This document presents information on analyzing organophosphorus and organochlorine pesticides. It describes the effects of pests and insects on various foods, the history of pesticide use in agriculture, and provides details on multi-residue methods for determining these pesticides. The methods involve extracting samples using solvents like acetone and dichloromethane, clean up using chromatography columns, and analyzing extracts using gas chromatography with detectors like FPD or ECD. Calibration is done against standard solutions to quantify residues in samples.

1. Presented by: Facilitated to:
Mr. L. Sanathoiba Singha
M. Pharm 1st Semester
Department of Pharmaceutical Analysis.
Mrs. Akkamma H. Godihal
Assistant Professor
Department of Pharmaceutical Analysis.
A presentation on
Karnataka College of Pharmacy
Bengaluru-64, Karnataka.
1

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EFFECTS OF PESTS AND INSECTS ON VARIOUS FOOD
• Damage to existing crops.

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• Damage to crops stored after harvest.

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• Damage to food products.

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• Damage to fresh foods like fruits and vegetables.

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USE OF PESTICIDE IN AGRICULTURE
• In China as early as 3000 BC a mixture of lime, wood ash and chalk was used as insecticide for crops.

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• Leonardo da Vinci noted that harmful insects can be destroyed with arsenic.

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• In England, as early as the beginning of 19th century, lime-sulfur solution was applied to fruit trees to protect
them against powdery mildew.

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 It was only in the 20th century that organic chemistry started interacting with agriculture.
 It was in the 1930s that the first modern pesticides like DDT (Dichlorodiphenyltrichloroethane), BHC (Benzene
hexachloride), organophosphates, etc were introduced.

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• Main classes of pesticides:
 Herbicides • Insecticides

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• Fungicide

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• Pesticide use has increased 50-fold since 1950 and around 2.5 million tons of industrial pesticides per year
are used nowadays.
Figure 1. World market of pesticides since 1990. Values are expressed in millions of
U.S. dollars. (From European Crop Protection Association (ECPA) Review 2005–2006,
http:==www.ecpa.be.)

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PESTICIDE CYCLE

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• Pesticides may ultimately affect non-target organisms such as humans, bees, wildlife, etc.

