UGANDA STANDARD  
       US ISO 6651
First Edition
   2009 -09-04
 
Reference numbe r 
   US ISO 6651:2001
 © UNBS 200 9 
  
Animal feeding stuffs — Semi-quantitative determination of 
aflatoxin B 1 — Thin-layer chromatographic methods 
 US ISO 6651:2001  
 
© UNBS  2009 – All rights reserved Compliance with this standard does not, of itself confer immunity from legal obligations 
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNBS 2009  
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
form or by any means, electronic or mechanical, in cluding photocopying and microfilm, without prior written 
permission from UNBS. 
Requests for permission to reproduce this document should be addressed to 
The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
Kampala  
Uganda 
Tel: 256 41 505 995 
Fax: 256 41 286 123 
E-mail: unbs@infocom.co.ug
    US ISO 6651: 2001  
© UNBS  2009 - All rights reserved National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry  of Tourism, Trade and 
Industry established by the Act of Parliament of 198 3, of the Laws of Uganda.  UNBS is mandated to co-
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex A limentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agr eements of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a gi ven field or area and consists of representatives of 
consumers, traders, academician s, manufacturers, governme nt and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committ ee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Uganda Standard, US ISO 6651:2001, Animal feeding stuffs — Semi-quantitative determination of 
aflatoxin B 1 — Thin-layer chromatographic methods, is identical with and  has been reproduced from an
International Standard ISO 6651:2001,  Animal feeding stuffs — Semi-quantitative determination of 
aflatoxin B 1 — Thin-layer chromatographic methods, and has been adopted as a Uganda Standard.
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." INTERNATIONAL
STANDARDISO
6651
Third edition
2001-12-15
Reference number
ISO6651:2001(E)
©ISO2001Animal feeding stuffs — Semi-quantitative
determinationofaflatoxinB1—T hi n -l a y er
chromatographic methods
Alimentsdes animaux — Dosagesemi-quantitatif del'aflatoxine B1—
Méthodes par chromatographie sur couchemince
ISO 6651:2001(E)
ii ©ISO 2001 –AllrightsreservedPDF disclaimer
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Printedin SwitzerlandISO 6651:2001(E)
©ISO 2001 – Allrights reserved iiiContents Page
1Scope .......................................................................................................................... ..................................... 1
2Normative reference ............................................................................................................. ............................ 1
3Principle ...................................................................................................................... ...................................... 1
4Reagents ....................................................................................................................... ................................... 2
5Apparatus ...................................................................................................................... ................................... 3
6Sampling ....................................................................................................................... .................................... 4
7Procedure ...................................................................................................................... ................................... 5
8Expressionof results andcalculations ............................................................................................. .............. 11
9Interlaboratory tests ........................................................................................................... ............................. 12
10Testreport ..................................................................................................................... ............................... 12
Annex
AResults ofinterlaboratory tests.................................................................................................. ..................... 13ISO 6651:2001(E)
iv ©ISO 2001 –AllrightsreservedForeword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO
member bodies). The work of preparing International Standards is normally carried out through ISO technicalcommittees. Each member body interested in a subject for which a technical committee has been established hasthe right to be represented on that committee. International organizations, governmental and non-governmental, inliaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical
Commission (IEC) on all matters of electrotechnical standardization.
InternationalStandards are draftedinaccordancewiththerules givenintheISO/IEC Directives,Part3.Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an InternationalStandardrequiresapproval by at least 75% of themember bodiescastingavote.
Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of
patent rights. ISO shall not beheld responsiblefor identifyinganyor all suchpatent rights.
International Standard ISO 6651 was prepared by Technical Committee ISO/TC 34,
Food products , Subcommittee
SC 10,Animalfeedingstuffs .
Thisthirdeditioncancels and replaces thesecondedition (ISO 6651:1987), of whichit constitutes aminor revision.
