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है”ह”ह

IS 13427 (1992): Animal feeds and feeding stuffs -Determination of aflatoxin B-1 content [FAD 5: LivestockFeeds, Equipment and Systems]

IS 13427 : 1992ISO 6651 : 1987

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Indian Standard

ANIMAL FEEDS AND FEEDING STUFFS ­DETERMINATION OF AFLATOXIN 81 CONTENT

UDC 636·085: 643 [ 516-9 ]

C> BIS 1992

BUREAU OF INDIAN STANDARDSMANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG

NEW DELHI 110002

July 1992 Price Group 5

AMENDMENT NO.1 AUGUST 1993TO

IS 13427 : 1992/ ISO 6651 : 1987 ANIMAL FEEDS ANDFEEDING STUFFS - DETERMINATION OF

AFLATOXIN 81 CONTENT

( Page 8, clause 9.1 ) - Addtbe following note underclause9.1 :

'NOTE - la the Indiao context tbe collaborative study on the same feed samplesbelweeD labonaoriel haveindiQted lbe variation oC:

a) ~ of lbe meaD ~alue Cor meanvalueof analoxin Bi between 101020 ppb, and

b) ~ of meaavaluefor the meaavalueof anatoxinBi above 20 ppb.'

(FADS)Reproerapby Unit. BIS. NewDelhi.lodi.

IS 13427 : 1992ISO 88&1 : 1987

Indian Standard

ANIMAL FEEDS AND FEEDING STUFFSDETERMINATION OF AFLATOXIN 8. CONTENT

NATIONAL FOREWORD

This Indian Standard which is identical with ISO 6651 : 1987 'Animal feeding stuffs­Determination of aflatoxin B·1 content', issued by the International Organization for Standardi­zation ( ISO ), was adopted by the Bureau of Indian Standards on the recommendation of theLivestock Fe$ds Sectional Committee ( FAD 5) and approval of the Food and] AgricultureDivision Council.

In the adopted Indian Standard, some terminology and certain conventions are not identicalwith those used in Indian Standards. Attention is especially drawn to the following:

a) Wherever the words 'International Standard' appear, referring to this standard, theyshould be read as 'Indian Standard'.

b) Comma ( , ) has been used as a decimal marker, while in Indian Standards, thecurrent practice is to use point ( . ) as the decimal marker.

CROSS REFERENCE,In this Indian Standard, the following International Standard is referred to:

ISO 6498 : 1983 Animal feeding stuffs - Preparation of test sample.The Technical Committee has reviewed the provisions of ISO 6498 : 1983 'Animal feedingstuffs - Preparation of test sample' to which reference is made in the text and has decidedthat this standard is acceptable for use in conjunction with this standard.

ADDITIONAL INFORMATION

The standard prescribes two methods:Method A One dimensional thin layer chromatography, andMethod B Two dlmenslcnet thin layer chromatography.

The extraction procedure is the same for both these methods.Method A is recommended for straight feeding stuffs and raw materials.Method B is recommended for compounded feeds such as poultry and cattle feeds and alsofor raw materials such as cottonseed and its oilcakes and deoiled meals.

The following Note may be included under clause 5.8:'NOTE- Thelolvent., namelv, diethyl ether ( &.3 ) and chloroform ( &.1 ) should be made free from traces ofmoisture by shaking with anhydrous sodium sulphate'.

The following may be included as an alternative developing solvent for Methods A and B inplace of chloroform/acetone ( 90+10 ) if silica gel used is of Indian make:

'6.8.6 Toluene/amyl alcohol/methanol ( 90 : 32 : 2 ):

Include 6.8.8 as an alternate for choice of solvent under clause 8.5.1.1.Sampling of the product for aflatoxin analysis shall be done in accordance with IS 13426 : 1992'Animal feeds and feeding stuffs - Methods of sampling for aflatoxin analysis'.

