1976L0372 — EN — 03.05.1994 — 003.001 — 1
Thisdocumentismeantpurelyasadocumentationtoolandtheinstitutionsdonotassumeanyliabilityforitscontents
►B SEVENTHCOMMISSIONDIRECTIVE
of1March1976
establishingCommunitymethodsofanalysisfortheofficialcontroloffeedingstuffs
(76/372/EEC)
(OJ L 102, 15.4.1976, p. 8)
Amended by:
Official Journal
No page date
►M1CommissionDirective 81/680/EEC of 30 July 1981 L 246 32 29.8.1981
►M2CommissionDirective 92/95/EEC of 9 November 1992 L 327 54 13.11.1992
►M3CommissionDirective 94/14/EC of 29 March 1994 L 94 30 13.4.1994
▼B
SEVENTHCOMMISSIONDIRECTIVE
of1March1976
establishingCommunitymethodsofanalysisfortheofficialcontrol
offeedingstuffs
(76/372/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 on the intro-
duction of Community methods of sampling and analysis for the
official control of feedingstuffs(1), as last amended by the Act of
Accession(2), 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 CommissionDirectives 71/250/EEC of 15 Jun e 1971(
3), 71/
393/EEC of 18 November 1971(4), 72/199/EEC of 27 April 1972(5),
73/46/EEC of 5 December 1972(6), 74/203/EEC of 25 March 1974(7)
and 75/84/EEC of 20 December 1974(8) have already established a
number of Community methods of analysis; whereas the progress of
work since then makes it advisable to adopt a seventh set of methods;
Whereas the measures provided for inthis Directive are inaccordan ce
with the opinion of the Standing Committee for Feedingstuffs,
HAS ADOPTED THIS DIRECTIVE:
Article 1
The Member States shall require that analyses for official controls of
feedingstuffs, as regards their content of aflatoxin B1, be carried out in
accordance with the methods described in the Annex to this Directive.
Article 2
The Member States shall, not later than 1 October 1976, bring into
force the laws, regulations or administrative provisions necessary to
comply with this Directive. They shall forthwith notify the Commissionthereof.
Article 3
This Directive is addressed to the Member States.1976L0372 — EN — 03.05.1994 — 003.001 — 2
(1) OJ No L 170, 3. 8. 1970, p. 2.
(2) OJ No L 73, 27. 3. 1972, p. 14.
(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.
(6) OJ No L 83, 30. 3. 1973, p. 21.
(7) OJ No L 108, 22. 4. 1974, p. 7.
(8) OJ No L 32, 5. 2. 1975, p. 26.▼M1
▼B
▼B
ANNEX
DETERMINATIONOFAFLATOXINB1
A. ONE-DIMENSIONAL THIN LAYER CHROMATOGRAPHIC METHOD
1.Purposeandscope
The method makes it possible to determine the level of aflatoxin B1in raw materials and straight feedingstuffs. This method can not be
applied inthe presen ce of citrus pulp. The lower limit of determi-
nation is 0,01 mg/kg (10 ppb).
In the presence of interfering substances it is necessary to repeat
the analysis using method B (high performance liquid chromato-
graphy).
2.Principle
The sample is subjected to extractionwith chloroform. The extract
is filtered, and an aliquot portion taken and purified by column
chromatography onsilica gel. The eluate is evaporated an d the
residue redissolved ina specific volume of chloroform or of a
mixture of benzene and acetonitrile. An aliquot portion of this solu-
tion is subjected to thin-layer chromatography (TLC). The quantity
of aflatoxinB1is determined under UV irradiation of the chromato-
gram, either visually or by fluorodensitometry, by comparison with
known quantities of standard aflatoxin B1. The identity of the afla-
toxinB1extracted from the feedingstuff must be confirmed by the
procedure indicated.
3.Reagents
NB:All the reagents must be of ‘analytical reagent’ quality unless
otherwise stated.
3.1. Acetone.
3.2. Chloroform, stabilized with 0·5 to 1·0 % of 96 % ethanol (v/v).
3.3. N-hexane.
3.4. Methanol.3.5. Anhydrous diethyl ether, free from peroxides.3.6. Mixture of benzene and acetonitrile: 98/2 (v/v).3.7. Mixture of chloroform (3.2) and methanol (3.4): 97/3 (v/v).3.8. Silica gel, for columnchromatography, particle size 0·05 to
0·20 mm.
3.9. Absorbent cotton wool, previously defatted with chloroform, or
glass wool.
3.10. Sodium sulphate, anhydrous, granular.3.11. Inert gas, e.g. nitrogen.3.12. 1 N Hydrochloric acid.3.13. 50 % (v/v) sulphuric acid.3.14. Kieselguhr (hyflosupercel), washed inacid.3.15. Silica gel G-HR or equivalent, for TLC.3.16. Standard solution with about 0·1 µg of aflatoxin B
1per ml in
chloroform (3.2) or the benzene/acetonitrile mixture (3.6), prepared
and checked as indicated in Section 7.
