Rapid TLC method of Aflatoxin analysis

3. Rapid TLC method of Aflatoxin analysis

(Modified Romer's method)

Reagents :

 i) 0.2 M NaOH (dissolve 8 g NaOH in water and

make up volume to 1 lit)

ii) 0.41 M Ferric Chloride (dissolve 66.5 g anhydrous

FeCl3 in water and make up volume to 1 lit)

iii) 0.03 % H2SO4 (0.3 ml conc. H2SO4 + 999.7 ml

water)

iv) Potassium wash solution (dissolve 1.12 g KOH

and 10 g KCl in water and make up volume to 1

lit)

Solvents :

 i) Acetone

ii) Chloroform

iii) Developing solvent

Chloroform : Acetone : Water (88 : 12 : 1)

Standard : Aflatoxin B1 - 1 μg/ml in Benzene : Acetonitrile (98:2)

Procedure :

 Take 25 g sample in a conical flask, add 100 ml distilled water and

blend for 2 minutes

 Add 150 ml acetone and blend again for 2 minutes

 Filter through Whatman no. 1 filter paper and transfer 75 ml of filtrate

to a conical flask containing 3 g cupric carbonate

 Prepare ferric gel by adding 85 ml of 0.2 M NaOH to 15 ml of 0.41 M

FeCl3. Add this mixture to the flask containing extract and cupric

carbonate

 Mix the contents slowly by swirling movements

 Filter through Whatman no. 1 filter paper

 Take 100 ml of filtrate in a 250 ml separating funnel

 Add 100 ml of 0.03 % H2SO4 and 10 ml of chloroform. Mix the

contents slowly

 Collect the chloroform layer into a 100 ml beaker

 Add again 10 ml of chloroform to the separating funnel and repeat the

above step. Combine both the chloroform extracts

 Take 100 ml potassium wash solution in a separate separating funnel

 Add the chloroform extract to the second separating funnel and mix it

slowly

 Collect the chloroform layer through anhydrous sodium sulfate bed

drop by drop to remove moisture

 Dry the chloroform extract in an oven at 50°C

 Dissolve the dried residue in 0.2 ml chloroform and spot on TLC plate

along with the standard

 Compare the flourescence intensities of the sample and standard spots

and identify the ones matching with each other

 Calculate the aflatoxin content in the following way

          Aflatoxin content (ppb) =                       S x C x D   x 1000

T x W

Where, S = Standard which compares with the sample in

fluorescent intensity

C = Concentration of standard (1 μg / ml)

D = Dilution factor in ml

T = Sample which compares with standard in fluorescent

intensity

                              W = Effective weight [25 x  75 x 100     = 4.286 g]

250 x 175

4. Analysis of Ochratoxin

(By thin layer chromatography)

Reagents : 

Sodium bicarbonate and diatomaceous earth mixture :

Add 25 ml of 5 % aqueous NaHCO3 to 50 g

diatomaceous earth (Celite 545), mix well and store in

tightly closed container

Solvents :

 i) Chloroform

ii) Hexane

iii) Acetic acid : benzene ( 2 : 98 )

iv) Acetic acid : benzene (1 : 99 )

Standard : 

Ochratoxin A 2 μg / ml in acetic acid : benzene (1 : 99)

Apparatus :

 i) Wrist action / horizontal shaker

ii) Hot water (steam) bath

iii) TLC plates (precoated silica gel plates or

equivalent)

iv) Developing tank / chamber

v) UV viewing cabinet

Procedure :

 Take 25 g of sample in a 250 ml glass stoppered conical flask, add

12.5 ml water and mix

 Add 125 ml chloroform and shake for 1 hour

 Filter through Whatman No.1 filter paper and collect the filtrate

 Plug the bottom of a glass column (2 cm x 30 cm) with glass wool,

put 6 g of NaHCO3 - Celite mixture and tamp firmly with a glass rod

 Add 50 ml of chloroform extract to the column and elute until

meniscus reaches top of the NaHCO3 - Celite column

 Wash the column with 70 ml hexane followed by 70 ml chloroform

and discard washings

 Elute Ochratoxin with 100 ml acetic acid : benzene ( 2 : 98)

 Collect the eluate and evaporate on steam bath

 Dissolve the residue in 5 - 10 ml chloroform, transfer to a small vial

( 10 ml capacity) and evaporate on steam bath

 Dissolve the residue in 0.5 ml acetic acid : benzene (1 : 99) by

vigorous shaking

 Spot on TLC plate along with the standard (5, 10, 15 and 20 μ1 or in

any other suitable range)

 Develop the plate using toluene : ethyl acetate : formic acid ( 5 : 4 :