Annex Aof this InternationalStandardis for informationonly.INTERNATIONALSTANDARD ISO 6651:2001(E)
©ISO 2001 – Allrights reserved 1Animal feedingstuffs — Semi-quantitative determinationof
aflatoxinB1— Thin-layer chromatographic methods
1 Scope
1.1ThisInternationalStandardspecifiestwomethodsforthedeterminationofaflatoxin B1inanimalfeedingstuffs.
These methods can only be usedfor semi-quantitative determinations.
1.2MethodA is applicable to the following simple feeding stuffs:
— oilseeds and oilseed residues, and in particular groundnut, copra, linseed, soya, babassupalm;
—m a n i o c m e a l ;
— maize germ expeller;
— cereals and cereal products;— peameal;
— potato pulpandflour.
In the presence of substances interfering with the determination by method A, it is recommended that the
determination be carried out in accordance with method B.
1.3MethodB is applicableto mixed feeding stuffs and tosimplefeedingstuffsnot mentionedin1.2.
This method is not applicable to feeding stuffs containing citruspulp.
2 Normative reference
The following normative document contains provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of thesepublications do not apply. However, parties to agreements based on this International Standard are encouraged toinvestigatethepossibilityofapplyingthemostrecenteditionofthenormativedocumentindicatedbelow.Forundatedreferences, the latest edition of the normative document referred to applies. Members of ISO and IEC maintainregisters of currently valid International Standards.
ISO 6498,
Animalfeedingstuffs— Preparation of test samples
3 Principle
A test portion is extractedwith chloroform then filtration. Analiquot portion is purifiedona silica gelcolumn.The eluate is evaporated and the residue is dissolved in a specified volume of chloroform or a mixture of benzene
andacetonitrile.
Thin-layer chromatography, one-dimensional for methodA and two-dimensional for method B, is carried out on an
aliquot portionof thissolution.
The aflatoxin B
1content is determined either visually or by fluorodensitometry, by examination of the chromatogram
underultravioletlightandcomparisonwithknownquantitiesofstandardaflatoxinB1appliedtothesameplateasthe
test portion extract.ISO 6651:2001(E)
2 ©ISO 2001 –AllrightsreservedThe identify ofaflatoxinB1is confirmed byformation of the hemiacetal derivative.
4R e a g e n t s
Use only reagents of recognized analytical quality, and distilled or deonized water or water of at least equivalent
purity.
4.1Chloroform , stabilized with to of (volume fraction) ethanol.
4.2n-Hexane.
4.3Diethyl ether , anhydrous, free from peroxides.
4.4Benzene/acetonitrile ,( )mi x t u r e .
Mix 98volumesof benzenewith 2volumesof acetonitrile.
4.5Chloroform/methanol ,( )mixtu re .
Mix 97volumesofchloroformwith3 volumes of methanol.
4.6Developing solvents .
The solvents should be used in covered tanks. When saturated tanks are specified, this is achieved by lining the
tanks withabsorbent paper andallowing the interiorstobecomesaturated withsolvent vapour.
4.6.1 Chloroform/acetone ,( )mix ture .
Mix 90volumesofchloroformwith10volumesofacetone,inanunsaturatedtank.
4.6.2 Diethyl ether/methanol/water ,( )mi x t u r e .
Mix 96volumesof diethylether, 3 volumes of methanoland1 volumeof water, in anunsaturated tank.
4.6.3 Diethyl ether/methanol/water ,m i x t u r e .
Mix 94volumesofdiethyletherwith 4,5 volumes ofmethanol and1,5volumes ofwater,ina saturatedtank.4.6.4 Chloroform/methanol ,) m i x t u r e .
Mix 94volumesof chloroformwith6 volumes of methanol, in asaturated tank.
4.6.5 Chloroform/methanol ,) m i x t u r e .
Mix 97volumesof chloroformwith3 volumes of methanol, in asaturated tank.
4.7Silicagel , for columnchromatography, of particlesize to .
4.8Silicagel , G-HR or equivalent, for thin-layer chromatography.
