WHO guidelines

Determination of pesticide residues

➢ Chlorides

➢ Phosphates

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Content

WHO guidelines for the standardization of crude drugs and extracts

Determination of pesticide residues

➢ Chlorides

➢ Phosphates

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Objectives

• At the end of this lecture, student will be able to

✓ Discuss the principle and procedure involved in the determination

of pesticide residues like

➢ Chlorides

➢ Phosphates

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Determination of pesticide residues

Methods for the determination of pesticide residues

✓ Chromatography (mostly column and gas), Coupled with MS

✓ Samples are extracted by a standard procedure

✓ These techniques are not universally applicable

✓ Some pesticides –

➢ Satisfactorily carried through the extraction and clean-up procedures

➢ Recovered with a poor yield & some are lost entirely

➢ After chromatography, the separations may not always be complete

➢ Pesticides may decompose or metabolize

Determination of pesticide residues
Methods for the determination of pesticide residues
✓ Spectrum of pesticides to be tested –

➢ Dependent on the specific pesticides used on the herbal material
➢ History of use of persistent pesticides in the region
➢ If the pesticide is known or can be identified- established method

for particular pesticide residue should be employed

Determination of pesticide residues
General aspects of analytical methodology
✓ Samples should be tested as quickly as possible after collection

✓ If stored – should be preferably in airtight containers under
refrigeration

✓ Water content – limited to 15% and below
✓ Light cause degradation of many pesticides
✓ Type of container or wrapping material used should not interfere
✓ Solvents and reagents used

➢ Purified solvents or to be distilled

➢ Blank determinations – should be carried out
✓ Simplest and quickest procedure should be used
✓ Process of concentrating solutions – avoid loss of pesticide residues

Determination of pesticide residues
Determination of total chlorine and phosphorus
Most pesticides contain organically bound chlorine or phosphorus
Procedure
Preparation of samples

Herbal material to fine powder

Extract with a mixture of water and acetonitrile
(Most pesticides soluble in this mixture, while most cellular constituents are sparingly soluble and hence removed)

Then transfer pesticide to light petroleum

Pesticides with chlorine – further purification required
Pesticides with phosphorus – further purification by column chromatography

Eluted with mixtures of light petroleum and ether

Determination of pesticide residues

Preparation of the column

✓ Use Florisil R grade 60/100 PR

✓ Activated at 650 °C

✓ Prepare a Florisil column (external diameter 22 mm) with 10 cm of

activated Florisil topped with about 1 cm of anhydrous sodium sulfate

✓ Pre-wet column with 40–50 ml of light petroleum

✓ Place a graduated flask under the column to receive the eluate

Determination of pesticide residues
Method

Grind the material

Place 20–50 g of the ground sample into a blender

Add 350 ml of acetonitrile with a water content of 35%

Blend for 5 minutes at high speed, filter

Transfer the filtrate to a 250-ml measuring cylinder and record the volume

Transfer the measured filtrate to separating funnel + 100 ml of light petroleum

Shake vigorously

Determination of pesticide residues
Method

Add 10 ml of sodium chloride (40%) and 600 ml of water

Separating funnel in horizontal position, mix vigorously for 30–45 seconds

Allow to separate, discard the aqueous layer

Wash the solvent layer with two 100-ml portions of water

Transfer the solvent layer to a 100-ml glass-stoppered cylinder

Record the volume

Add about 15 g of anhydrous sodium sulfate & shake vigorously

Determination of pesticide residues
Method

Transfer the extract directly to a Florisil column

Flow rate not more than 5 ml/minute

Rinse the cylinder with two 5 ml of light petroleum & transfer to the column

Elute at the same rate with 200 ml of ether/light petroleum (TS1)

Change the receiver & elute with 200 ml of ether/light petroleum (TS2)

Again change the receiver and elute with 200 ml of ether/light petroleum (TS3)