15. ORGANOPHOSPHORUS PESTICIDE ANALYSIS
INTRODUCTION:
P
O
R 1 O O R 3
O R 2
• Esters of phosphoric acid.
• Acts on acetylcholinesterase enzyme.
• Research on organophosphorus compounds is marked by
the works of Lassigne (1820); investigated the interaction
of alcohol and phosphoric acid.
• Lange and Krueger (1932) were the first to report the
strong bioactivity of organophosphorus compounds.
• Kept as secret during World War II for possible use in
chemical warfare.
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• Used as:
 Insecticides e.g. fenitrothion and chlorpyrifos Herbicides e.g. glyphosate and glufosinate
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MULTI RESIDUE METHOD FOR DETERMINATION OF ORGANOPHOSPHORUS PESTICIDES
Apparatus
A.
i. A gas chromatograph equipped with flame photometric detector operated in phosphorus mode (FPD-P), 526 mm
filter.
ii. Operating conditions:
 Injector temperature 220°C
 Baseline noise should be < 2%
iii. Columns :
 SPB-5 (Supelco, Bellefonte, PA) or any equivalent 30m x 0.53mm
 N2 carrier gas
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B. Chromatographic cleanup columns:
 Ready to use Extrelut-3, needle is fixed at column end as flow regulator.
 Glass column 30cm x 20 mm with glass septum (Carbon-celite cleanup).
C. Chopper grinder.
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D. High speed blender.
E. Rotary vacuum evaporator.
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Reagents
i. Reference standard solutions: prepared using heptane or n-hexane (benzene is added if solubilization is
difficult)
ii. Solvents: Acetone, acetonitrile, benzene, dichloromethane, methanol, n-hexane (All HPLC grade).
iii. Silanized glass wool.
iv. Celite 545.
v. Active carbon.
vi. Sodium Chloride.
vii. Cotton wool washed with acetone and n-hexane.
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Pesticide standard solutions:
i. Stock solutions of 1mg/ml using ethyl acetate.
ii. Working solution of 0.5μg/ml.
Extraction:
Foods are divided into three main groups according to moisture and fat content-
i. Group I (Vegetables and fruits):
 50g of chopped sample is added into high speed blender jar.
 100ml of acetone is added.
 Blended for 2 mins at high speed.
 Filtered with Buchner funnel.
 Washed with 50ml acetone.
 Extract brought to 150-200ml volume with acetone : water
(2:1).
ii. Group II (Milk):
 100ml milk is taken in a separatory funnel.
 150ml acetone is added.
 Extracted first with 100ml methylene chloride.
 Then again with 50ml of same.
 Extract is dried over anhydrous sodium sulfate at 50-60°C,
under reduced pressure.
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iii. Group III (Grains):
 50gm chopped sample into high speed blender jar.
 50 ml distilled water added.
 Filtered with Buchner funnel.
 Washed with 50ml acetone.
 Extract brought to 150-200ml volume with acetone : water
(2:1).
Partition
• For all groups half the volume of food extract equivalent to 25g of sample is placed in a separatory
funnel.
• 100ml of dichloromethane, 100ml of acetone and 10g of sodium chloride is added.
• Shaken vigorously till most sodium chloride is dissolved.
• Layers are allowed to separate.
• Aqueous layer is transferred to a second separatory funnel.
• Organic layer is dried through sodium sulfate.
• 200ml of dichloromethane is added to the second separatory funnel.
• The organic layer is dried.
• All organic layers are collected and concentrated just to dryness in rotary vacuum evaporator.
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Chromatographic column clean up
i. Group I and II
 Glass column is filled with 2g celite.
 4g of carbon celite (1:4) is added.
 Topped with glass wool plug.
 Column is washed with 20ml benzene.
 Sample is transferred quantitatively with small portions of benzene.
 Pesticide is eluted with 60ml of acetonitrile-benzene (1:1).
 Concentrated just to dryness in rotary vacuum evaporator.
 Suitable amount of benzene is added and analyzed by GC.
ii. Group II
 Sample is transferred quantitatively to disposable Extrelut-3 with 3ml of n-hexane.
 Solution is allowed to drain into filling material.
 Waited for 10mins to obtained even distribution.
 Eluted three times with 5ml acetonitrile-n-hexane (1:1).
 Concentrated just to dryness in rotary vacuum evaporator.
 Suitable amount of benzene is added and analyzed by GC.
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Determination
Residues are quantified by height or area measurement from solution of known concentration of authentic
compounds.
Compounds Column
SPB-5 SP2250-SP2401
Acephate 0.26 0.97
Chlorpyriphos 1.18 0.82
Ethion 1.73 1.09
Monocrotophos 0.64 0.9
Paraoxon 1.06 1.08
Parathion 1.19 19
Pirimiphos-methyl 1.11 0.97
Table. GC retention times (min) for some organophosphorus insecticides relative to parathion-methyl.
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Calculation
Residue level
mg
kg
=
h1 v1 V
h2 v2 m
𝑐
Where,
h1= peak height of the sample
h2= peak height of the standard
v1= μl of the standard injected
v2= μl of the extract injected
V= final volume of the sample extract (ml)
m= mass in g of the sample
c= concentration in ppm of reference solution.
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ORGANOCHLORINE PESTICIDE ANALYSIS
INTRODUCTION:
• Insecticides
• Characterized by three kinds of chemicals:
 DDT (Dichlorodiphenyltrichloroethane) analogs
 BHC (Benzene hexachloride) isomers.
 Cyclodiene compounds.
• Have a broad spectrum of activity on different families of
insect pests of fruits, vegetables and cotton.
• Most have been banned due to their toxicity owing to its
insolubility in water and very low vapor pressure.
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MULTI RESIDUE METHOD FOR DETERMINATION OF ORGANOCHLORINE PESTICIDES IN
MILK, FISH AND EGGS
Apparatus
i. Refrigerated centrifuge; able to rotate at 3000rpm at
15°C.
ii. SPE (Solid phase extraction) automate
iii. Rotary vacuum evaporator.
iv. Gas chromatograph system with injection device
and electron capture detector.

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v. Capillary column- nonpolar dimethyl polysiloxane, thickness 0.25μg, 50m x 0.32mm.
vi. Pre-column- 1.5m x 0.32mm.
vii. Volumetric pipettes.