3.17. Standard solution for qualitative testing purposes containing about
0·1 µg of aflatoxinB1and B2per ml inchloroform (3.2) or the
benzene/acetonitrile mixture (3.6). These concentrations are given
as a guide. They must be adjusted so as to obtain the same intensity
of fluorescence for both aflatoxins.
3.18. Developing solvents:1976L0372 — EN — 03.05.1994 — 003.001 — 3
▼M2
▼B
▼B
3.18.1. Chloroform (3.2)/acetone (3.1): 9/1 (v/v), unsaturated tank;
3.18.2. Diethyl ether (3.5)/methanol (3.4)/water: 96/3/1 (v/v/v), unsaturated
tank;
3.18.3. Diethyl ether (3.5)/methanol (3.4)/water: 94/4·5/1·5 (v/v/v), satu-
rated tank;
3.18.4. Chloroform (3.2)/methanol (3.4): 94/6 (v/v), saturated tank;3.18.5. Chloroform (3.2)/methanol (3.4): 97/3 (v/v), saturated tank.
4.Apparatus
4.1. Grinder-mixer.4.2. Shaking apparatus or magnetic stirrer.
4.3. Fluted filter papers, Schleicher and Schüll No 588 or equivalent,
diameter: 24 cm
4.4. Glass tube for chromatography (internal diameter: 22 mm, length:
300 mm), with a PTFE cock and a 250-ml reservoir.
4.5. Rotary vacuum evaporator, with a 500-ml round-bottom flask.4.6. 500-ml conical flasks, with ground-glass stoppers.4.7. TLC apparatus.4.8. Glass plates for TLC, 200 × 200 mm, prepared as follows (the
quantities indicated are sufficient to cover five plates). Put 30 g of
silica gel G-HR (3.15) into a conical flask. Add 60 ml water,
stopper and shake for a minute. Spread the suspension on the plates
so as to obtaina un iform layer 0·25 mm thick. Leave to dry inthe
air and then store in a desiccator containing silica gel. At the time
of use, activate the plates by keeping them in the oven at 110 ºC
for one hour.
Ready-to-use plates are suitable if they give results similar to those
obtained with the plates prepared as indicated above.
4.9. Long-wavelength (360 nm) UV lamp. The intensity of irradiation
must make it possible for a spot of 1 ng of aflatoxin B
1to be still
clearly distinguished on a TLC plate at a distance of 10 cm from
the lamp.
4.10. 10 ml graduated tubes with polyethylene stoppers.
4.11. UV spectrophotometer.
4.12. Fluorodensitometer (optional).
5.Procedure
5.1.Preparationofthesample(seeunder‘Observations’,PartC,point
1).
Grind the sample so that the whole of it will pass through a sieve
with a 1-mm mesh (in accordance with recommendation ISO R
565).
5.2.Extraction
Put 50 g of ground, homogenized sample into a 500-ml conical
flask (4.6). Add 25 g of Kieselguhr (3.14), 25 ml of water and
250 ml of chloroform (3.2). Stopper the flask, shake or stir for 30
minutes with the apparatus (4.2) and filter through a fluted filter
paper (4.3). Discard the first 10 ml of the filtrate and then collect
50 ml.
5.3.Columnclean-up
Insert into the lower end of a chromatography tube (4.4) a cotton or
glass wool plug (3.9), fill two-thirds of the tube with chloroform
(3.2) and add 5 g of sodium sulphate (3.10).
Check that the upper surface of the sodium sulphate layer is flat,
thenadd 10 g of silica gel (3.8) insmall portion s. Stir carefully
after each addition to eliminate air bubbles. Leave to stand for 15
minutes and then carefully add 15 g of sodium sulphate (3.10). Let
the liquid fall until it is just above the upper surface of the sodium
sulphate layer.1976L0372 — EN — 03.05.1994 — 003.001 — 4
▼B
Mix the 50 ml of extract collected in 5.2 with 100 ml of n-hexane
(3.3) and quantitatively transfer the mixture to the column. Let the
liquid fall until it is just above the upper surface of the sodium
sulphate layer. Discard this washing. Then add 100 ml of diethy-
lether (3.5) and again allow it to fall to the upper surface of the
sodium sulphate layer. During these operations see that the rate of
flow is 8 to 12 ml per minute and that the column does not run dry.
Discard the liquid that comes out. Thenelute with 150 ml of the
chloroform/methanol mixture (3.7) and collect the whole of the
eluate.
Evaporate the latter almost to dryness at a temperature not
exceeding 50 ºC and under a stream of inert gas (3.11) with the
rotary evaporator (4.5). Quantitatively transfer the residue, using
chloroform (3.2) or the benzene-acetonitrile mixture (3.6), to a
10 ml graduated tube (4.10). Concentrate the solution under a
stream of inert gas (3.11) and then adjust the volume to 2 ml with
chloroform (3.2) or the benzene/acetonitrile mixture (3.6).
