Quality control methods for

 
Preparation of samples 
Grind or reduce not less than 100g of crude medicinal plant material to a 
moderately fine powder (sieve no. 355/180). The larger the sample size, i.e. 
500g-1kg or more, the greater the possibility of detecting pockets of 
contamination. 
Weigh 50g of the powdered material, transfer to a conical glass-stoppered flask, 
and add 170 ml of methanol R and 30 ml of water. Using a mechanical device, 
shake vigorously for not less than 30 minutes. Filter through a medium-porosity 
filter-paper. If a special clean-up procedure is required (see below), collect 100ml 
of filtrate (A) from the start of flow; otherwise discard the first 50ml and collect 
40ml of filtrate (B). 
In order to eliminate interfering plant pigments use a special clean-up 
procedure: transfer 100 ml of filtrate A to a 250-ml beaker and add 20 ml of zinc 
acetate/aluminium chloride TS and 80 ml of water. Stir, allow to stand for 5 
minutes, add 5 g of a filter aid, such as diatomaceous earth, mix and filter 
through a medium-porosity filter-paper. Discard the first 50ml and collect 80ml 
of filtrate (C). 
Transfer either filtrate B or C to a separating funnel. Add 40ml of sodium 
chloride (100 g/l) TS and 25 ml of light petroleum R, and shake for 1 minute. 
Allow the layers to separate and transfer the lower layer to a second separating 
funnel. Extract twice with 25 ml of dichloromethane R and shake for 1 minute. 
Allow the layers to separate and combine each of the lower layers in a 125-ml 
conical flask. Add several boiling chips and evaporate almost to dryness on a 
water-bath. Cool the residue, cover the flask and keep it for the determination by 
thin-layer chromatography or for a further clean-up procedure by column 
chromatography. 

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Quality control methods for medicinal plant materials 
If necessary, remove further interfering compounds using a column 300 mm 
long with an internal diameter of 10 mm, a stopper and either a medium-pore 
sintered disc or a glass-wool plug. Prepare a slurry by mixing 2 g of silica gel R 
with 10 ml of a mixture of 3 volumes of ether R and 1 volume of light petroleum 
R, pour into the column and wash with 5 ml of the same solvent mixture. Allow 
the adsorbent to settle and add to the top of the column a layer of 1.5 g of 
anhydrous sodium sulfate R. Dissolve the residue from above in 3 ml of 
dichloromethane R and transfer it to the column. Rinse the flask twice with 1-ml 
portions of dichloromethane R and add them to the column, eluting at a rate not 
faster than 1 ml/min. Then add successively to the column 3 ml of light 
petroleum R, 3 ml of ether R and 3 ml of dichloromethane R, and elute at a rate 
not faster than 3 ml/min. Discard the eluates. Add to the column 6 ml of a 
mixture of 9 volumes of dichloromethane R and 1 volume of acetone R and elute 
at a rate not faster than 1 ml/min, preferably without using vacuum. Collect this 
eluate in a small vial, add a few boiling chips and evaporate just to dryness on a 
water-bath. 
Method 
To either of the residues obtained above, add 0.2 ml of a mixture of 98 volumes 
of chloroform R and 2 volumes of acetonitrile R, close the vial and shake 
vigorously until the residues are dissolved, preferably using a vortex mixer. 
Carry out the test as described in section 6, "Thin-layer chromatography", using 
silica gel G as the coating substance and a mixture of 85 volumes of chloroform 
R, 10 volumes of acetone R and 5 volumes of 2-propanol R as the mobile phase. 
Apply separately to the plate 2.5 µl, 5 µl, 7.5 µl and 10 µl of aflatoxin mixture TS, 
then apply three volumes, each of 10 µl, of the sample residues. Further 
superimpose on one of these spots 5 µl of aflatoxin mixture TS. Place the plate in 
an unsaturated chamber and develop. After removing the plate from the 
chromatographic chamber, allow it to dry in air and examine the chromatogram 
in a dark room under ultraviolet light (365 nm). 
Four clearly separated blue fluorescent spots are obtained from the aflatoxin 
mixture. Observe any spot obtained from the solutions of the residues that 
coincides in hue and position with those of the aflatoxin mixture. Any spot 
obtained from the solutions of the residues with the superimposed aflatoxin 
mixture should be more intense than the corresponding spot for the test 
solution, and should show no sign of separation or tailing, which would be a 
sign of dissimilar compounds.