1) in an unequilibrated chamber

 Air dry the plate, view under long wave UV light (365 nm) and

compare the intensity of greenish blue fluorescent spots of the sample

with that of standard spots and identify the spot, matching each other

 Calculate the Ochratoxin A content using the formula

                                   Ochratoxin A μg / kg = S x Y x V

Z x W

Where, S = Volume in μl of ochratoxin A standard spot

comparable to Z μl of sample spot

Z = Volume in μl of sample spot comparable to S μl of

ochratoxin A standard

Y = Concentration of ochratoxin Astandard (2 μg / ml)

V = Volume (μl) of the dissolved residue before spotting

W= Effective weight of the sample

25 x 50

150

Confirmation

Expose the developed plate to NH3 fumes. Greenish blue fluorescence

of Ochratoxin will turn to bright blue.

Reference

AOAC. 1995. Official methods of analysis. 16th ed. Assoc. Off. Anal.

Chem., Washington, D.C.

5. Analysis of T-2 toxin

(By thin layer chromatography)

Reagents : 

i) 30 % ammonium sulphate (dissolve 30 g (NH4)2

SO4 in water and make up volume to 100 ml)

ii) Celite 545

iii) Potassium wash solution (dissolve 1.12 g KOH

and 10 g KCl in water and make up volume to 1 lit)

iv) Sodium sulphate

v) Silica gel

vi) Methanol : H2SO4 ( 1 : 1 v/v )

Solvents : 

i) Methanol : water (1 : 1 v/v)

ii) Chloroform

iii) Diethyl ether

iv) Hexane

v) Benzene

vi) Acetone : Benzene (5 : 95 v/v)

vii) Developing solvent mixture - Toluene : ethyl

acetate formic acid (6 : 3: 1 v/v)

Standard :

T-2 toxin 50 μg / ml in Benzene or diethyl ether

Apparatus : 

i) Wrist action / horizontal shaker

ii) TLC plates (precoated silica gel plates

or equivalent)

iii) Developing tank / chamber

iv) UV viewing cabinet

Procedure :

 Take 50 g of sample in a glass stoppered conical flask

 Add 250 ml of methanol : water (1 : 1) and shake for 1 hour

 Filter using whatman No.1 filter paper and collect 60 ml of extract

into a beaker

 Add 240 ml 30 % (NH4)2 SO4 and stir vigorously for 1 minute

 Add 20 g of celite and stir for 1 minute

 Filter and collect 200 ml of filtrate

 Transfer filtrate to a separating funnel

 Add 10 ml of chloroform and shake vigorously for 1 minute

 Allow the layers to separate and collect the bottom layer into another

separating funnel

 Repeat the extraction with another 10 ml of chloroform

 Combine both the extracts and add 100 ml of potassium wash solution

 Swirl gently for 30 seconds and let layers separate

 Drain the lower chloroform layer through a bed of Sodium sulphate

(in a funnel) to dry and collect 10 ml of clear filtrate

 Column Preparation : Plug the bottom of a glass column ( 2 cm x

30 cm ) with glass wool and add 5 g anhydrous sodium sulphate. Fill

the column to half level with chloroform and add 10 g silica gel.

Wash sides of column with chloroform and stir to eliminate air

bubbles. Drain off chloroform leaving about 7 cm above the upper

level of silica gel. Add 15 g anhydrous sodium sulphate without

disturbing the silica gel. Drain off chloroform to the upper level of

sodium sulphate

 Wash the column serially with 50 ml of diethyl ether and 10 ml of

chloroform and discard the washings

 Mix 10 ml of sample extract with 30 ml of hexane and add to the

column and slowly drain until solvent is about 1 cm above Sodium

sulphate

 Add in succession 30 ml benzene and 40 ml acetone : benzene (5: 95)

and discard both the washings

 Elute T-2 with diethyl ether until 30 ml of eluate is collected and

evaporate the eluate

 Dissolve the residue in 0.5-1.0 ml diethyl ether. Spot on TLC along

with the standard (5-20 μ1 or any other suitable range) and develop

the plate in toluene : ethyl acetate : formic acid (6 : 3 : 1)

 Air dry the plate and spray with methanol : H2SO4 (1 : 1)

 Dry at 110°C for 10 minutes and observe blue fluorescence under

long wave UV light (365 nm)

 Compare the intensities of the blue fluorescent spots of the sample

with those of standard and identify the ones matching each other

 Calculate the T-2 content of sample using the following formula

T - 2 μg / kg =    S x Y x V

                            Z x W

Where, S = Volume in μl of T - 2 standard spot comparable to

Z μl of sample spot

Z = Volume in μl of sample spot comparable to S μl of

T - 2 standard

Y = Concentration of T - 2 standard (50 μg / ml)

V = Volume (μl) of the dissolved residue before spotting

W= Effective weight of the sample

       50 x 60 x 200     x 10

                                                                  250   300    20

References

Romer, T.R., Boling, T.M. and Mc Donald, J.L. 1978. Gas liquid

chromatographic determination of T-2 toxin and diacetoxyscirpenol in

corn and mixed feeds. JAOAC. 61 : 801 - 807.

