7. Rapid TLC Method of Multi-Mycotoxin Analysis (Modified Tapia Method)
Reagents :i) 4 % KCl (4 g KCl in 100 ml di. water)
ii) 5 N HCl (405.9 ml Conc. HCl in 1 l di. water)
iii) Na2So4 ( anhydrous )
iv) 20 % KOH (20 g KOH in 100 ml di. water)
v) 20 % H2SO4 in ethanol (20 ml Conc. H2SO4 + 80 ml ethanol)
Solvents :
- Acetonitrile
- Hexane
- Chloroform
- Chloroform : Acetone : Water (88 : 12 : 1)
- Toluene : Ethyl acetate : Formic acid (5 : 4 : 1)
Standards :
- Aflatoxin B11 µg/ml in Acetonitrile: Benzene (2 : 98)
- Ochratoxin A 2 µg/ml in Acetonitrile: Benzene (2: 98)
- Zearalenone 50 µg / ml in Benzene
- T-2 toxin 50 µg / ml in Ethyl acetate
Sterigmatocystin Citrinin Oosporein
Procedure :
- Take 25 g sample, add 85 ml acetonitrile, 15 ml 4 % KCl and 2 ml 5 N HCl and blend at high speed for 3 minutes
- Filter through Whatman no. 1 filter paper
- Transfer 50 ml filtrate into a 250 ml separating funnel
- Add 50 ml water, followed by 50 ml hexane and shake well
- Collect the lower layer, add 50 ml hexane again and repeat the above step
- Collect the lower layer into another separating funnel and extract with two 10 ml portions of chloroform
- Drain the chloroform layer through anhydrous Sodium sulphate and evaporate in oven at 50 oC
- Dissolve the residue in 0.2 ml chloroform and spot on TLC plate along with the standards
- Develop plate in chloroform : acetone : water (88 : 12 : 1) in one direction and toluene : ethyl acetate : formic acid (5 : 4 : 1) in the second direction
- Spray zone of spots, corresponding to sterigmatocystin with 20 % aqueous KOH
- Spray zone of spots, corresponding to T-2 toxin with 20 % H2So4 in ethanol and heat at 110C
- View the spots, identify and quantify the toxins as done with individual toxins Effective weight of the sample = 25 x 50 = 12.255 g
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