5.4.Thin-layerchromatography
Spot on a TLC plate (4.8), 2 cm from the lower edge and at inter-
vals of 2 cm, the volumes indicated below of the standard solution
and the extract:
— 10, 15, 20, 30 and 40 µl of the standard aflatoxin B1solution
(3.16);
— 10 µl of the extract obtained in 5.3 and, superimposed on the
samepoint , 20 µl of the standard solution (3.16);
— 10 and 20 µl of the extract obtained in 5.3.
Develop the chromatogram in the dark with one of the developing
solvents (3.18). The choice of the solvent must be made before-
hand, by depositing 25 µl of the qualitative standard solution
(3.17) on the plate and checking that, when developed, aflatoxin
B1and B2are completely separated.
Let the solvents evaporate in the dark and then irradiate with UV
light, placing the plate 10 cm from the lamp (4.9). The spots of
aflatoxinB1give a blue fluorescence.
5.5.Quantitative determinations
Determine either visually or by fluorodensitometry as indicated
below.
5.5.1. Visual measurements
Determine the quantity of aflatoxin B1inthe extract by matchin g
the fluorescence intensity of the extract spots with that of one of
the standard solution spots. Interpolate if necessary. The fluores-
cence obtained by the superimposition of the extract on the
standard solution must be more intense than that of the 10 µl of
extract and there must not be more than one visible spot. If the
fluorescence intensity given by the 10 µl of extract is greater than
that of the 40 µl of standard solution, dilute the extract 10 or 100
times with chloroform (3.2) or the benzene/acetonitrile mixture
(3.6) before subjecting it again to thin-layer chromatography.
5.5.2. Measurements by fluorodensitometry
Measure the fluorescence intensity of the aflatoxin B1spots with
the fluorodensitometer (4.12) at an excitation wavelength of
365 nm and an emission wavelength of 443 nm. Determine the
quantity of aflatoxin B1inthe extract spots by comparisonof their
fluorescence intensities with that of the standard aflatoxin B1spots.
5.6.Confirmationoftheidentity ofaflatoxin B1
Confirm the identity of the aflatoxin B1inthe extract by the
processes indicated below.
5.6.1. Treatment with sulphuric acid
Spray sulphuric acid (3.13) onto the chromatogram obtain ed in5.4.
The fluorescence of the aflatoxin B1spots must turnfrom blue to
yellow under UV irradiation.
5.6.2. Two-dimensional chromatography involving the forma-
tionof aflatoxinB1-hemiacetal (aflatoxinB2a)
NB:The operations described below must be carried out following
carefully the diagram infigure 3.1976L0372 — EN — 03.05.1994 — 003.001 — 5
▼B
5.6.2.1. Applicationof the solution s
Score two straight lines on the plate (4.8) parallel to two contig-
uous sides (6 cm infrom each side) to limit migrationof the
solvent fronts. Spot the following solutions on the plate using capil-
lary pipettes or microsyringes:
—onpointA: a volume of purified extract of the sample, obtained
in 5.3, containing about 2·5 nm of aflatoxin B1;
—onpointsBandC: 25 µl of the standard solution (3.16).
5.6.2.2. Development
Develop the chromatogram indirectionI, inthe dark, usin g the
developing solvent (3.18.1) (1 cm layer in an unsaturated tank)
until the solvent front reaches the solvent limit line.
Remove the plate from the tank and allow to dry in the dark at
ambient temperature for five minutes. Then spray hydrochloric
acid (3.12) along a band 2·5 cm high, covering points A and B
(indicated by the hatched area in figure 3) until it darkens,
protecting the rest of the plate with a glass sheet. Allow to react
for 10 minutes in the dark and dry with a stream of air at ambient
temperature.
Next, develop the chromatogram indirectionII, inthe dark, usin g
the developing solvent (3.18.1) (1 cm layer in an unsaturated tank)
until the solvent front reaches the solvent limit line. Remove the
plate from the tank and allow to dry at ambient temperature.
5.6.2.3. Interpretation of the chromatogram
Examine the chromatogram under UV light (4.9) and check for the
following features.
(a) Appearance of a blue fluorescent spot of aflatoxin B1origi-
nating from the standard solution applied at C (migration in
the directionI).
(b) Appearance of a blue fluorescent spot of unreacted (with the
hydrochloric acid) aflatoxinB1and a more intense blue fluores-
cent spot of aflatoxin B1-hemiacetal, both originating from the
standard solution applied at B (migration in direction II).
(c) Appearance of spots matching those described in (b), origi-
nating from the sample extract applied at A. The position of
these spots is defined first by the migration distance of the afla-
toxinB1from point A in direction I (the same as that travelled
by the standard applied at C), and then by the migration
distances from there in direction II of the aflatoxin B1-hemia-
cetal (same as those travelled by the standard applied at B).
The fluorescence intensities of hemiacetal spots originating
from the extract and from the standard applied at B should
match.