4.9Diatomaceous earth (Hyflosupercel), acid-washed.
4.10Sodiumsulfate , anhydrous granules.
4.11Trifluoroacetic acid .
4.12Inertgas , for example nitrogen.0,5% 1,0% 96%
98+2
97+3
90+10
96+3+1
(94+4,5+1,5)
(94+6
(97+3
0,05mm 0,20mmISO 6651:2001(E)
©ISO 2001 – Allrights reserved 34.13Sulfuricacid , solution (volume fraction).
4.14Aflatoxin B1, standard solution containing about of aflatoxin B1per millilitre, in the chloroform (4.1) or
inthebenzene/acetonitrile mixture (4.4).
WARNING —Aflatoxinsare highly carcinogenic and must be handled with great care.Prepare and check the solution as follows.4.14.1 Preparationof stocksolutionand determinationof concentrationPrepare a solution of aflatoxin B
1in the chloroform (4.1) or the benzene/acetonitrile mixture (4.4) such that the
concentrationisbetween and .Determinetheabsorptionspectrumbetween and bymeansofthespectrometer (5.9).
Measure the absorbance ( ) at in the case of the chloroform solution, or at in the case of the
benzene/acetronitrile mixture solution.
Calculate the concentration of aflatoxin B
1, inmicrograms per millilitre of solution, from theformulae:
a) forthe chloroform solution
b) forthe solutioninthe benzene/acetonitrilemixture
4.14.2 Dilution
Dilute the stock solution (4.14.1), as appropriate, away from daylight, to obtain a standard solution with a
concentration ofaflatoxinB1of about .
If kept in arefrigerator at , this solutionis stablefor 2 weeks.4.14.3 Testingofchromatographicpurityof the solutionOnto a plate (5.7), apply a spot of of the standard aflatoxin B
1solution of concentration to
(4.14.1). Develop the chromatogram as indicated in 7.5.1. Under ultraviolet light, the chromatogram shall show onlyonespot andno fluorescenceshall beperceptibleintheoriginal deposition zone.
4.15Aflatoxin B1and B2(see the warning in 4.14), solutions for qualitative testing, containing about of
aflatoxinB1andB2per millilitre, in the chloroform (4.1) orinthebenzene/acetonitrile mixture(4.4).
Theseconcentrationsaregivenasaguide.Theyshallbeadjustedsoastoobtainthesameintensityoffluorescence
f orbothafl ato x ins(s ee7.5.1).
5A p p a r a t u s
Usuallaboratory equipment and, in particular, the following.
5.1Grinder/mixer .50%
0,1g
8g/ml 10g/ml 330nm 370nm
A363nm 348nm
312A1000
22300
312A1000
19800
0,1g/ml
4C
5l8 g/ml 10g/ml
0,1gISO 6651:2001(E)
4 ©ISO 2001 –Allrightsreserved5.2Sieve, of aperturesize .
Fordetails, seeISO 5651).
5.3Shakingapparatus ormagneticstirrer .
5.4Chromatographic tubes , made of glass (internal diameter , length ), with a
polytetrafluoroethylenetapanda reservoir, plugged at thebottom end withcottonorglass wool.
5.5Rotaryvacuumevaporator ,with a round-bottomedflask.
5.6Apparatus for thin-layer chromatography (TLC) , i.e. that necessary for the preparation of the plates (5.7)
and applicationofspots(capillarypipettesormicrosyringes),a developingtank,andspraying apparatusforapplyingthe sulfuric acid (4.13) totheplates.
5.7GlassTLCplates , ,preparedasfollows(thequantitiesindicatedaresufficienttocoverfive
plates).
Place of the silica gel (4.8) in a conical flask, add of water, stopper and shake for . Spread the
suspension on the plates so as to obtain a uniform layer thick. Leave to dry in the air and then store in adesiccator containingsilica gel. At the timeof use, activatetheplates by keepingthem in anoven at for .