For the purpose of deciding whether a particular requirement of this standard is complied with,the final value, observed or calculated, expressing the result of a. text or analysis, shall berounded off in accordance with IS 2 : 1960 'Rules for rounding off numerical values (revised )'.The number of significant places retained in the rounded off value should be the same as thatof the specified value in this standard.

1

1 Scope

This International Standard specifies two methods for thedetermination of the aflatoxin B, content of animal feedingstuffs.

2 Field of application

2.1 Method A is applicable to the following simple feedingstuffs :

- oilseedsand oilseed residues, and in particular ground­nut, copra, linseed, soya, sesame, babassu palm;

manioc meal;

maize germ expeller;

cereals and cereal products;

pea meal;

potato pulp and flour.

In the presence of substances interfering with the determina­tion by method A, it is recommended that the determination becarried out in accordance with method B.

2.2 Method B ;s applicable to mixed feeding stuffs and tosimple feeding stuffs not mentioned in 2.1.

This method is not applicable to feeding stuffs containing citruspulp.

2.3 The lower limit of detection of aflatoxin 8, is 0,01 mg/kg.

3 Reference

ISO 6498, Animal ftHJding stuffs - Preparation of testsamples.

4 Principle

Extraction of the test portion with chloroform, filtration, andpurification of an aliquot portion on ·a silica gel column.

IS 13427 : 1992180 88&1 : 1887

Evaporation of the eluBte and dissolution of the residue in aspecified volume of chloroform or mixture of benzene andacetonitrile.

Thin-layer chromatography, one-dimensionalfor method Aandtwo-dimensional for method S, of an aliquot portion of thissolution.

Determination of the aflatoxin B1 content, either visually orbyfluorodensitometry, by examination of the chromatogramunder ultraviolet light and comparison with known quantities ofstandard aflatoxin 81 applied to the same plate asthe test por­tion extract.

Confirmation of the identity of aflatoxin B1 by formation of thehemiacetal derivative.

5 Reagents

All reagents shall be of recognized analytical quality. Thewaterused shall be distilled water or water of at least equivalentpurity.

6.1 Chloroform, stabilized with 0,5 to 1,0 % of 96 " (YI JI)ethenol.

5.2 n-HeX8ne.

&.3 D'.thy' ether, anhydrous, free from peroxides.

5.4 Benzene/Bcetonltril., (98 + 2) mixture.

Mix 98 volumes·of benzene with 2 volum. of acetonitrile.

6.6 Chloroform/methanol, (97 + 3) mixture.

Mix 97 volumes of. chloroform with 3 volumes of methanol.

~.8 Developing IOlventa.1)

6.1.1 Chloroform/.eeton., (90 + 10)mixture.

Mix 90 volum. of chloroform with 10volumes of acetone, inan unsaturated tank.

1) The aoIventllhould be used in covered tanks. When saturated tamesare specified, this i8achieved by lining the tanks with ablorbent paper andallowing the interiors to btfcome uturated With solvent v.pollr.

,

18 13427 : 1892180 8861 : 1887

&.8.2 D'.thyl ether/methanol/weter, (96 + 3 + 1) mix­ture.

Mix 98 volumesof diethyl ether, 3 volumesof methanoland 1volume of water, in an unsaturated tank.

&.8.3 Diethyl ether/methanol/water, (94 + 4,6 + 1,6)mixture.

Mix 94 volumesof diethyl ether with 4,5 volumesof methanoland 1,5 volumes of water, in a saturatedtank.

&.8.4 Chloroform/methanol, (94 + 6) mixture.

Mix 94 volumesof chloroform with 8 volumesof methanol, in asaturated tank.

&.8.& Chloroform/methanol, (97 + 3) mixture.

Mix 97 volumesof chloroform with 3 volumesof methanol, in asaturated tank.

&.7 Silica gel, for column chromatography, of particle size0,06 to 0,20 mm.

&.8 Silica gel, G-HR or equivalent, for thin-layerchromatog­raphy.

6.9 Dletomaceous earth (HyfI08uperceU, acid-washed.

&.10 Sodium sulfate, anhydrousgranules.