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Reagents
• Pesticide standard solution in hexane
• Acetone
• Diethyl ether Petroleum ether
• n-hexane
• Acetonitrile
• Methanol
• Methyl chloride
• Dodecane
• Sodium sulfate anhydrous
• Filter paper
• Nonpolar SPE cartridge
• Polar SPE cartridge
• Mobile phase- Helium 99.99% purity;
filtered for oxygen and water

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General fat extraction
i. For milk:
 50ml milk + 5ml methanol + 0.5g sodium oxalate, in separating funnel and shaken.
 20ml diethyl ether is added and shaken again.
 Repeated with 25ml petroleum ether.
 Centrifuged for 5 mins at 1500rpm for 5 mins.
 Organic phase is transferred into another separating funnel.
 Aqueous phase is extracted twice with 50ml portions of ether and petroleum ether (1:1).
 Combined solvent extracts washed over sodium sulfate anhydrous layer.
 Evaporated using rotary vacuum evaporator at 35°C.
ii. For fish:
 50ml of n-hexane + 20g of sample.
 Mixed and centrifuged for 5 mins at 1500rpm.
 Upper phase is decanted.
 Extraction is repeated with 50ml n-hexane.
 The two extracts are kept together.
 Solvent evaporated at 35°C to 1ml.
 Evaporation is finished with a gentle stream of nitrogen.

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iii. For eggs:
 A beaker is taken.
 15g sand + 15g sodium sulfate anhydrous + 10g sample is taken in the beaker and mixed.
 A glass column with cotton wool swab is taken.
 Sodium sulfate anhydrous is poured till 2cm of the column.
 The contents of the beaker are poured in.
 Eluted with 170ml n-hexane and acetone (2:1).
 Solvent evaporated at 35°C to 1ml.
 Evaporation is finished with a gentle stream of nitrogen.
Pesticide extraction:
• For each product pesticides are extracted from fat by cryogenic extraction.
• 0.5g fat extract in centrifuge tube.
• 3ml of acetonitrile and methylene chloride (75:25) is added.
• Mixed vigorously.
• Centrifuged for 20 mins at 3000rpm and 15°C.
• Upper layer kept supernatant.
• Bottom slowly heated to melt fat.
• Extraction repeated with 3ml of same solvent mixture.
• Solvent evaporated at 35°C to 2ml.
• Evaporation is finished with a gentle stream of nitrogen. (Solution A)

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Clean up
i. C18 SPE:
 Cartridge is processed twice with 5ml petroleum + 5ml acetone + 5ml methanol.
 Eluted to meniscus.
 Solution A is loaded into the cartridge.
 Eluted to meniscus (kept for 3 mins in contact).
 Container of Solution A is washed with 10ml acetonitrile and loaded into the cartridge and eluted.
 Elutant is evaporated at about 35°C with dodecane.
 Diluted in n-hexane. (Solution B)
ii. Florisil SPE:
 Cartridge is processed with 10ml n-hexane and eluted to meniscus.
 Solution B is loaded.
 Eluted with 10ml petroleum ether-diethyl ether (98:2) and 12ml of petroleum ether-diethyl ether (85:15).
 The two fractions are mixed together.
 Evaporated together with 100μl dodecane.
 Final extract dissolved in appropriate volume of n-hexane for GC analysis. (Solution C)

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Gas chromatographic determination
 Operation conditions:
 Helium as mobile phase at 23 psi.
 Initial oven temperature at 100°C, holding time 2 min.
 Initial injector temperature at 50°C.
 Detector temperature at 320°C.
 Injection volume 1μl.
Calculation
Residue level
mg
kg
=
h1 v1 V
h2 v2 m
𝑐
Where,
h1= peak height of the sample
h2= peak height of the standard
v1= μl of the standard injected
v2= μl of the extract injected
V= final volume of the sample extract (ml)
m= mass in g of the sample
c= concentration in ppm of reference solution.

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REFERENCES:
 Tadeo, J. L. Analysis of Pesticides in Food and Environmental Samples. CRC Press, New York. 2008.
3:10:19:20.
 Ministry of Health and Family Welfare, Government of India. fssai Manual of Methods of Analysis of Foods
Pesticide Residues. 2012, Lab manual 11. 70-77:116-120.
 Matolcsy, G, Nadasy, M and Andriska, V. Pesticide Chemistry. Elsevier Science Publishing Co. Inc., New York.
1988 (32). 108:15-16.
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