Ready-to-use plates are suitable if they give results similar to those obtained with the plates prepared as indicated
above.
5.8Long-wavelength ( ) ultraviolet lamp .
The intensity of irradiation shall make it possible for a spot of of aflatoxin B1to be clearly distinguished on a
TLC plateat adistanceof from the lamp.
WARNING — Ultraviolet light isdangerousto theeyes.Protectivegoggles shall beworn.
5.9Spectrometer , suitable for makingmeasurements in the ultraviolet regionof thespectrum.
5.10Fluorodensitometer (optional).
5.11Fluted filter paper .
5.12Graduatedtube , of capacity , with apolyethylene stopper.
5.13Conicalflask , of capacity , with a ground glass stopper.
5.14Pipette,ofca p ac ity .
5.15Analytical balance .
6 Sampling
Take the laboratory sample from the material to be sampled in accordance with the International Standard for the
material concerned unless sampling for the determination of aflatoxin is excluded from its field of application. If noappropriate International Standard exists, agreement shall be reached between the parties concerned, taking intoaccount the characteristicsof thematerial beingsampled.
1) ISO 565, Test sieves —Metalwire cloth,perforated metal plate andelectroformed sheet —Nominalsizesof openings .1,0mm
22mm 300mm
250ml
500ml
200mm200mm
30g 60ml 1min
0,25mm
110C1 h
360nm
1,0ng
10cm
10,0ml
500ml
50mlISO 6651:2001(E)
©ISO 2001 – Allrights reserved 57 Procedure
7.1 Preparationoftestsample
7.1.1If thesamplecontainsmorethan of fat, it shall bedefattedwithlight petroleum beforegrinding.
In suchcases, the analytical resultsshall be expressedinterms of themassof thenon-defattedsample.
7.1.2Grind the laboratory sample so that is completely passes through the sieve (5.2). Mix thoroughly. See
ISO 6498.
7.2 Testportion
Weigh, to the nearest , of the prepared test sample into the conicalflask (5.13).
7.3 Extraction
Add to the test portion (7.2) of the diatomaceous earth (4.9), of water, and of the chloroform (4.1)accurately measured from a measuring cylinder. Stopper the flask, and shake or stir for using the shakingapparatus (5.3). Filter through the fluted filter paper (5.11), taking care to discard the first of the filtrate, andsubsequently collect at least of thefiltrate.
7.4 Columnclean-up
7.4.1 Preparationof the column
Fill two-thirds of the chromatographic tube (5.4) with the chloroform (4.1) and add of the sodium sulfate (4.10).
Checkthattheuppersurfaceofthesodiumsulfatelayerisflat,thenadd ,insmallportions,ofthesilicagel(4.7).Stircarefullyaftereachadditiontoeliminateairbubbles. Leavetostandfor andthencarefullyadd of thesodium sulfate (4.10). Open the tap and allow the liquid to flow until it is just above the upper surface of the sodiumsulfate layer. Close the tap.
7.4.2 Purification
Transfer,bymeansofthepipette(5.14), of thefiltratecollectedin7.3toa conicalflask,andadd
of the
n-hexane (4.2). Mix and quantitatively transfer the mixture to the column, rinsing the flask with the n-hexane.
Openthetapandallowtheliquidto flowatarateof to untilitislevelwiththeuppersurfaceofthesodium sulfate layer. Close the tap. Discard the liquid collected and pour of the diethyl ether (4.3) into thecolumn. Again open the tap and allow the liquid to flow until it is level with the upper surface of the sodium sulfatelayer. During theseoperations, ensurethat thecolumn does not rundry.
Elute with of the chloroform/methanol mixture (4.5) and collect the whole of the eluate in the flask of
the rotary evaporator (5.5). Evaporate to dryness on the rotary evaporator, preferably under a stream of inert gas(4.12), at atemperature not exceeding , and under reducedpressure.
If arotary evaporator is not available, adda boiling aid andevaporatealmost todrynessona water bath.