&.11 Trifluoroacetlc acid.

6.12 Inert ga., for example nitrogen.

&.13 Sulfuric acid, 50 % (V/ JI) solution.

&.14 Aflatoxin 8" standard solution containing about0,1 J.lg of aflatoxin B, per millilitre, in the chloroform (5.1)or inthe benzene/acetonitrile mixture (6.4).

WARNING - Afilltoxins are highly carclno9t!'nlc andmust be handled with great care.

P~pare and check the solution as follows.

5.14.1 Preperetlon of stock solution and determinationof concentration

Prepare a solution of aflatoxin 8 , in the chloroform (5.1) or thebenzene/acetonitrile mixture (5.4) such that the concentrationis between8 and 10 ~g/ml. Determine the absorptionspectrumbetween 330 and 370 nm by means of the spectrophotometer(8.9).

Measure the absorbance (A) at 363 nm in the case of thechloroform solution, or at 348nm in the case of thebenzene/acetonitrile mixture solution.

Calculate the concentration of-aflatoxin 8" in micrograms permillilitre of solution, from the formulae :

a) for the chloroform solution :

312 x A x 1·000

22~

b) for the solution in the benzene/acetonitrile mixture:

312 x A x 1 000

191m

6.14.2 Dilution

Dilute the stock solution (5.14.1), as appropriate, away fromdaylight, to obtain a standardsolution with a concentration ofaflatoxin 8, of about 0,1 ~g/ml.

If kept in a refrigerator at 4 °C, this solution is stable for 2weeks.

5.14.3 Testing of chromatographic purity of the solution

Onto aplate (6.7),apply a spot of 5 ~I of the standard aflatoxin8, solution of concentration 8 to 10 IJg/ml (5.14.1). Developthe chromatogram as indicatedin 8.5.1. Underultraviolet light,the chromatogram shall ·show only one spot and nofluorescence shall be perceptible in the original depositionzone.

5.1& Aflatoxin 8, and 82 (see the warning in 6.14), solu­tions for qualitative testing, containing about 0,1 IJg of afla­toxin 8, and B2 per millilitre, in the chloroform (5.1) or in thebenzene/acetonitrile'mixture(5.4).

These concentrations are given as a guide. They shall be.ad­justed so as to obtain the same intensity of fluorescence forboth aflatoxins (see 8.5.1).

8 Apparatus

Usual laboratory equipment, and'in particular :

8.1 Grinder/mixer.

8.2 Sieve, of aperturesize1,0 mm.1)

8.3 Shaking apparatus or magnetic stirrer.

1) See ISO 565, Test sieves - Woven metal wiftl Cloth, perforated plate lind e1ectroformed sheet - Nomi"., sizes of openings.

4

6.4 Chromatographic tubes, made of glass (internaldiameter 22 mm, length 300mm), wi~h a polytetrafluoro­ethylene tap and a 250 ml reservoir, plugged at the bottom endwith cotton or glass wool.

6.& Rotary vacuum evaporator, with a 600 ml round­bottomed flask.

6.8 Apparatu8 for thin-layer chromatography (TLCJ, i.e.that necessary for the preparation of the plates (6.7) and ap­plication of spots (capillary pipettes or microsyringes), adeveloping ~ank, and spraying apparatus for applying thesulfuric acid (5.13J to the plates.

6.7 GI8S. TLC plate., 200mm x 200mm, prepared asfollows (the quantities indicated are sufficient to COJer fiveplates).

Place 30 9 of the silica gel (5.8) in a conical flask,- add 60 ml ofwater, stopper and shake for 1 min. Spread the suspension onthe plates so as to obtalna uniform layer 0,25 mm thick. Leaveto dry in the air and then store in a desiccator containing silicagel. At the time of use, activate the plates bVkeeping them inan oven at 110°C for 1 h.

Ready-to-use plates are suitable if they give results similar tothose obtained With the plates prepared as indicated above.

