College of Medical Technology

Applied Pharmacognacy (DT307)- third year

Drug Technology Department

Determination of pesticide residues

By
Nesrin Atiah Elhussadi

2020
1. Introduction

2. Determination of phosphates

3. Qualitative and quantitative determination of organochlorine pesticides

4. Analysis of esters of organophosphorus compounds

5. Determination of specific pesticide residues in medicinal plant

material

6. Determination of specifi c organochlorine, organophosphorus

:
Medicinal plant materials may contain pesticide residues, which accumulate as a result of agricultural practices, such as spraying,
treatment of soils during cultivation and administration of fumigants during storage. It is therefore recommended that Potentially
hazardous contaminants and residues in herbal medicines every country producing medicinal plant materials should have at least
one control laboratory capable of performing the determination of pesticides using a suitable method.
Classification of pesticides
Different classifications of pesticides exist. A classification based on the chemical composition or structure of the pesticide is the
most useful for analytical chemists, for example:
1. • chlorinated hydrocarbons and related pesticides: hexachlorocyclohexane (HCH) or benzene hexachloride (BHC), lindane,
methoxychlor
2. • organophosphorus pesticides: carbophenothion (carbofenotion), chlorpyrifos and,methylchlorpyrifos, coumaphos
(coumafos), demeton, dichlorvos, dimethoate,, ethion, fenchlorphos (fenclofos), malathion, methyl parathion, parathion
3. • carbamate insecticides: carbaryl (carbaril)
4. • dithiocarbamate fungicides: ferbam, maneb, nabam, thiram, zineb, ziram
5. • amino acid herbicides: glyphosate
6. • inorganic pesticides: aluminium phosphide, calcium arsenate
7. • pesticides of plant origin: tobacco leaf extract, pyrethrum fl ower, and pyrethrum,extract; derris and Lonchocarpus root and
rotenoids.
Determination of phosphates .2
:The phosphomolybdate method

It is based on the reaction of phosphate ions with ammonium molybdate to form a molybdophosphate complex, which is
subsequently reduced to form a strongly blue-coloured molybdenum complex. The intensity of the blue colour is measured
spectrophotometrically. This method is applicable for the determination of any phosphates that have undergone a prior
separation procedure. Extracts of most uncontaminated materials contain about 0.05–0.1 mg/kg of phosphorus. Therefore, no
contamination with organophosphate pesticides can be assumed for results in this range.

Ammonium
molybdate

Phosphate
ions
MOLYBDOPHOSPHATE

Spechtrophotometry
800-900 nm
Apparatus
The determination is made with a spectrophotometer capable of measuring absorbance at 820 nm using an absorption cell with a
path-length of 1 cm.
Method
Place 7 ml of the solution obtained after combustion in a calibrated 10-ml testtube. Add 2.2 ml of sulfuric acid (300 g/l) TS and
mix the solution well. Add 0.4 ml of ammonium molybdate (40 g/l) TS and swirl the mixture. Then add 0.4 mlof
aminonaphtholsulfonic acid TS and swirl again. Heat the solution to 100 °C for 12 minutes (― 2 minutes), cool, and transfer a
portion to a 1-cm cell. Measure the absorbance at 820 nm using water in the reference cell. Prepare standard dilutions with a
known content of phosphate and measure the absorbance as described above. Plot the absorbances against the phosphate content of
the dilutions in μg per ml and interpolate the phosphate content of the solutions of the material tested

7ml of soln
After
combustion 2.2 ml of H2SO4
10 ml TT

Cool and transfer to 1cm cell

Messure at 820 nm

Heat 100 °C for 12 min

0.4ml of ammonium molybdate 0.4 mlof aminonaphtholsulfonic acid

Qualitative and quantitative determination of organochlorine pesticides

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, using a high-speed mixer. Filter the homogenized mixture through a porcelain
filter (Buchner funnel, diameter 70 mm) 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 fi ltrate in ml.