Quantitatively transfer the residue, using the chloroform (4.1) or the benzene/acetonitrile mixture (4.4), to the
graduated tube (5.12). Again evaporatethe solution, for example on a water bath, preferably under a stream of inertgas (4.12), and adjust thevolume to withthechloroform(4.1) or the benzene/acetonitrilemixture(4.4).5%
0,01g 50g
25g 25ml 250ml
30min
10ml
50ml
5g
10g
15min 10g
50ml 250ml 100ml
8ml/min 12ml/min
100ml
150ml 500ml
50C
10ml
2,0mlISO 6651:2001(E)
6 ©ISO 2001 –Allrightsreserved7.5 Thin-layerchromatography
7.5.1 MethodA— One-dimensionalthin-layer chromatography
7.5.1.1 Choice of solvent
The choice of solvent (4.6.1, 4.6.2, 4.6.3, 4.6.4 or 4.6.5) shall be made beforehand to ensure that aflatoxins B1and
B2arecompletelyseparatedwhentheplate is developed, whichdepends on the batch of plates inuse.
Place of the qualitative solution(4.15) ontheprepared plates (5.7) (oneplatefor each solvent tobechecked).Follow the procedure in 7.5.1.2 for development, evaporation and irradiation.Two distinct spots are produced bya suitable solvent.
7.5.1.2 Procedure
Onto a TLC plate (5.7), and using a capillary pipette or microsyringe, apply from the lower edge, and at
intervals of , the volumes indicatedbelow of thestandard aflatoxinB
1solutionandtheextract:
— , , , and of thestandardaflatoxinB1solution(4.14);
— of the extract obtained in 7.4.2 and, superimposed on the same point, of the standard aflatoxin B1
solution (4.14);
— and of theextract obtainedin7.4.2.
Develop the chromatograminthedark using the developingsolvent chosen (see 7.5.1.1).Remove the plate from the tank, allow the solvents to evaporate from the plate in the dark and then examine under
ultraviolet light, placingthe plate fromthelamp (5.8). The spots of aflatoxin B
1show abluefluorescence.
7.5.2 MethodB— Two-dimensionalthin-layer chromatography
7.5.2.1 Application ofthe solutions (seeFigure1)
Trace two straight lines on a plate (5.7) parallel to two contiguous sides ( and from each side
respectively), to establish the limit of migration of the solvent fronts. Apply the following solutions to the plate using
capillary pipettes or microsyringes:
— at point A: of thepurified sampleextract obtained in 7.4.2;
— at point B: of thestandardaflatoxinB1solution(4.14);
— at point C: of the standardaflatoxin B1solution (4.14);
— at point D: of the standardaflatoxin B1solution (4.14);
— at point E: of thestandardaflatoxinB1solution(4.14).
Dry inaslow stream of air or inert gas (4.12). The spots obtainedshall have adiameterof about .
7.5.2.2 Development (seeFigure1)
Develop the chromatogram in direction I, in the dark, using the developing solvent (4.6.3) ( layer in a saturated
tank) until the solvent front reaches the limit line. Remove the plate from the tank and allow to dry, in the dark, atambient temperature forat least .25l
20mm
20mm
10l1 5l2 0l3 0l4 0l
10l 20l
10l2 0l
10cm
50mm 60mm
20l
20l
10l
20l
40l
5mm
1cm
15minISO 6651:2001(E)
©ISO 2001 – Allrights reserved 7Then develop the chromatogram in direction II, in the dark, using the developing solvent (4.6.1) ( layer in an
unsaturatedtank)untilthesolvent front reachesthelimitline.Removetheplatefromthetankandallowtodry, inthedark,at ambienttemperature.
