1.2.1.6 Spot tests for toxic constituents
Nitrates
Reagents
• Diphenylamine
• Concentrated sulphuric acid
• Distilled water
Procedure
Place the material to be tested in a white spot plate. Add 2 to 3 crystals of diphenylamine and a drop of water. Add a drop of concentrated sulphuric acid.
Positive results
Presence of nitrate will produce a deep blue colour.
Cyanogenic glycosides (HCN) in feeds
Procedure
Prepare sodium picrate paper by dipping strips of filter paper into 1% picric acid solution
Take a small amount of ground feed sample in a test tube. Insert a piece of moistured sodium picrate paper in tube, taking care that it does not come in contact with sample. Add few drops of CHCl
Results
(chloroform) and stopper tube tightly If cyanogenic glycosides present in the feed, sodium picrate paper gradually turns orange, then brick red.
Note: Test is delicate and rapidity of change in colour depends upon amount of free HCN present. This test works well with fresh plant materials but relatively less sensitive to dry substances, particularly ground.
Aflatoxin
Reagents
• Methanol
• N-hexane
• Benzene
• Anhydrous sodium sulphate
• Green basic cupric carbonate
Procedure
Take 100 g of dry ground sample in a mixer and add 300 ml of solvent methanol: water (7:3). Mix the content at higher speed for 5 minutes. Allow to settle and then filter through double layer of muslin cloth using vacuum. Take 100 to 150 ml liquid filtrate in a separating funnel. Add 30 ml benzene and shake for one minute and add 200 ml distilled water. Allow to settle and discard lower layer. Place the upper layer into a beaker and evaporate to complete dryness. Re-suspend in 0.5 ml benzene. Spot 50 µl on Whatman filter paper No.
4. Allow the spot to dry and place it under a long wave UV light.
Results
Development of a blue fluorescence colour on it clearly indicates that the sample contains aflatoxins. This method can detect aflatoxins at 10 to 15 ppb. Notice: If the sample has a high fat soluble pigments, add 50 ml hexane to the separating funnel, shake for one minute and add 50-100 ml water. Take lower layer and discard the upper layer. Then proceed further with addition of 30 ml benzene.
Compounds other than aflatoxin present in benzene fraction and may produce fluorescence of their own or partially mask the fluorescence of the aflatoxin. To prevent this problem, collect benzene layer into a 50 ml beaker containing 10 g anhydrous sodium sulphate and
5 g of green basic cupric carbonate. Shake gently and filter through porcelain filter into a 50 ml flask and evaporate to dryness. Re-suspend in 0.5 ml benzene and follow the above procedure.