6.Calculationoftheresults
6.1.Fromthevisualmeasurements
The content in micrograms of aflatoxin B1per kg of sample (ppb)
is givenby the formula:
S/C1Y/C1V
W/C1X
inwhich:
Y and X are respectively the volumes in microlitres of the standard
solutionof aflatoxinB1(3.16) and of the extract having a similar
intensity of fluorescence;
S = concentration in micrograms of aflatoxin B1per ml inthe
standard solution (3.16);
V = final volume of the extract in microlitres, allowing for any
dilutionthat was n ecessary;
W = weight in grammes of the sample corresponding to the
volume of extract subjected to columnclean -up.1976L0372 — EN — 03.05.1994 — 003.001 — 6
▼B
6.2.Fromthefluorodensitometricmeasurements
The content in micrograms of aflatoxin B1per kg of sample is
givenby the formula:
S/C1V
W/C1Y
inwhich:
Y = volume inmicrolitres of the extract spotted onthe plate (10
or 20 µl);
S = quantity in nanograms of aflatoxin B1inthe extract spot
(proportional to the value of Y taken), deduced from the
measurements;
V = final volume of the extract in microlitres, allowing for any
dilutionthat was n ecessary;
W = weight in grammes of the sample corresponding to the
volume of extract subjected to columnclean -up.
7.Preparationandtestingofthestandardsolution(3.16)
7.1.Determinationoftheconcentration ofaflatoxinB1
Prepare a standard solution of aflatoxin B1inthe chloroform (3.2)
or the benzene/acetonitrile mixture (3.6) with a concentration of 8
to 10 µg/ml. Determine the absorption spectrum between 330 and
370 nm with the aid of the spectrophotometer (4.11).
Measure the optical density (A) at 363 nm in the case of the
chloroform solution; or at 348 nm in the case of the solution in
the benzene/acetonitrile mixture.
Calculate the concentration in micrograms of aflatoxin B1per ml of
solutionfrom the formulae below:
312/C1A/C11000
20600for the chloroform solution;
312/C1A/C11000
19800for the solution in the benzene/acetonitrile mixture.
Dilute as appropriate, away from day light, to obtaina workin g
standard solution with a concentration of aflatoxin B1of about
0·1 µg/ml. If kept ina refrigerator at 4 ºC, this solutionis stable
for two weeks.
7.2.Testingofchromatographicpurity
Spot ona plate (4.8) 5 µl of the stan dard solutionof aflatoxinB1containing 8 to 10 µg/ml (7.1). Develop the chromatogram as indi-
cated in 5.4. In UV light the chromatogram should show only onespot and no fluorescence must be perceptible in the original deposit
zone.
8.Repeatability
The difference between the results of two parallel determinations
carried out on the same sample by the same analyst should not
exceed:
— 25 % related to the highest result for contents of aflatoxin B
1from 10 and up to 20 µg/kg;
— 5 µg, in absolute value, for contents greater than 20 and up to
50 µg/kg;
— 10 % related to the highest value for contents above 50 µg/kg.
9.Reproducibility
See under ‘Observations’, Part C, point 2.1976L0372 — EN — 03.05.1994 — 003.001 — 7
▼M2
B. DETERMINATION OF AFLATOXIN B1. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHIC METHOD
1.Purposeandscope
This method is for the determination of aflatoxin B1inan imal
feedingstuffs including those containing citrus pulp. The lower
limit of determination is 0,001 mg/kg (1 ppb).
2.Principle
The sample is extracted with chloroform. The extract is filtered and
analiquot portionis purified ona Florisil cartridge followed by a
C18cartridge. The final separation and determination is achieved by
high performance liquid chromatography (HPLC) using a reversed
phase C18column, followed by post-column derivatization with
iodine in water, and fluorescence detection.
Note:mycotoxins are extremely toxic sustances. Manipulations
should be performed ina design ated fume cupboard. Special
precautions should be taken when toxins are in a dry form because
of their electrostatic nature and resulting tendency to disperse in
working areas.
3.Reagents
3.1. Chloroform, stabilized with 0,5 to 1,0 % of ethanol, by mass. See
observation10.2.
3.2. Methanol, HPLC grade for preatation of 3.6.
3.3. Acetone.3.4. Acetonitril, HPLC grade.3.5. Eluting solvents: Prepare one day before use, or remove air in the
solvents ultrasonically.
3.5.1. Mixture of acetone (3.3) and water, 98 + 2 (v+v).3.5.2. Mixture of water and methanol (3.2), 80 + 20 (v+v).3.5.3. Mixture of water and acetone (3.3), 85 + 15 (v+v).3.6. Mobile phase for HPLC
Mixture of water, methanol (3.2) and acetonitril (3.4), 130 + 70 + 40
(v+v+v).
NBThe composition of the mobile phase solvent may need to be
adjusted, depending on the characteristics of the HPLC column
used.
3.7. Saturated iodine solution: add 2 g of iodine to 400 ml of water.
Mix for at least 90 min and filter through a membrane filter
(4.15). Protect the saturated solutionfrom light to preven t photode-
gradation.
3.8. Acid washed Celite 545, or equivalent.3.9. Florisil cartridge (Waters SEP-PAK), or equivalent.3.10. C
18cartridge (Waters SEP-PAK), or equivalent.
