Quality control methods for

Qualitative and quantitative determination of organochlorine pesticides 
 
Recommended procedure 
 
Preparation of sample 
Place 20 g of powdered plant material (sieve no. 180), accurately weighed, in a 
500-ml beaker (tall form), mix with 98 ml of water and allow to macerate for at 
least 30 minutes. Add 200 ml of acetone R; the resulting volume of extraction 
solvent will be 295 ml. Extract for 5 minutes, while cooling and using a high-
speed mixer. Filter the homogenized mixture through a porcelain filter (Büchner 
funnel, diameter 70mm) fitted with a filter-paper, using a slight vacuum, into a 
250-ml graduated cylinder, allowing the process to last no longer than 1 minute, 
and then measure the volume (V) of the filtrate in ml. 
Method 
Transfer the filtrate prepared as above to a 500-ml separating funnel. Add a 
quantity of sodium chloride R equivalent in grams to one-tenth of the volume of 
the filtrate, then add 100 ml of dichloromethane R. Shake vigorously for 5 
minutes, allow the phases to separate and discard the lower (aqueous) layer. Dry 
the acetone-dichloromethane phase, transfer it to a 500-ml conical flask, add 25 g 
of anhydrous sodium sulfate R and swirl occasionally. Next, filter the solution 
into a 500-ml flask with a ground-glass stopper using a glass funnel (diameter 
100 mm) containing purified glass-wool and anhydrous sodium sulfate R. Rinse 
the separating funnel, the conical flask and the glass funnel twice with 10 ml of 
ethyl acetate R. Add 5 ml of 2,2,4-trimethylpentane R, and concentrate the crude 
extract to about 2 ml in a rotary vacuum evaporator in a water-bath at 30-40°C. 
Expel the remaining solvent in a gentle stream of air. 
To purify by gel chromatography, macerate 50g of suitable beads (e.g. S-X3 bio-
beads) in an elution mixture of cyclohexane R and ethyl acetate R (1:1) and pour 
them into a chromatographic column (length 600 mm, diameter 25 mm) adapted 
for use with a vacuum pump. Rinse the gel bed with the elution mixture under 
air-free conditions. Dissolve the extract in the flask with 5.0 ml of ethyl acetate R. 
Add 2 g of anhydrous sodium sulfate R, swirl gently and add 5.0 ml of 
cyclohexane R. Filter the completely dissolved crude extract through a rapid 
filter into a 10-ml test-tube with a ground-glass stopper and close the tube 
immediately. Then transfer 5.0 ml of the filtrate onto the gel column. Elute with 
the elution mixture at an average rate of 5.0 ml/minute. Plant material 
components leave the gel column first, followed by the active ingredients of 
pesticides. Fractionation must be determined for each column, using appropriate 
reference substances. 

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Quality control methods for medicinal plant materials 
Discard the first fraction (about 100 ml) containing the impurities. Collect the 
organochlorine pesticides appearing in the next eluate (about 70ml) in a flask 
with a ground-glass stopper. Add l0ml of 2,2,4-trimethylpentane R and 
concentrate the solution to about 5 ml in a rotary vacuum evaporator and a 
water-bath at 30-40°C. Pipette another 5 ml of 2,2,4-trimethylpentane R into the 
flask and carefully evaporate the solution to about 1 ml (do not allow to become 
completely dry). 
Calculate the amount of plant material in g in the purified extract using the 
following formula: 
g
in 
weight 
sample
x 
590
V
where V = volume of filtrate. 
To purify further, transfer 1 g of previously deactivated silica gel for column 
chromatography (70-230 mesh) containing 1.5% of water, to a chromatographic 
column (length 25 cm, internal diameter 7 mm). Put 10 mm of anhydrous 
sodium sulfate R on top of the content of the column and cover with purified 
glass-wool. Before use rinse the column with 5 ml of hexane R. Allow the solvent 
to reach the surface of the column filling, then transfer quantitatively, by means 
of a pipette, the purified extract obtained by gel chromatography from the flask 
to the prepared silica gel column and rinse with 1 ml of hexane R. Set the flask 
aside for subsequent elutions. 
Using a 10-ml volumetric flask as the receiver, elute any residues of 
polychlorinated biphenyls from the column with 10 ml of hexane R (eluate 0). 
Add 2 ml of an elution mixture composed of toluene R/hexane R (35:65) to the 
flask and swirl. Quantitatively transfer the solution to the column. Using 
another 10-ml volumetric flask as the receiver, elute the majority of the 
organochlorine pesticides from the silica gel column using 6 ml of the same 
elution mixture. Dilute the contents of the flasks to volume with the elution 
mixture (eluate 1). 
Rinse the flask with 2 ml of toluene R and transfer it quantitatively to the 
column. Collect the eluate in a third 10-ml volumetric flask. Add 8 ml of toluene 
R to the flask, swirl and transfer the solution to the silica gel column; elute the 
remaining organochlorine pesticides using the same receiver. Dilute the contents 
of the flask to volume with toluene R (eluate 2). 
Evaluate the test solutions by capillary gas chromatography using an electron 
capture detector (ECD). Confirm the findings obtained for the main column (first 
separation system) with a second capillary column of different polarity (second 
separation system). 

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Quality control methods for medicinal plant materials