6.8 Long-wavelength (360 nm) ultraviolet lamp.

The intensity of irradiation shall make it possible for a spot of1,0 ng of aflatoxin B1 to be clearly distinguished on a TLC plateat a distance of 10 cm from the lamp.

WARNING - Ultraviolet light is dangerous to the eye••Protective goggle. shall be worn.

6.9 Spectrophotometer, suitable for making measure­ments in the ultraviolet region of the spectrum.

6.10 Fluorodensltometer (optional).

6.11 Fluted filter peper.

6.12 Graduated tube, of capaci!'( 10,0ml, with apolvethvlene stopper.

8.13 Conlca. flask, of capacity 600ml, with a ground glassstopper.

6.14 Pipette, of capacity 60ml.

8.16 Balance.

7 Sampling

Take the laboratory sample from the material to be sampled inaccordance with the International Standard for the materialconcerned unless 8Impling for the determination of aflatoxin isexcluded from its field of application. If no appropriate Inter-

5

IS 13427 : 1892180 8861 : 1887

national Standard exists, agreement shall be reached betweenthe parties concerned, taking into account the characteristicsof the matenal being 88mpled.

8 Procedure

8.1 Preparetion of the test 8amp'e

8.1.1 If the sample contains more than 5 % of fat, it shall bedefatted with light petroleum before grinding.

In such cases, the analytical results shall be expressed in termsof the mass of the non-defatted sample.

8.1.2 Grind the laboratory sample so that it completely passesthrough the sieve (6.2). Mix thoroughly. (See ISO6498.)

8.2 Test portion

Weigh, to the nearest 0,01 g, 50 9 of the prepared test sampleinto the conical flask (6.13).

8.3 Extraction

Add to the test portion (8.2) 25 g of the diatomaceous earth(6.9),25 ml of water, and 250ml of the chloroform (5.1) ac­curately measured from a measuring cylinder. Stopper theflask, and shake or stir for ~ min using the apparatus (6.3).Filter through the fluted filter paper ,(6. 11), taking care todiscard the first 10ml of the filtrate, and subsequently collect atleast 60 ml of the filtrate.

8.4 Column clean-up

8.4.1 Preparation of the column

Fill two-thirds of the chromatographic tube (6.4) with thechloroform (5.1) and add 5 g of the sodium sulfate (5.10).Check that the upper surface of the sodium sulfate layer is flat,then add 10g, in small portions, of the silica gei (5.7). Stircarefully after each addition to eliminate air bubbles. Leave tostand for 16 min and then carefully add' 10 9 of the sodiumsulfate (5.10). Open the tap and allow the liquid to flow until itis just above the upper surface of the sodium sulfate layer.Close the tap.

.8.4.2 Purification

Transfer, by means of the pipette (6.14), 60 ml of the filtratecollected in 8.3 to a 2&)'ml conical flask, and add 100ml of then-hexane (5.2). Mix and quantitatively transfer the mixture tothe column, rinsing the flaskwith the n-hexan8. Open the tapand allow the liquid to flowat a rate of 8 to 12milmin until it islevel with the upper surface of the sodium sulfate layer. Closethe tip. Discard the liquid collected and pour 100 ml of thediethyl ether (5.3) 'into the column. Again open the tap andallow the liquid to flow until it is level with the upper surface ofthe sodium sulfate layer. During these operations, ensure thatthe column does not run dry.

IS 13427 : 1992180 8861 : 1887

Elute with 150ml of the chloroformImethanol mixture (5.6)and collect the whole of the eluate in the 500 ml flask of therotary evaporator (8.5). Evaporate to dryness on the rotaryevaporator, preferably under a stream of inert gM (5.12), at atemperature not exceeding 60°e, and underreduced pressure.

NOTE - If8 rotary evaporator ianot available, add a boiling aid andevaporate almost to dryness on a water bath.