3.11. Inert gas e.g. nitrogen.
3.12. AflatoxinB1standard solution in chloroform, concentration 10 µg/
ml. Check the concentration of the solution as follows: determine
the absorption spectrum of the solution between 330 and 370 nm
by means of the spectrophotometer (4.23). Measure the absorbance
(A) at the maximum near 363 nm. Calculate the concentration of
aflatoxinB1inmicrograms per millilitre of solutionfrom the
formula:
Concentration èmg =ml ðÞ ¼312/C2A/C21000
22300¼13;991/C2A
3.12.1. Aflatoxine B1stock standard solution in chloroform.
Transfer quantitatively 2,5 ml of the aflatoxin B1standard solution
(3.12) to a 50 ml volumetric flask and adjust to the mark with1976L0372 — EN — 03.05.1994 — 003.001 — 8
▼M2
chloroform (3.1). Store this solutionina cool place (4 ºC) inthe
dark, well sealed and wrapped in aluminium foil.
3.13. AflatoxinB1calibraltionsolution s HPLC.
NBUse alcid-washed glassware for preparationof these solution s
(see 4, Apparatus).
3.13.1. Calibrationsolution4n g/ml.
Allow the volumetric flask with stock standard solution (3.12.1) to
warm up to room temperature inthe alumin ium foil (a few hours).
Transfer 400 µl of the stock standard solution (200 ng aflatoxin B1)
into a 50 ml volumetric flask, and evaporate the solution to dryness
in a current of inet gas (3.11).
Dissolve the residue obtained in approximately 20 ml of water/
acetone mixture (3.5.3), make up to the mark with the water/
acetone mixture and mix well.
3.13.2. Calibrationsolution3 n g/ml.
Transfer quantitatively 7,5 ml of the calibration solution (3.13.1)
into a 10 ml volumetric flask, make up to the mark with the
water/acetone mixture (3.5.3), and mix well.
3.13.3. Calibration solution, 2 ng/ml.
Transfer quantitatively 25 ml of the calibration solution (3.13.1) to
a 50 ml volumetric flask, make up to the mark with the water/
acetone mixture (3.5.3) and mix well.
This solution is also referred to as the reference standard, to be
used for repetitive injection during HPLC (5.5).
3.13.4. Calibrationsolution1 n g/ml.
Transfer quantitatively 2,5 ml of the calibration solution (3.13.1) to
a 10 ml volumetric flask, make up to the mark with the water/
acetone mixture (3.5.3) and mix well.
3.14. Ampoule containing a mixture of aflatoxins B1,B2,G1and G2concentrations approximately 1, 0,5, 1 and 0,5 µg/ml respectively,
in1 ml chloroform.
3.14.1. Chromatographic test solution.
Transfer the content of the ampoule (3.14) into a glass-stoppered
test-tube or screw-capped vial. Transfer 40 µl of this solution into
a glass-stoppered test-tube (acid-rinsed) (4.22). Evaporate the
chloroform in a stream of inert gas (3.11) and redissolve into
10 ml of the water/acetone mixture (3.5.3).
3.15. Reagents for confirmatory test (6).3.15.1. Sodium chloride saturated solution.3.15.2. Sodium sulphate, anhydrous, granular.
4.Apparatus
Caution: Use of non acid-washed glassware for aqueous aflatoxin
solutions may cause losses. Particular care should be taken with
new glassware and disposable glassware such as autosampler vials
and Pasteur pipettes. Therefore laboratory glassware coming into
contact with aqueous solutions of aflatoxins should be soaked in
dilute acid (e.g. sulfuric acid = 2 mol/l) for several hours, then
rinsed well with distilled water to remove all traces of acid (e.g.
three times, check with pH-paper). Inpractice, this treatmen t is
necessary for the round bottomed flask (4.4), the volumetric flasks,
measuring cylinders, vials or tubes used for calibration solutions
and final extracts (particularly vials for autosamplers), and Pasteur
pipettes, if these are used to transfer calibration solutions or
extracts.
4.1. Grinder-mixer.
4.2. Sieve of aperture size 1,0 mm, (ISO R 565).4.3. Mechanical shaker.4.4. Rotary vacuum evaporator, equipped with a 150 to 250 ml round
bottomed flask.1976L0372 — EN — 03.05.1994 — 003.001 — 9
▼M2
4.5. High performance liquid chromatograph, injector with a loop
suitable for the injection of 250 ml. See the manufacturers instruc-
tions for partial or complete loop filling.
4.6. HPLC analytical column: 3 µm or 5 µm C18packing.
4.7. Pulse-free pump for delivery of the iodine post-column reagent.
4.8. Valco zero dead volume Tee, stainless steel (1/16 ″× 0,75 mm).
4.9. Spiral reaction coil; Teflon or stainless steel. Dimensions of
3 000 × 0,5 mm to 5 000 × 0,5 mm have beenfoun d to be appro-
priate in combination with 5 µm or 3µm HPLC columns.
4.10. Thermostatically controlled water-bath adjusted to 60 ºC, capable
of temperature regulationto better than0,1 ºC.
