
4.2. Glassware must be of resistant borosilicate type and it is recommended
to use apparatus which is reserved exclusively for trace element deter-
minations.
4.3. Platinum crucible and (optional) quartz crucible.4.4. Atomic absorption spectrophotometer meeting the requirements of the
method with regard to sensitivity and precision in the required range.
5. PROCEDURE
5.1.Samplescontainingorganicmatter
5.1.1.Ashing and preparation of the solution for analysis (*)
(i) Place 5 to 10 g of sample weighed to the nearest 0·2 mg in a quartz
or platinium crucible (4.3) (see Note (b)), dry in an oven at 105 ºC
and introduce the crucible into the cold muffle furnace (4.1). Close
the furnace (see Note (c)) and gradually raise the temperature to
450 to 475 ºC over about 90 minutes. Maintain this temperature
for 4 to 16 hours (e.g. overnight) to remove carbonaceous material
and then open the furnace and allow to cool (see Note (d)).
Wash the crucible out with a total of about 5 ml of hydrochloric
acid (3.1) and add the latter slowly and carefully to the beaker
(there may be a vigorous reaction due to CO
2formation). Add
hydrochloric acid (3.1) dropwise with agitation until all efferves-
cence has stopped. Evaporate to dryness, occasionally stirring with
a glass rod.
Next add 15 ml of 6 N hydrochloric acid (3.2) to the residue
followed by about 120 ml of water. Stir with the glass rod, which
should be left in the beaker, and cover the beaker with a watch-
glass. Bring gently to the boil and maintain at boiling point until
no more ashcan be seen to dissolve. Filter on ash -free filter paper
and collect the filtrate in a 250 ml volumetric flask. Wash the
beaker and filter with 5 ml of hot 6 N hydrochloric acid (3.2) and
twice with boiling water. Fill the volumetric flask up to the mark
withwater (HCl concentration about 0·5 N).
(ii) If the residue in the filter appears black (carbon), put it back in the
furnace and ash again at 450 to 475 ºC. This ashing, which only
requires a few hours (about three to five hours), is complete when
the ash appears white or nearly white. Dissolve the residue with
about 2 ml of hydrochloric acid (3.1), evaporate to dryness and
add 5 ml of 6 N hydrochloric acid (3.2). Heat, filter the solution
into the volumetric flask and make up to the mark with water
(HCl concentration about 0·5 N).
Notes:
(a) In determining trace elements it is important to be alert to the risks
of contamination, particularly by zinc, copper and iron. For this
reason, the equipment used in preparing the samples must be free
of these metals.
To reduce the general risk of contamination, work in a dust-free
atmosphere with scrupulously clean equipment and carefully
washed glassware. The determination of zinc is particularly sensi-
tive to many types of contamination, e.g. from glassware, reagents,
dust, etc.
(b) The weight of sample to be ashed is calculated from the approxi-
mate trace element content of the feedingstuff in relation to the
sensitivity of the spectrophotometer used. For certain feedingstuffs
low in trace elements it may be necessary to start witha 10 to 20 g
sample and make up the final solution to only 100 ml.1978L0633 — EN — 19.08.1981 — 001.001 — 12
(*) Green fodder (freshor dried) is liable to contain large amounts of vegetable silica,
which may retain trace elements and must be removed. For samples of these feeding-stuffs, therefore, the following modified procedure must be followed.
Carry out operation 5.1.1 (i) as far as the filtration. Wash the filter paper containing
the insoluble residue twice with boiling water and place it in a platinum crucible (4.3).Ignite in the muffle furnace (4.1) at a temperature below 550 ºC until all carbonaceousmaterial has completely disappeared. Allow to cool, add a few drops of waterfollowed by 10 to 15 ml of hydrofluoric acid (3.4) and evaporate to dryness at about
150 ºC. If any silica remains in the residue, redissolve it in a few millilitres of hydro-
fluoric acid (3.4) and evaporate to dryness. Add five drops of sulphuric acid (3.5) andheat until no more white fumes are given off. After the addition of 5 ml of 6 N hydro-chloric acid (3.2) and about 30 ml of water, heat, filter the solution into the 250 mlvolumetric flask and make up to the mark with water (HCl concentration about 0·5 N).
Proceed then with the determination from point 5.1.3.
▼B
(c) Ashing must be carried out in a closed furnace without injection of
air or oxygen.
(d) The temperature indicated by the pyrometer must not exceed
475 ºC.