Quantitatively transfer the residue, using the chloroform (5.1)or the benzene/acetonitrile mixture (5.4)", to the 10 mlgraduated tube (6.12). Again evaporate the solution, for exam­ple in 8 water bath, preferably under a stream of inert gas(5.12), and adjust the volume to 2,0 ml with the. chloroform(6.1) or the benzene/acetonitrile mixture (5.4).

8.& Thln.;.18yer chromatography

8.&.1 Method A - One-dlmenslon.1 thin-I.yerchrom8togr.phy

8.&.1.1 Choice of solvent

The choice of solvent (5.8.1, 5.6.2, 5.6.3, 5.8.4 or 5.6.5) shallbe made beforehand to ensure that aflatoxin. B1 and 82 arecompletely aeparated· when the plate is develop8d, whichdepend. on "the batch of plates in use.

Place 25~ of the qualitative solution (5.15) on the preparedplates(8.7) (one plate for eachsolvent to be checked).

Follow the procedure in 8.5.1.2 for development, evaporationand irradiation.

Two distinct spots are producedby a suitablesolvent.

8.&.1.2 Procedure

Onto a TLC plate (8.7), and using a capillary pipette orrnicroeyringe, apply20 mm from the lower tK;Ige,' and at inter­vals of 20mm, the volumes indicated below of the standardaflatoxin 8, solution and the extract :

- 10, 16, 20, 30 and 40 J.lI of the standard aflatoxin 8,solution (5.14);

- 10 ~I of the extract obtained in 8.4.2 and, superim­posed on the same point, 20 ~I of the standard afla­toxin 8,lOIution (6.14);

- 10end 20~ of the extl'8etobtained in 8.4.2.

Develop the chromItogl'lm in the dark using the developinglOIvent chosen (888 8.6.1.1).

Remove the plate from the tank, .11ow the solvents to8VIIpOI1It8from the pIefe in the darkand then examine underulti1Molet light, placing the plate 10 emfrom the lamp i8.8).Thespots of aflatoxin 8, Ihcwv a blue fluorescence.

8

8.&.2 Method B - Two-dimension.' thln-I.v.rchrom.togrephy

8.6.2.1 Application of the solutions (seefigure 1)

Trace two straight lines on a plate (6.7) parallel to two con­tiguous sides (50 and 60 mmfrom each side respectively), toestablish the limit of migration of the solventfronts. Apply thefollowing solutions on the .plate using capillary pipettes ormicr08yringes :

- at point A : 20 J,ll of the purified sample extract ob­tained in 8.4.2;

- at point a : 20 JJI of the standard aflatoxin B1 solution(5.14);

- at point C·: 10 IJI of the standard aflatoxin 81 solution(5.14);

- at point D ~ 20 IJI of the standard aflatoxin 81 solution(5.14);

- at point E : 40III of the standard aflatoxin 8 1 solution(6.14).

Dry in a slow stream of air or inert gas (5.12). The spots ob-tainedshall havea diameterof about 5 mm. .

8.&.2.2 Development (see figure 1)

Develop the chromatogram in direction I, in the dark, using thedeveloping solvent (5.8.3) (1 cm layerin a saturated tank) untilthe solvent front reaches the limit line. Remove the plate fromthe tank and allow to dry, in the dark, at ambient temperaturefor at least 15 min.

Then develop the chromatogram in direction II,. in the dark,using the developing solvent (6.6.1) (1 cm layer in an unsatu­rated tank) until the solvent front reaches the limit line. Re­move the plate from the tank and allow to dry, in the dark, atambient temperature.

8.&.2.3 Interpretation of the chromatogram (see figure 2)

Examine the chromatogram under ultraviolet light by placingthe plate 10cm from the lamp (8.8). Locatethe position of theblue fluorescentspots a, C, 0 and E of the aflatoxin 8, fromthe 8t8~dard solution and trace two imaginary lines pasSingthrough these spots and at right angles to the directions ofdevelopment. The point P of intersectionof these lines is thelocation at which to expect the aflatoxin 8, spot originatingfrom the test portion extract applied at point A (see figure 1).However, the aetuallocation of the aflatoxinB1spot maybeIt apointQ at the intersection of two imaginary straightlinea fonningan angle of lbout 1000 between them and passing throughpoints B and C respectively.