4.11. Fluorescence detector, with excitation at 365 nm and emission at
435 nm wavelenths. (For filter instrument: emission wavelength
> 400 nm). Detection of at least 0,05 ng aflatoxin B1shall be
possible. Some back pressure may be advisable (e.g. restrictor,
Teflon or stainless steel coil connected to the outlet of the
detector), to suppress air bubbles inthe flow-cell.
4.12. Strip chart recorder.4.13. Electronic integrator (optional).4.14. Fluted filter paper diameter: 24 cm, Macherey-Nagel 617 1/4 or
equivalent.
4.15. Membrane filter with a pore size of 0,45 µm, Millipore HAWP
04700 or equivalent.
4.16. 500 ml glass stoppered conical flask.4.17. Glass column (internal diameter approximately 1 cm, length
approximately 30 cm) equipped with a Luer tip.
4.18. Luer chloroform-resistant stopcock (e.g. Bio-rad 7328017, Analyti-
chem A1 6078, J.T. Baker 4514 or equivalent).
4.19. Chemically resistant syringe, 10 ml Luer connector.4.20. Syringe suitable for HPLC injection of 250 µl (see 4.5).
4.21. 100 µl microsyringe for preparation of calibration solutions (check
that the accuracy is within2 % by weighin g).
4.22. 10 ml glass stoppered calibrated tubes.
4.23. Spectrophotometer, suitable for making measurements in the UV
regionof the spectrum.
4.24. Equipment for confirmatory test (6).
4.24.1. Acid-rinsed 100 ml separating funnel with Teflon stopcock.4.24.2. Heating block, 40 to 50 ºC.
5.Procedure
5.1.Preparationofthesample.
Grind the sample so that it passes through the sieve (4.2).
5.2.Testportion.
Weigh 50 g of the prepared test sample into the conical flask
(4.16).
5.3.Extraction
Add 25 g of Celite (3.8), 250 ml of chloroform (3.1) and 25 ml of
water to the test portion(5.2). Stopper the flask, an d shake for 30
minutes on a mechanical shaker (4.3). Filter through a fluted filter
paper (4.14). Collect 50 ml of the filtrate. If necessary, take an
aliquot of the filtrate and dilute to 50 ml with chloroform so that
the concentration of aflatoxin B
1is not greater than 4 ng/ml.1976L0372 — EN — 03.05.1994 — 003.001 — 10
▼M2
5.4.Clean-up (the procedure should be carried out without significant
interruptions).
Caution:
— protect the laboratory, where the analyses are done, adequately
from daylight. This canbe achieved effectively by usin g:
(i) UV absorbing foil on the windows in combination with
subdued light (no direct sunlight);
(ii) Curtains or blinds in combination with artificial light (fluor-
escent tubes are acceptable);
— Aflatoxin containing solutions must be protected from light as
much as possible (keep inthe dark, use alumin ium foil).
5.4.1. Florisil SEP-PAK purification
5.4.1.1. Preparationof the column -cartridge assembly
Attach a stopcock (4.18) to the shorter stem of a Florisil cartridge
(3.9) (see figure 1). Wash the cartridge and remove aid by taking
10 ml chloroform (3.1) and passing 8 ml via the stopcock rapidly
through the cartridge using a syringe (4.19). Attach the longer stem
of the cartridge to a glass column (4.17) and pass the reamining
2 ml chloroform through the cartridge into the column. Close the
stopcock. Remove the syringe.
5.4.1.2. Purification
Add the filtrate collected in5.3 to the column -cartridge assembly
and drain by gravity. Rinse with 5 ml of chloroform (3.1), followedby 20 ml of methanol (3.2). Discard the eluates. During these
operations, ensure that the column-cartridge assembly does not run
dry.
Elute aflatoxinB
1with 40 ml of the acetone-water mixture (3.5.1)
and collect the whole of the eluate in the round bottomed flask of
the rotary evaporator (4.4). Concentrate the eluate on the rotary
evaporator at 40 ºC to 50 ºC until no more acetone is distilled.
(NBapproximately 0,5 ml of liquid remains in the flask at this
point. Experiments have shown that further evaporation is not
harmful and that when 0,5 ml of liquid remains, there is then no
significant amount of acetone. Residues of acetone might lead to
losses of aflatoxinB1onthe C18cartridge) Add 1 ml of methanol
(3.2), swirl the flask to dissolve aflatoxinB1onthe sides of the
flask, add 4 ml water, and mix. Disconnect and discard the
cartridge. Rinse the glass column with water and retain for the C18purificationstep.
5.4.2. C18SEP-PAK purification
5.4.2.1. Preparationof the column -cartridge assembly.
Attach a stopcock (4.18) to the shorter stem of a C18-cartridge
(3.10) (see figure 1). Prime the cartridge and remove any air by
passing 10 ml methanol (3.2) via the stopcock rapidly through the
cartridge with a syringe (4.19) (Air bubbles in the cartridge are
visible as light spots onthe otherwise greyish backgroun d). Take
10 ml water, and pass 8 ml through the cartridge (Avoid introduc-
tion of air into the cartridge, when switching from methanol to
water). Attach the longer stem of the cartridge to a glass column
(4.17) and pass the remaining 2 ml water through the cartridge in
the column. Close the stopcock. Remove the syringe.