5.1.2.Spectrophotometric determination
5.1.2.1. Preparation of calibration solutions
For each of the elements to be determined, prepare from the working
standard solutions given in points 3.7.1, 3.8.1, 3.9.1 and 3.10.1 a range
of calibration solutions, eachcalibration solution h aving an HCl
concentration of about 0·5 N and (in the cases of iron, manganese and
zinc) a lanthanum chloride concentration equivalent to 0·1 % La (w/v).
The trace element concentrations selected must lie within the range of
sensitivity of the spectrophotometer used. The tables below show, by
way of example, the compositions of typical ranges of calibration solu-
tions; depending, however, on the type and sensitivity of
spectrophotometer used it may be necessary to select other concentra-
tions.
Iron
µ g Fe/ml 0 0·5 1 2 3 4 5
ml working stan-
dard solution
(3.7.1)
(1 ml = 100 µg Fe) 0 0·5 1 2 3 4 5
+ ml 6 N HCl (3.2) 7 7 7 7 7 7 7
+ 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml
withwater.
Copper
µ g Cu/ml 0 0·1 0·2 0·4 0·6 0·8 0·1
ml working stan-
dard solution
(3.8.1)
(1 ml = 10 µg Cu) 0 1 2 4 6 8 10
+ ml 6 N HCl (3.2) 8 8 8 8 8 8 8
Make up to 100 ml withwater.
Manganese
µ g Mn/ml 0 0·1 0·2 0·4 0·6 0·8 1·0
ml working stan-
dard solution
(3.9.1)
(1 ml = 10 µ g
Mn)0124681 0
+ ml 6 N HCl (3.2) 7 7 7 7 7 7 7
+ 10 ml lanthanum chloride solution (3.11) and make up to 100 ml with
water.
Zinc
µg Zn/ml 0 0·05 0·1 0·2 0·4 0·6 0·8
ml working stan-
dard solution
(3.10.1)
(1 ml = 10 µg Zn) 0 0·5 1 2 4 6 8
+ ml 6 N HCl (3.2) 7 7 7 7 7 7 7
+ 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml
withwater.1978L0633 — EN — 19.08.1981 — 001.001 — 13
▼B
5.1.2.2. Preparation of solution for analysis
For the determination of copper, the solution prepared from point 5.1.1
can normally be used directly. If necessary to bring its concentration
within the range of the calibration solutions, an aliquot portion may bepipetted into a 100 ml volumetric flask and made up to the mark with
0·5 N hydrochloric acid (3.3).
For the determination of iron, manganese and zinc, pipette an aliquot
portion of the solution prepared from point 5.1.1 into a 100 ml volu-
metric flask, add 10 ml of lanthanum chloride solution (3.11) and
make up to the mark with 0·5 N hydrochloric acid (3.3) (see also point
8 ‘Observation’).
5.1.2.3. Blank experiment
The blank experiment must include all the prescribed steps of the
procedure except that the sample material is omitted.
The calibration solution ‘0’ must not be used as the blank.
5.1.2.4. Measurement of the atomic absorption
Measure the atomic absorption of the calibration solutions and of the
solution to be analyzed using an oxidizing air-acetylene flame at the
following wavelengths:
Fe: 248·3 nm
Cu: 324·8 nm
Mn: 279·5 nmZn: 213·8 nm
Carry out eachmeasurement four times.
5.2.Mineralfeedingstuffs
If the sample contains no organic matter, prior ashing is unnecessary.
Proceed as described in point 5.1.1 (i) starting from the second para-
graph. Evaporation with hydrofluoric acid may be omitted.
6. CALCULATION OF RESULTS
Using a calibration curve, calculate the trace element concentration in
the solution to be analyzed and express the result in milligrams of trace
element per kilogram of sample (ppm).
7. REPEATABILITY
The difference between the results of zwo parallel determinations
carried out on the same sample by the same analyst should not exceed:
—5′mg/kg, in absolute value, for contents of the trace element
concerned up to 50 mg/kg;
— 10 % of the higher result for contents of the trace element
concerned from 50 and up to 100 mg/kg;
— 10 mg/kg, in absolute value, for contents of the trace element
concerned from 100 and up to 200 mg/kg;
— 5 % of the higher result for contents of the trace element concerned
above 200 mg/kg.
8. OBSERVATION
The presence of large quantities of phosphates may interfere with the
determination of iron, manganese and zinc. Suchinterference must be
corrected by addition of lanthanum chloride solution (3.11). If,
however, in the sample the weight ratio
CaþMg
P2, addition of
lanthanum chloride solution (3.11) to the solution for analysis and to
the calibration solutions may be omitted.1978L0633 — EN — 19.08.1981 — 001.001 — 14