8.1.2.4 Supplementary chromltography

TrICeon I new plate (8.7)two strIight linesperellel to two con·tiguous sidel, • indicated in figure 1, Ind eppIy, at point A,20 IJI of tM purified test portion emld obtained in 8.4.2end,

superimposed on it, 20 III of the standard aflatoxin B, solution(6.14). Develop as indicated in 8.6.2.2. Examine thechromatogram underultraviolet light and checkthat

- the aflatoxinB1 spots from theextractandthestandardsolution an; superimposed;

- the fluorescence of this spot is more intense than thatof the aflatoxin 8 1 spot developed at point Q on the·firStplate.

8.8 Determination

Two methods of determination may be used, i.e. visualmeasurement or fluorodensitometric measurement.

8.8.1 Vlsu.1 me.lurement

8.6.1.1 Method A

Determine thequantityof aflatoxinB, in theextractby compar­ing the intensityof fluorescence of the extract spotswith thatof the standard solution spots. Interpolate if necessary.

The fluorescence obtained by the superimposition of the ex­tract on the standard solutionshall bemoreintense thanthat ofthe 10 IJI of extractandshall beperceptible asonly onespot. Ifthe intensity of fluorescence given by the 10 IJI of extract isgreater thanthatof the 40 ~ of standard solution,dilutethe ex­tract 10 or 100 times with the chloroform (5.1) or with thebenzene/acetonitrile mixture (6.4) before repeating thin-layerchromatography.

8.6.1.2 Method 8

Determine the quantityof aflatoxinB, in the extractby compar­ing the intensityof the extractspot with that of spotsC, D andE from the standard solution. Interpolate if necessary.

If the intensityof fluorescence given by the 20 III of extract is'greater than that of the40 IJI of standard solution,dilutethe ex­tract 10 or 100 times with the chloroform (5.1) or with thebenzene/acetonitrile mixture (5.4) before repeating thin-layerchromatography.

8.8.2 Me.surement by fluorodensltometry

Measure the intensityof fluorescence of the eflatoxin8, spotswith the fluorodensitometer (6.10) at an excitation wavelengthof 366nm and an emission wavelength of 443nm.

Determine, in the case of method A, the quantity of aflatoxinB, ttl the extract spots by comparison with the intensity offluorescence of the spots from the standard solution, and, inthe case of method8, the quantityof aflatoxinB1in the extractspot by comparison with the intensityof fluorescence of spotsC, 0 and E from the standard solution.

8.7 Confirmation of the Identity of aflatoxin 8,

Confirm the identity of the aflatoxin 8, in the extract by the.presumptive test with sulfuricacid (see 8.7.1)and, if the resultof this test is positive, by the actualconfirmation test (8.7.2). If

7

18 13427 : 1892180 8861 : 1·887

the resultof the presumptive test with sulfuricacidIs~ive,there is no need to proceed with the actualconfirmation since,in this case, no aflatoxin 8, il present.

8.7.1 Presumptive test with sulfuric acid

Spray the sulfuric acid (6.13) on to the chromatogram 0b­tained in 8.6.1 or 8.5.2. The fluorescence of the aflatoxin B,spots shall tum from blue to yellow underultravioletlight.

8.7.2 Confirmation te.t : formation of aflatoxinB1-hemiacetal (aflatoxin 82a)

In the case of simpleandonlyslightlypigmented feeds, usetheone-dimensional thin-layer chromatographic method describedin 8.7.2.1. In the ca. of simple pigmented feeds, mixed feeds,or in cases of doubt, use the two-dimensional thin-layerchromatographic method described in 8.7.2.2.