7.5.2.3 Interpretationofthechromatogram (seeFigure2)
Examinethechromatogramunderultravioletlightbyplacingtheplate fromthelamp(5.8).Locatetheposition
of the blue fluorescent spots B, C, D and E of the aflatoxin B
1from the standard solution and trace two imaginary
lines passing through these spots and at right angles tothedirections of development. The point Pof intersectionofthese lines is the location at which to expect the aflatoxin B
1spot originating from the test portion extract applied at
point A(seeFigure 1). However, theactual locationoftheaflatoxin B1spot may beat apointQ at theintersectionof
two imaginary straight lines forming an angle of about between them and passing through points B and Crespectively.Dimensionsinmillimetres
Figure 1— Application of solutions
1cm
10cm
100ISO 6651:2001(E)
8 ©ISO 2001 –Allrightsreserved7.5.2.4 Supplementary chromatography
Trace on a new plate (5.7) two straight lines parallel to two contiguous sides, as indicated in Figure1, and apply, at
point A, of the purified test portion extract obtained in 7.4.2 and, superimposed on it, of the standardaflatoxin B
1solution (4.14). Develop as indicated in 7.5.2.2. Examine the chromatogram under ultraviolet light and
check that
— the aflatoxin B1spotsfrom the extract and the standard solution are superimposed, and
— the fluorescence of this spot is more intense than that of the aflatoxin B1spot developed at point Q on the first
plate.
7.6 Determination
7.6.1 Visualmeasurement
7.6.1.1 Method A
Determine the quantity of aflatoxinB1in the extract by comparing the intensity of fluorescence of the extract spots
withthat of the standardsolutionspots. Interpolateif necessary.
The fluorescence obtained by thesuperimposition of the extract onthestandard solution shallbemoreintense than
thatofthe ofextractandshallbeperceptibleasonlyonespot.IftheintensityoffluorescencegivenbytheDimensions in millimetres
Figure 2—Interpretationof thechromatogram
20l 20l
10l 10lISO 6651:2001(E)
©ISO 2001 – Allrights reserved 9of extract is greater than that of the of standard solution, dilute the extract 10 or 100times with the chloroform
(4.1) or withthebenzene/acetonitrile mixture (4.4) beforerepeatingthin-layer chromatography.
7.6.1.2 MethodB
Determined the quantity ofaflatoxinB1in the extract by comparing the intensity of the extract spot with that of spots
C, D and Efromthestandardsolution. Interpolate if necessary.
Iftheintensityoffluorescencegivenbythe ofextractisgreaterthanthatofthe ofstandardsolution,dilute
the extract 10 or 100 times with the chloroform (4.1) or with the benzene/acetonitrile mixture (4.4) before repeatingthin-layer chromatography.
7.6.2 Measurement of fluorodensitometry
Measure the intensity of fluorescence of the aflatoxin B
1spots with the fluorodensitometer (5.10) at an excitation
wavelength of and an emission wavelength of .
Determine, inthe case ofmethodA, the quantity of aflatoxinB1in the extract spots by comparison with the intensity
of fluorescence of the spots from the standard solution and, in the case of method B, the quantity of aflatoxin B1in
theextract spot by comparison withtheintensity of fluorescence of spots C, D and E fromthestandardsolution.
7.7 Confirmationofthe identity ofaflatoxin B1
7.7.1 General
Confirm the identity of the aflatoxinB1in the extract by the presumptive test with sulfuric acid (see 7.7.2) and, if the
result of this test is positive, by the actual confirmation test (7.7.3). If the result of the presumptive test with sulfuric
acid is negative, there is no need to proceed with the actual confirmation since, in this case, no aflatoxin B1is
present.
7.7.2 Presumptivetest with sulfuricacid
Spray the sulfuric acid (4.13) onto the chromatogram obtained in 7.5.1or 7.5.2. Thefluorescence of the aflatoxin B1
spots shall turn frombluetoyellow under ultraviolet light.