5.4.2.2. Purification
Transfer the extract collected in 5.4.1.2 quantitatively to the glass
column (4.17), rinsing the flask twice with 5 ml water/methanol
mixture (3.5.2) and drain by gravity. During these operations,
ensure that the column-cartridge assembly does not run dry.
(When air bubbles develop in the constriction near the cartridge,
stop the flow and tap the top of the glass column, to remove the
air bubbles. Then continue). Elute with 25 ml water/methanol
mixture. Discard the eluate. Elute the aflatoxinB1with 50 ml
water/acetone mixture (3.5.3), and collect the whole of the eluate
ina 50 ml volumetric flask. Make up to the mark with water an d
mix: the resulting test solution is used for chromatography (5.5).
Caution: Filtration of the final extract prior to HPLC is normally
not necessary. If considered necessary, cellulose filters
are not to be used, because they may lead to losses of
aflatoxinB1. Teflonfilters are acceptable.1976L0372 — EN — 03.05.1994 — 003.001 — 11
▼M2
5.5.Highperformanceliquidchromatography
(See Figure 2 for setting-up of the equipment). Allow sufficient
time for conditioning and stabilizing the instruments.
Note1:
The flow-rates givenfor the mobile phase an d the post-column
reagent are indicative only. They may need to be adjusted
depending on the characteristics of the HPLC column.
Note2:
The detector response to aflatoxin B1depends on the temperature,
therefore compensation should be made for drift (see Figure 3). By
injecting a fixed amoung of aflatoxin B1reference standard (3.13.3)
at regular intervals (i.e. every third injection), the aflatoxin B1peak
values between these reference standards can be corrected using the
mean response, provided that the difference between responses of
consecutive reference standards is very small (< 10 %). Therefore
injections must be made without interruptions. If interruption is
necessary, the last injection before interruption and the first injec-
tion after interruption must be the reference standard (3.13.3).
Because the calibration curve is linear and passes through the
origin, the amounts of aflatoxin B1inthe sample extracts are deter-
mined directly by reference to the adjacent standards.
5.5.1. HPLC pump settings
Set the HPLC pump (4.5) to give a flow of 0,5 or 0,3 ml/minfor a
5 µm or a 3 µm HPLC column(4.6) respectively usin g the mobile
phase (3.6).
5.5.2. Post-columnpump settin gs
Set the pump (4.7) to give a flow of 0,2 to 0,4 ml/minof the
iodine-saturated water solution (3.7). As a rough guide: Flows of
approximately 0,4 or 0,2 ml/minare advisable incombin ation
with flows of 0,5 and 0,3 ml/min of the mobile phase (3.6) respec-
tively.
5.5.3. Fluorescence detector
Set the fluorescence detector (4,11) to exc. = 365 nm and em
= 435 nm (filter instrument; > 400 nm). Adjust the detector
attenuator to obtain approximately 80 % full scale deflection of
the recorder penfor 1 n g of aflatoxinB1.
5.5.4. Injector
For all solutions, inject 250 µl amounts following the instructions
of the manufacturer of the injector.
5.5.5. Check of chromatographic separation
Inject the chromatographic test solution (3.14.1). Valleys should be
less than5 % of the sum of peak heights of the adjacen t peaks.
5.5.6. Check of the stability of the system
Before each series of analyses, respectively inject the reference
standard (3.13.3), until stable peak areas are achieved ( NBPeak
responses for aflatoxin B1between consecutive injections should
not differ by more than 6 %). Proceed without delay with the check
of linearity (5.5.7).
5.5.7. Check of linearity
Inject the aflatoxin B1calibrationsolution s (3.13.1 to 3.13.4). Every
third injection use the reference standard (3.13.3), for correction of
drift inrespon se ( NBPeak responses for this reference standard
must not differ by more than 10 % in 90 minutes). Correct for drift
according to the formula in 7. The calibration graph should be
linear and pass through the origin, within twice standard error of
Y-estimate. Values found must not differ by more than 3 % from
the nominal values. If these requirements are fulfilled, continue
without delay. If not, identify and correct the sources of the
problem before continuing.
5.5.8. Injection of sample extracts
Inject the purified sample extracts (5.4.2.2). After every two sample
extracts repeat the injection of the reference standard (3.13.3)
according to the following sequence: reference standard, extract,
extract, reference standard, extract, extract, reference standard etc.1976L0372 — EN — 03.05.1994 — 003.001 — 12
▼M2
6.Confirmatorytest
6.1.Furthertreatment oftheextract (5.4.2.2)
Add 5 ml sodium chloride solution(3.15.1) to the fin al extract
obtained at 5.4.2.2.. Extract three times each with 2 ml chloroform
(3.1) for one minute, in the separating funnel (4.24.1). Pour the
combined chloroform extracts over approximately 1 g sodium
sulphate (3.15.2) into a 10 ml test tube. A small funnel (diameter:
4 cm) can be used with a piece of cottonwool in the constriction,
covered with a approximately 1 g sodium sulphate.