8.7.2.1 One-dimensional thin-layer chromatography

Tracea straight line on a plate (8.7) to divideit into two equalparts. Apply on each part, 20 mm from the lower edge and atintervals of 15 mm, the volumes indicated below of the stan­dardaflatoxin 8 , solutionand the extract :

- 25 III of the standard aflatoxin B, solution (5.14);

- a volumeof the extractobtained in8.4.2 containing ap­proximately 2,5 ng of aflatoxin 8,;

- 25 ~I of the standard aflatoxi~ B1 solution (6.14) and,superimposed on it, a volume of the extract obtained in8.4.2 containing approximately 2,6 ng of aflatoxin 8,.·

Apply to one of the two halves-of the plate, superimposed onthe spots previously applied, 1 to 2 ~I of the trifluoroacetic acid(5.11). Dry in a stream of air at ambient temperature.

Develop the chromatogram, in the dark, using one of thedeveloping solvents (6.8). The choice of the solvent shall bemade beforehand. The solvent system shall ensure that theaflatoxinB1-hemiacetal (aflatoxin82a) is clearty separated frominterfering substances. The solvent front shall ·travel about120mm.

Allow the solvents to evaporate in the dark, and then spraysulfuric acid (5.13) on to the part of the plate not previouslytreated with the trifluoroacetic acid. Examine the' plate underultraviolet light.

.The identity of aflatoxin 8, is confirmedif

a) the Rf value of the aflatoxin 8, derivative originatingfrom the extract corresponds with that from the standardsolution;

b) the aflatoxinB, derivative originating from the standardsolution, superimposed on the extract, has a fluorescencemore intense than the aflatoxin B1 derivative originatingfrom the extract.

Since fluorescent spots from the extract, having the 11I118 Rfvalue 81 the aflatoxin B,-hemiacetal, might lead to • false

· IS 13427 : 1882180 88&1 : 1887

positive interpretation of the chromatogram, their presenceshouldbechecked on the part of the platetreatedwith sulfuricacid.

In cases of doubt, confirmation by two-dimensional thin-layerchromatography (8.7.2.2) shall be used.

8.7.2.2 Two-dimensional thin-layer chromatography (seefigure 3)

8.7.2.2.1 Application of the solutions

Trace two straight lines on a plate (6.7), parallel to two con­tiguous sid. 160 mm from eachside), to establish the limit ofmigration of the solvent fronts. Apply the following solutionson the plate using capillary pipettes or microsyringes :

- at point A: a volume of purified extract from thesample, obtained in 8.4.2, containing about 2,5 ng of afla­toxin 8" and a drop (1 to 2 IJI) of the trifluoroacetic acid(5.11);

- at points 8 and C : 25 IJI of the standard aflatoxin B1

solution (5.14), anda drop of the trifluoroaceticacid (5.11).

Dry in a streamof air at ambient temperature.

8.7.2.2.2 Development

Develop the chromatogram in direction I, in the dark, usingthedeveloping solvent (5.6.2) (1 cm layer in an unsaturated tank)until the solvent front reaches the limit line. Remove the platefrom the tank and allow to dry, in the dark, at ambienttemperature, for 5 min.

Then develop the chromatogram in direction II, in the dark,using the developing solvent (5.6.1) .(1 cm layer in an unsatu­rated tank) until the solvent front reaches the limit line. Re­move the plate from the tank and allow to dry at ambienttemperature.

8.7.2.2.3 Interpretation of the chromatogram

Examine the chromatogram under ultraviolet light from thelamp (6.8) and check for the following features :

8) appearance of a blue fluorescent spot of aflatoxinB1-hemiacetal, andsometimes a weakbluefluorescentspotof aflatoxinB1which hIS not reacted with the trifluoroaceticacid, originatingfrom the stlJldard solution appliedat pointC (migration in direction I) and from the standard solutionappliedat point B Imigration in direction II);

b) .ppearance of spots similar to those described in 8),originatingfrom the sample-extract appli~ at point A. Theposition.of these spots is definedby those originating fromthe standard solution appliedat points Band C. The inten­sities of fluorescence of the aflatoxin B1-hemiacetal spotsoriginatingfrom the extract and from the standard solutionapplied at points 8 and C should be c;omparable.