7.7.3 Confirmationtest7.7.3.1 Formation of aflatoxin B
1-hemiacetal(aflatoxin B2a)
Inthecaseofsimpleandonlyslightlypigmentedfeeds,usetheone-dimensionalthin-layerchromatographicmethod
described in 7.7.3.2. In the case of simple pigmented feeds, mixed feeds, or in cases of doubt, use the two-dimensional thin-layer chromatographic methoddescribedin7.7.3.3.
7.7.3.2 One-dimensionalthin-layer chromatography
Trace a straight line on a plate (5.7) to divide it into two equal parts. Apply to each part, from the lower edge
andat intervals of , the volumes indicatedbelow of thestandard aflatoxin B
1solutionand theextract:
— of the standard aflatoxin B1solution (4.14);
— a volumeof the extract obtainedin7.4.2containingapproximately of aflatoxinB1;
— of the standard aflatoxinB1solution (4.14) and, superimposed on it, a volume of the extract obtained in
7.4.2containingapproximately of aflatoxinB1.40l
20l4 0 l
365nm 443nm
20mm
15mm
25l
2,5ng
25l
2,5ngISO 6651:2001(E)
10 ©ISO 2001 –AllrightsreservedApply to one of the two halves of the plate, superimposed on the spots previously applied, to of the
trifluoroacetic acid(4.11). Dryina stream of air at ambient temperature.
Developthechromatogram,inthedark,usingoneofthedevelopingsolvents(4.6).Thechoiceofthesolventshallbe
made beforehand. The solvent system shall ensure that the aflatoxin B1-hemiacetal (aflatoxin B2a) is clearly
separated frominterfering substances. The solvent front shall travel about .
Allowthesolventstoevaporateinthedark,andthenspraysulfuricacid(4.13)ontothepartoftheplatenotpreviously
treatedwith thetrifluoroacetic acid. Examinetheplateunder ultraviolet light.
The identify ofaflatoxinB1is confirmed if
a) the value of the aflatoxinB1derivative originating from the extract corresponds with that from the standard
solution;
b) the aflatoxin B1derivative originating from the standard solution, superimposed on the extract, has a
fluorescence more intense than the aflatoxin B1derivative originating fromtheextract.
Since fluorescent spots from the extract, having the same value as the aflatoxin B1-hemiacetal, might lead to a
false positive interpretation of the chromatogram, their presence should be checked on the part of the plate treatedwithsulfuricacid.
Incases of doubt, confirmationby two-dimensional thin-layer chromatography (7.7.3.3) shall be used.
7.7.3.3 Two-dimensionalthin-layerchromatography (see Figure 3)
7.7.3.3.1 Application of thesolutions
Tracetwostraightlinesonaplate(5.7),paralleltotwocontiguoussides( fromeachside),toestablishthelimit
of migration of the solvent fronts. Apply the following solutions to the plate using capillary pipettesor microsyringes:
— atpoint A:avolumeof purifiedextractfromthesample,obtainedin7.4.2,containingabout of aflatoxinB
1
and adrop ( to ) of thetrifluoroacetic acid (4.11);
— at points BandC: of thestandard aflatoxinB1solution(4.14) anda dropof the trifluoroacetic acid(4.11).
Dry inastreamof airatambienttemperature.
7.7.3.3.2 Development
Develop the chromatogram in direction I(see Figure 3), in the dark, using the developing solvent (4.6.2) ( layer
inanunsaturatedtank)untilthesolventfrontreachesthelimiteline.Removetheplatefromthetankandallowtodry,in the dark, at ambient temperature for .
Then develop the chromatogram in direction II, in the dark, using the developing solvent (4.6.1) ( layer in an
unsaturated tank) until the solvent front reaches the limit line. Remove the plate from the tank and allow to dry atambient temperature.