Rukmini, C. and Bhat, R.V. 1978. Occurrence of T-2 toxin in Fusarium

infested sorghum from India. J. Agric. Food Chem. 26 : 647-649.

6. Analysis of Zearalenone

(By thin layer chromatography)

Reagents :

 i) Aluminium chloride solution (dissolve 20g

AlCl3 6H2O in 100 ml methanol)

ii) Celite 545

Solvents : 

i) Chloroform : water mixture ( 10 : 1 v/v)

ii) Hexane

iii) Chloroform

iv) Diethyl ether

v) Benzene

vi) Acetone : benzene ( 5 : 95 v/v)

vii) Acetonitrile

viii) Developing solvent -

Methanol : Chloroform (5 : 95 v/v) or

Acetic acid : Benzene (5 : 95 v/v)

Standard :

Zearalenone 50 μg / ml in Benzene

Apparatus :

i) Wrist action / horizontal shaker

ii) Hot water (steam) bath

iii) TLC plates (precoated silica gel plates or

equivalent)

iv) Developing tank / chamber

v) UV viewing cabinet

Procedure :

 Take 50 g of sample in a glass stoppered conical flask and add 300 ml

of chloroform : water (10 : 1) and 25 g of celite

 Shake for 1 hour and filter using Whatman No. 1 filter paper

 Column Preparation : Plug the bottom of a glass column

(2 cm x 30 cm) with glass wool and add 5 g anhydrous sodium

sulphate. Fill the column about half full with chloroform and add 10 g

silica gel. Wash sides of column with chloroform and stir to

eliminate air bubbles. Drain off chloroform leaving about 7 cm above

the upper level of silica gel. Add 15 g anhydrous sodium sulphate

without disturbing the silica gel. Drain off chloroform to the upper

level of sodium sulphate

 Transfer 50 ml of sample extract together with 150 ml hexane into the

silica gel column

 Drain until the solvent reaches top of the column and discard the

washings

 Wash the column serially with 150 ml of diethyl ether and 150 ml of

benzene and discard both the washings

 Elute Zearalenone with 250 ml of acetone : benzene ( 5 : 95 )

 Add few silica chips to the eluate and evaporate on steam bath,

preferably under gentle stream of N2

 Dissolve residue in 10 ml of hexane and transfer quantitatively to a

separating funnel

 Repeat the above step for 3 times using 10 ml of hexane each time

 Finally rinse the residue with 10 ml of acetonitrile and add to the

hexane washes present in the separating funnel. Shake well and let

phases separate

 Collect the lower acetonitrile phase into a 100 ml beaker

 Add another 5 ml acetonitrile to the hexane washes present in the

separating funnel and repeat the above step. Combine both the

acetonitrile fractions.

 Evaporate the combined acetonitrile fractions on steam bath ( under

stream of N2)

 Transfer the residue to a small vial (about 10 ml capacity) using about

5 - 10 ml of chloroform and evaporate as in the previous step

 Add about 0.5 ml benzene to the residue and shake vigorously

 Spot on TLC plate (5, 10, 15, 20 μl or other suitable volumes) along

with the standard and develop the plate in methanol : chloroform

(5 : 95) or acetic acid : benzene (5 : 95)

 Air dry the plate and spray the spots with aluminium chloride

solution, heat at 130° C for 5 min and examine under longwave UV

light (365 nm)

 Compare the intensities of the blue fluorescent spots of sample with

those of the standard and identify the ones, matching with each other.

Calculate the Zearalenone content of the sample in the following way

Zearalenone μg / kg = S x Y x V

                                     Z x W

Where, S = Volume in μl of Zearalenone standard spot

comparable to Z μl of sample spot

Z = Volume in μl of sample spot comparable to S μl of

Zearalenone standard

Y = Concentration of Zearalenone standard (1 μg / ml)

V = Volume (μl) of the dissolved residue before spotting

W= Effective weight of the sample

50x   50

         300

References :

AOAC, 1995. Official methods of analysis. 16th ed. Assoc. Off. Anal.

Chem. Washington, D.C.