Wash the sodium sulphate layer with a few ml of chloroform and
collect the washing in the same test tube. Evaporate the chloroform
extract to dryness in the same test-tube using the heating block
(4.24.2) and redissolve in 1 ml of chloroform.
6.2.Preparationofderivative andthinlayerchromatography:
See Annex to Council Directive 76/372/EEC method A, point 5.6.2.
7.Calculationofresults
Calculate the aflatoxinB1content (µm/kg) present in the sample,
using the formula:
aflatoxinB 1content in èmg =kg¼m/C2Vext
Vm/C2M/C2Vf
Vc
where:
m = amount of aflatoxin B1in ng represented by the B1peak of the sample, calculated as follows:
m¼P sampleðÞ
Ps t1ðÞ þPs t2ðÞ/C22rs tðÞ
P(sample) = peak area of aflatoxinB1for the sample
P(st1) = peak area of aflatoxinB1resulting from the
preceeding injection of reference standard (3.13.3)
P(st2) = peak area of aflatoxinB1resulting from the
following injection of reference standard (3.13.3)
r(st) = injected amount of aflatoxin B1inthe referen ce
standard (3.13.3) in ng
Vm= volume of the injected sample extract in ml
Vext= final volume of sample extract in ml, allowing for
any dilution that was made (5.3)
M = mass of sample ing
Vf= volume of filtrate transferred to Florisil cartridge
(5.4.1.2) inml
Vc= volume of chloroform, used for the extractionof the
sample inml
If the procedure is followed as inthis protocol, the formula reduces
to:
aflatoxinB1content in µg/kg = 20 × m.
7.1. Calculations of the results may also be done by peak height
measurement.
8.Repeatability:
see under 10.1
9.Reproducibility:
see under 10.11976L0372 — EN — 03.05.1994 — 003.001 — 13
▼M2
10.Observations
10.1.Precision
A collaborative study(1), carried out at the international level on
mixed feeding stuffs gave the results for repeatability and reprodu-
cibility indicated in Table 1. The term repeatability (r) used here isdefined as the largest ratio which is not significant at the 95 %
probability level for comparisonof two readin gs of the same
sample in the same laboratory under similar conditions. The term
reproducibility (R) is similarly defined for comparing two different
laboratories. Inaccordan ce to ISO 3534 — 1977, 2.35(
2)a nd
CommissionDecision89/610/EEC(3) r and R are also given in
Table 1 in terms of coefficients of variation.
Table1
Repeatability (r) and reproducibility (R) expressed as ratios
andcorrespondingcoefficientsofvariation
(15 laboratories)
Level r R CVr(*) CVR
(µg/kg) (%) (%)
8 & 14 1,4 1,7 11 18
(*) CV = coefficient of variation
10.2.Stabilization ofchloroform(3.1)
The adsorptioncharacteristics of the florisil cartridge may be
changed if stabilizers other than ethanol are used. This should be
verified inaccordan ce with 10.3 whenthe chloroform described is
not available.
10.3.Accuracy
The correct applications of the method shall be verified by making
replicate measurements on certified reference materials. If these are
not available, the performance of the method should be verified by
recovery experiments made on the fortified blank samples. The
deviationof the meanfrom the actual value, expressed as a percen -
tage of the actual value, shall not lie outside the limits −20 to
+10 %.1976L0372 — EN — 03.05.1994 — 003.001 — 14
(1) Egmond, H.P. van, Heisterkamp, S.H. and Paulsch, W.E. (1991). Food Additives and
Contaminants 8, 17-29.
(2) ISO 3534-1977.
(3) OJ No L 351, 2. 12. 1989, p. 39.
▼M2
Figure1:Column-cartridgeassembly
Figure 2: Flow diagramof the LC system with iodine post-column
derivatization1976L0372 — EN — 03.05.1994 — 003.001 — 15
▼M2
Figure 3: Compensation for drift in aflatoxin B1response by
injecting referencestandard(3.13.3)atregularintervals
C. OBSERVATIONS CONCERNING METHODS A AND B
1.Defatting
Samples containing more than 5 % fats must be defatted with light petroleum
(bp 40 to 60 ºC) after the preparations indicated in 5.1.
Insuch cases, the an alytical results must be expressed interms of the weight
of the non-defatted sample.
2.ReproducibilityoftheresultsforMethodA
The reproducibility of the results, i.e. the variationbetweenthe results
obtained by two or more laboratories on the same sample has been estimated
at:
± 50 % of the meanvalue for meanvalues of aflatoxinB, from 10 an d up to
20 µg/kg;
± 10 µg/kg onthe meanvalue for meanvalues greater than20 an d up to
50 µg/kg;
± 20 % of the meanvalue for meanvalues above 50 µg/kg.1976L0372 — EN — 03.05.1994 — 003.001 — 16
▼M3
▼B
Appendix toAnnex
Figure1
Figure21976L0372 — EN — 03.05.1994 — 003.001 — 17
▼B
Figure31976L0372 — EN — 03.05.1994 — 003.001 — 18