7.7.3.3.3 Interpretationofthechromatogram
Examinethe chromatogramunderultraviolet light fromthelamp (5.8) andcheck for thefollowingfeatures.
a) AppearanceofabluefluorescentspotofaflatoxinB
1-hemiacetal,andsometimesaweakbluefluorescentspotof
aflatoxin B1which has not reacted with the trifluoroacetic acid, originating from the standard solution applied at
point C (migrationindirection I) and from the standard solutionapplied at point B (migrationindirection II).
b) Appearance of spots similar to those described in a), originating from the sample extract applied at point A. The
position of these spots is defined by those originating from the standard solution applied at points B and C. The
intensitiesoffluorescenceoftheaflatoxin B1-hemiacetalspotsoriginatingfromtheextractandfromthestandard
solution applied at pointsB and C should be comparable.1l2l
120mm
Rf
Rf
60mm
2,5ng
1l2l
25l
1cm
5min
1cmISO 6651:2001(E)
©ISO 2001 – Allrights reserved 117.8 Numberofdeterminations
Carry out two determinations on the same test sample.
8 Expressionofresults andcalculations
8.1 Visualmeasurements
Theaflatoxin B1content, expressedin microgramsper kilogram of sample, isequalto
where
is the concentration, in micrograms of aflatoxin B1per millilitre of the standard solution (4.14)
(approximately );Dimensionsinmillimetres
Figure 3—Confirmation test
CV1V3
mV2
C
0,1g/mlISO 6651:2001(E)
12 ©ISO 2001 –Allrightsreservedis the mass, in grams, of the test portion corresponding to the volume of extract subjected to column
clean-up ( );
isthe finalvolume of the extract, in microlitres, taking into account anydilution that was necessary;
and are, respectively, the volumes, in microlitres, of the extract and of the standard aflatoxin B1solution
(4.14),applied totheplate, having similarintensitiesoffluorescence.
8.2 Fluorodensitometricmeasurements
The aflatoxinB1content, expressedinmicrograms per kilogram of sample, is equal to
where
is the mass, in grams, of the test portion corresponding to the volume of extract subjected to column clean-
up( );
is the mass, in nanograms, of aflatoxinB1in the extract spot (taking into account the volume ), deduced
fromthemeasurements;
is the finalvolume of the extract, in microlitres, taking into account any dilution that was necessary;
is the volume, in microlitres, of extract applied to the plate ( or ).
9 Interlaboratorytests
Details of interlaboratory tests on the precision of the method are summarized in annex A. The values derived from
this interlaboratory testmaynotbeapplicable toconcentration rangesand matrices other than those given.
10 Testreport
The test report shallspecify:
— all information necessary for the complete identification of the sample;— the sampling method used, if known;
— the test method used (A or B), withreference to this International Standard;
— the methodof determination (visual or fluorodensitometric);— all operating details not specified in this International Standard, or regarded as optional, together with details of
any incidents which may have influencedthetest result(s);
— the test result(s) obtained.m
10,0g
V1
V2V3
m1V1
mV2
m
10,0g
m1 V2
V1
V2 10l2 0lISO 6651:2001(E)
©ISO 2001 – Allrights reserved 13Annex A
(informative)
Results ofinterlaboratory tests
Three interlaboratory tests, two of which were carried out at the international level (Nos. 1 and 2), on mixed feeding
stuffs (methodB) gave the results indicated inTableA.1.
The11laboratoriesparticipatingintrial 2alsoanalysedthesamplebymethodA,itscompositionbeingsuitable,and
obtained resultsverysimilar to those when using method B, by visual or fluorodensitometric measurement.
Table A.1
ParameterTest
123
Number of laboratories 23 11 13Mean, 162,7 25,4 13,4Standard deviationofrepeatability( ), 16,9 2,7 1,7Coefficient of variation ofrepeatability, 10 11 13Repeatability limit ( ), 47,8 7,6 4,8Standard deviationofrepeatability( ), 45,2 6,8 4,0Coefficient of variation ofreproducibility, 28 27 30Reproducibilitylimit ( ), 128,0 19,2 11,3g/kg
srg/kg
%
2,83srg/kg
sRg/kg
%
2,83sRg/kgISO 6651:2001(E)
ICS 65.120
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©ISO 2001 –Allrightsreserved