Evaporate each eluate to a suitable volume for further testing

Determination of pesticide residues
Method

First elute

Chlorinated pesticides (aldrin, DDE, TDE, HCH, heptachlor, heptachlor

epoxide, lindane, methoxychlor), polychlorinated biphenyls (PCB), and

phosphated pesticides (carbophenothion, ethion and fenchlorphos)

Second elute

Chlorinated pesticides (dieldrin and endrin) and phosphated pesticides

(methyl parathion and parathion)

Third elute

Phosphated pesticide (malathion)

Determination of pesticide residues
Combustion of the organic matter

Combustion of the organic matter in oxygen – preparatory step for the
determination of chlorine and phosphorus

Pesticide extracted from sample and purified

Extract is concentrated, evaporated to dryness

Transferred to a sample holder

Burnt in suitable conical flask flushed with oxygen

Gases produced during combustion – absorbed in a suitable solution

Absorbed chlorine – determined as chloride (colorimetry)

Absorbed phosphorus – orthophosphate (colorimetry)

Determination of pesticide residues
Combustion Equipment

✓ Conical flask of borosilicate glass

✓ Stopper fused with platinum wire about 1 mm in diameter

✓ Free end of the wire attached to platinum gauze – holding the sample

Sample holder for chlorine-containing residues

✓ Halide-free filter-paper – solid

✓ Cone made from cellulose acetate film – liquid

Sample holder for phosphorus-containing residues

✓ Halide-free filter-paper

Combustion of chlorine-containing residues

Combustion of phosphorus-containing residues

Determination of pesticide residues
Determination of chloride

Procedure

Equipment

✓ Spectrophotometer at 460 nm

✓ Path-lengths of 2 cm and 10 cm

Method
15 ml of solution obtained after combustion in a 50-ml conical flask

Add 1 ml of ferric ammonium sulfate (0.25 mol/l), 3 ml of mercuric thiocyanate

Allow to stand for 10 minutes, transfer to a 2-cm cell

Measure the absorbance at 460 nm using water as reference cell

Determination of pesticide residues
Standard

Sodium chloride with 5 μg of chloride per ml

0 ml, 2 ml, 4 ml, 6 ml, 8 ml and 10 ml into a series of 50-ml conical flasks

Dilute to 15 ml with water

Develop the colour & measure the absorbance at 460 nm

Plot the absorbances against the chloride content of the dilutions in μg/ml

Interpolate the chloride content in the solution of the material under test

Determination of pesticide residues
Determination of phosphates
Procedure

Phosphomolybdate method

✓ Reaction of phosphate ions with ammonium molybdate to form a

molybdophosphate complex

✓ Subsequently reduced to form a strongly blue-coloured molybdenum

complex

✓ Intensity of blue colour measured spectrophotometrically

✓ Applicable for the determination of any phosphates

✓ Naturally occurring phosphates – removed during the clean-up

procedure

Determination of pesticide residues
Equipment

✓ Spectrophotometer, absorbance at 820 nm

✓ Path-length – 1 cm

Method

✓ Place 7 ml of the solution after combustion in a calibrated 10-ml test-tube

✓ Add 2.2 ml of sulfuric acid (300 g/l) & mix

✓ Add 0.4 ml of ammonium molybdate (40 g/l) & mix

✓ Add 0.4 ml of aminonaphtholsulfonic acid & mix

✓ Heat to 100 °C for 12 minutes

✓ Cool & transfer a portion to a 1-cm cell

✓ Measure the absorbance at 820 nm using water in the reference cell

Determination of pesticide residues

Standard

Standard dilutions with a known content of phosphate

Measure the absorbance at 820 nm

Plot absorbances against the phosphate content

Interpolate the phosphate content of the solutions of the material tested

Summary

✓ Chromatography (mostly column and gas), Coupled with MS

✓ Preparation of

➢ Sample

➢ Column

➢ Elution

➢ Combusted material

✓ Determination of chlorides (spectrophotometry) and phosphates

(phosphomolybdate method)

Thank You
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