98ml of water

20gm
200ml of acetone
Measure the volume

With in 1minute
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 fi ltrate, then add 100 ml of dichloromethane R. Shake vigorously for 5 minutes, allow the phases to separate and discard the lowe
(aqueous) layer. Dry the acetone-dichloromethane phase, transfer it to a 500-ml conical fl ask, add 25 g of anhydrous sodium sulfate R and swir
occasionally. Next, filter the solution into a 500-ml flask with a ground-glass 75 stopper using a glass funnel (diameter 100 mm) containing purif
ed glass-wool and anhydrous sodium sulfate R. Rinse the separating funnel, the conical fl ask and the glass funnel twice with 10 ml of ethy 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 a 30–40 °C.
Expel the remaining solvent in a gentle stream of air. To purify by gel chromatography, macerate 50 g 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, diamete 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 fla with 5 ml
of ethyl acetate R. Add 2 g of anhydrous sodium sulfate R, swirl gently and add 5 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 ml of the filtrate on
to the gel column. Elute with the elution mixture at an average rate of 5 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 Discard the fi rst
fraction (about 100 ml) containing the impurities. Collect the organochlorine pesticides appearing in the next eluate (about 70 ml) in a flask with a
ground-glass stopper. Add l0 ml 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 fl ask and carefully evaporate the solution to about 1 ml (do not allow
to become completely dry) Calculate the amount of plant material, in grams, in the purifi ed extract using the following formula:
V
---- sample weight in g
590
where V = volume of fi
ltrate.
Determination by gas chromatography
First separation system Use a vitreous silica column, 30 m long with an internal diameter of 0.25 mm,
packed with a chemically bound phase of 5% phenyl, 95% methyl-polysiloxane. Use the following temperature programme:
•heat at 60 °C for 0.5 minutes; increase the temperature at a rate of 30 °C per minute to 160 °C and maintain this temperature for 2
minutes;increase the temperature at a rate of 2 °C per minute to 250 °C and maintain this temperature for 5 minutes.
Use a “split/split-free” Inject a volume of 1 μl at a rate of
30 seconds (“split-free”). The detector temperature should be 300 °C.
Second separation system
Use a vitreous silica column, 15 m long with an internal diameter of 0.25 mm, packed with a chemically bound phase of 7%
cyanopropyl, 7% phenyl, 86% methylpolysiloxane.
Use the following temperature programme:
•heat at 60 °C for 12 seconds; increase the temperature at a rate of 30 °C per minute to 180 °C and maintain this temperature for 1
minute; increase the temperature at a rate of 2 °C per minute to 250 °C and maintain this
temperature for 5 minutes. Use an on-column injector to inject a volume of 1 μl of the sample solution. The detector temperature
should be 300 °C.
Use the “external standard” method for the qualitative and quantitative evaluation of the organochlorine pesticides in the test
solutions with reference solutions
of the following pesticides: and hexachlorocyclohexane (HCH); hexachlorobenzene; quintozene; aldrin; dieldrin; endrin; and
endosulfan;
endosulfan sulfate; heptachlor, heptachlorepoxide; camphechlor; TDE, DDE and DDT (both o,p’- and p,p’-
isomers); methoxychlor. 77
Measure the peak height of the pesticides obtained in the chromatograms and calculate the concentration of the residues in
using
mg/k the following
formula:
ht × 10 wr

w hr

where ht = peak
height obtained for
the test solution in
mm
w = quantity of
sample in the
purified extract (g)
Wr= quantity
Analysis of pesticide
of esters in ng in thecompounds
of organophosphorus reference solution injected
hr = peakmost
Although height obtained for the
organophosphorus reference
compounds solution
undergo in mm.
rapid decomposition, Member States may elect to test for them because of their
harmful nature if present in significant concentrations. Testing may be more relevant in the case of herbal medicines used at high
concentrations and frequency.
The extraction and the clean-up procedures can be performed as described above
Determination of specific pesticide residues in medicinal plant
material
General recommendations
For the total determination, mix thoroughly 1 kg of plant material. In order to obtain reliable chromatographic results, do one or more of the
following:
• repeat the separation using another column;
• use a different separation system;
• use a different detector system;
• apply a coupling technique;
• prepare a derivative;
•perform chromatography with a mixture of the sample and a reference
substance;
• change the sample preparation;
•use a fractionated elution during the column-chromatography clean-up
procedure of the plant extract and test every fraction by chromatography; and
• compare the distribution coeffi cient of the material with that of a reference
substance.
Prior to the quantitative determination of the material to be tested, check whether there is a linear relationship between the values obtained for
the reference substance and its concentration over the range 0.1–2 times the standard concentration. Otherwise, prepare another concentration
range or evaluate the results using a reference curve. n Store the reference solutions protected from light to prevent decomposition. Us glass
vessels closed with glass stoppers and keep them in a container saturated with the solvent employed to avoid any increase in concentration due
to evaporation Check the loss by evaporation by interim weighing of the vessels.