8.8 Number of determinations

Carryout two determinations on the same tilt umple.

8

9 Expression of results

9.1 Method of calculation and formulae

9.1.1 Vlsu.1 me.surements

Theaflatoxin B1content, expressed in micrograms per kilogramof -.ample, is equal to

C x V1 x V3

m x V2

where

C is the concentration, in micrograms of aflatoxin B1 permillilitre, of the standard solution (5.14) (approximately0,1 IJg/ml);

m is the mass, in grams,of the test portion correspondingto the volume of extract subjected to column clean-up(10,0 g);

V1 is the final volumeof the ~xtract, in microlitres, takin9into account anv dilution that was necessary;

V2and V3 are, respectively, the volumes, in microlitres,ofthe extract and of the standard aflatoxin B, solution (5.14),applied on I the plate, having similar intensities offluorescence.

9.1.2 Fluorodensitometric measurements

TheaflatoxinB1content, expressed in micrograms perkilogramof sample, is equal to

m1 x V1

m x V2

where

m is the mass, in grams, of the test portion correspondingto the volume of extract subjected to column clean-up(10,0g);

m1 is the mass, in nanograms, of aflatoxin B1 in the ex­tract spot (taking irto account the volume V2), deducedfrom the measurements;

V1 is the final volumeof the extract, in microlitres, takinginto account any dilution that was necessary;

V2 is the volume, in microlitres, of extract appliedon theplate (10or 20 J.1I).

9.2 Precision

Three round-robin trials, two of which were carried out at theinternational level 'Nos. 1 and 2), on mixed feeding stuffs(method B) gave the results indicated in the table.

The 11 IIborItories participating in trial 2 also analysed the.mple by method A, its composition being suitable, and ob­tained results very similar to those when using method B, byvisual or fluorodensitometric measurement.

18 13427 : 1882180 88&1 : 1887

10 Te.t report

The test repon shill show the method used (A or B), themethod of determination (visual or fluorodensitometricmeasurement)andthe r88ul18 obtained. It shall also mention alloperating conditions not specified in this International Stan-

dard, or regardedIIoptional, IIwell.sanycircumet8nCeS thetmay have influenced the results.

The test report shill include all the information necesaery forthe complete identific8tionof the sample.

P.remeter 88mp'.1 2 3

Number of laborltories 23 11 13

Mean 162,7 ~g/kg 25,4 ~g/kg 13,4~g/kg

Standard deviationof repeatabHity (s,) 18,9J,lg/kg 2,7 ~g/kg 1,7 J.lg/kg

Coefficient of variation ofrepeatability 10 % 11 " 13 "Repeatability (2,83s,)· 47,8 J.lg/kg 7,8 J,lg/kg 4,8 J,lg/kg

Standard deviationof reproducibility (sR) 46,2 ~g/kg 8,8 ~g/kg 4,0 ~g/kg

Coefficient of variationof reproducibility 28% XI ". 30"Reproducibility (2,83 sR) 128,0 ~g/kg 19,2~g/kg 11,3~g/kg

Dimensions in millimetres

II

I I~EIto!C

~I•

la I

, A . B-t--------------~--M_--j

I-l~

~20_ - 130 50=-- -- - -

oN

C)N...

Figure 1 - Appllc8tlon of soludon•

•

IS 13427 : 1882ISO 8861 : 1987

II

\ tE\ I\ fD\~lcr,1\I \I \I \I \I \ ~

tJtlCi I \_~~_ t4

~--r--'PI Q\I \I \

.....

Figure 2 - Interpretation of the chromatogram

-----+---.... C

I

140

Flgur. 3 - Conflrm.tlon te.t

10

Dimensions in millimetres

........

60

Reprograpby Unit, BIS, New Delhi, India

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Revilioa 01 ladiaD Stud.rd_

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AaaeDdaeDti 1IIaed SIDce PabUcatl••

Amend No. Date of Issue

BURBAU OF INDIAN STANDARDS

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