In this slide contains pesticide used in grains, limits as per FSSAI , general detection method for pesticide in Grains and extraction procedures. Presented by: P.Pavan Kalyan. (Department of pharmaceutical analysis). RIPER, anantapur.

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
Presented by
P. PAVAN KALYAN
(Reg.No:20L81S0705)
Under the guidance of
Dr. P. Ramalingam, Ph.D
Director – R&D Division
METHODS FOR ESTIMATION OF
PESTICIDES IN GRAINS
A Seminar as a part of curricular requirement for 1st year
M. Pharm 1st semester

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Contents
• Introduction.
• Uses and benefits of pesticides in grains.
• Pesticides used in grains.
• Limits of pesticides in grains as per fssai.
• General detection methods for pesticides in grains.
• Extraction procedures for pesticides from grains.

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• Definition
Pesticide is a substance or Mixture of
substance intended for Preventing, destroying, repelling or
lessening the damage caused by the pest.
• A Pest can be a insect,plant pathogen ,weed , bacteria,bird
etc….
• A Pesticide can be a chemical, biological agent, antimicrobial,
disinfectant etc….
• Many chemical Pesticides are poisonous to human and
animals.
Introduction

4. RIPER
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NAAC &
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
Classification of pesticides
Nematicide Phorate,DBCP Namatodes
Molucicide Na
pentachloridephenate
Molluscs
Fungicide Bordeaux mixture Fungus
Types of Pesticides Examples Used to kill
Herbicide Borax,nitrofen Weeds
Insecticide DDT,BHC Insects
Rodenticide Warfarin,zinc sulphate Rodents
Algaecide Coppersulphate,
,endothal
Algae
Bactericide Dichlorophen,oxalinic
acid
Bacteria
Piscicides Triflo methyl nitrophenol Fishes

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
Pesticides work in the following ways
• By blocking the cellular process of the target organisms in a
Purely mechanical way.
Eg :Spray oils , petroleum oils
• By destroying or altering the pests metabolism.
. Eg: cyanide
• By distrusting enzyme process or denature proteins.
Eg: inorganic copper compounds
• By simulting or interfearing with hormones.
Eg: phenoxy herbicides
Working of pesticides

6. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
Uses of Pesticides in grains
• Pesticides are used to control organisms that are considered to be
harmful, or pernicious to their surroundings.
• For example,
they are used to kill mosquitoes, that can transmit potentially
deadly diseases like West Nile virus, yellow fever, and malaria.
• They can also kill bees,wasps or ants, insecticides can protect
animals from illnesses that can be caused by parasites such
as fleas.
• Herbicides can be used to clear roadside weeds, trees, and brush.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
• Herbicides are commonly applied in ponds and lakes to control algae
and plants.
• Pesticides are used in grocery stores and food storage facilities to
manage rodents and insects that infest food such as grains.
• Each use of a pesticide carries some associated risk.
• Pesticide regulatory agencies such as the
United States Environmental Protection Agency (EPA)
Pest Management Regulatory Agency (PMRA) of Canada.
Continued…..

8. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
• They are used in public health programs to control vector born
diseases.
• They are used to protect the stored food grain.
• They protect the standing crops in the field.
• They do not increase the crop yield like fertilizer but by
protecting the crop from pests.
• They can be used to control household pests.
Benefits of pesticides in grains

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
• Several insecticides can be applied directly to grain, including:
• diatomaceous earth
• Malathion
• aluminum phosphide (phosphine)
• magnesium phosphide (phosphine)
• gaseous phosphine
• carbon dioxide
• methyl bromide
Pesticides used in garins

10. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
• There are many classes of synthetic pesticides.
• The main classes consist of
organochlorines,
organophosphates,
carbamates,
pyrethroids.
• Exposure to pesticides can cause acute (short term) or chronic (long
term) effects on animals and humans, especially in the reproductive,
endocrine, and central nervous systems.
synthetic pesticide

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
Class Examples Area of Effect
Organochlorines
DDT, toxaphene,
dieldrin, aldrin
Reproductive, nervous,
endocrine, and immune
system
Organophosphates
Diazinon, glyphosate,
malathion
Central nervous system
Carbamates
Carbofuran, aldicarb,
carbaryl
Central nervous system
Pyrethroids
Fenpropanthrin,
deltamethrin,
cypermethrin
Poorly understood

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
• These are most effective, important and commercially vaible
because these are inexpensive,cause no pollution and no
threat to human health.
• Naturally available agents in Nature
viruses – Nuclear polyhedrosis virus
Bacteria – Bacillus thuringiensis
. Fungi – metarhizium, beauria
• It also include the use of natural extracts obtained from plants
and microbes.
They are – Azadiractin from neem
Nicotine from tobacco
Bio-pesticides

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S.NO NAME OF
PESTICIDE
FOOD
COMODITY
TOLERANCE
LIMIT
mg/kg(ppm)
INSTUMENT
1 Carbaryl Foodgrain,
rice,maiz
0.075 LC-MS
2 Dichlorvos Food grains 0.125 LC-MS
Limits of pesticides in grains as per fssai
3 Hydrogen
cyanide
Food grains,
Milled food
grains
1.875
0.15
GC
4 Hydrogen
phosphate
Food grains 1.0 GC-MS
5 Inorganic
bromide
Food grains
Milled food
grains
1.25 GC

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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
6 Malathion Food grains 0.2 LC-MS
7 Chlorfenvinphos Food grains 0.005 LC-MS
8 Oxadiazon Rice 0.005 LC-MS
9 Tridemorph Wheat 0.005 LC-MS
10 Flufenacet Rice 0.01 LC-MS
11 2,4 dichloro
phenoxy acetic
acid
Food grains 0.01 LC-MS

15. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
• In the pesticide residue analysis, the commonly used detection
methods are mainly
• gas chromatography (GC) ,
• gas chromatography-mass spectrometry (GC-MS),
• high-performance liquid chromatography (HPLC),
• liquid chromatography tandem mass spectrometry (LC-
MS/MS) .
• Although LC-MS/MS and GC-MS show excellent detection
capability, the high cost still inhibits their widespread use.
Compared to them, HPLC with its convenience, efficiency, and
durability, is the most extensive means of pesticide detection.
General detection methods for
pesticides in grains

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AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
The traditional methods of extraction of pesticides mainly
• liquid-phase extraction (LPE) ,
• liquid-phase microextraction (LPME),
• Supercritical fluid extraction (SFE) ,
• QuEChERS method of extraction,
• Dispersive liquid-liquid microextraction (DLLME)
• In recent years, a rapid, simple, and convenient method is
dispersive liquid-liquid microextraction (DLLME)
Extraction procedures for pesticides
from grains.

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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
Sample Extraction (Step 1)
samples were grinded into fine powder, and 1 g powder was placed in
a 10 mL centrifuge tube, then a solution of 5 mL methanol containing
110 Micro litre [OMIM][Tf2N] was added into the centrifuge tube and
followed by ultrasonic treatment for 8 min.
DLLME Procedure (Step 2)
After centrifugation for 5 min, the obtained methanol solution
containing OPPs and ionic liquid was taken out into a centrifuge tube
including 5 mL distilled water, and a cloudy solution was formed in
the tube. Then, it was centrifuged for 5 min. Finally, 5 micro litres of
[OMIM][Tf2N] sedimentary facies formed in the bottom of the
centrifuge tube was injected to the HPLC system for analysis.
Extraction procedures of DLLME

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
QuEChERS Method of Extraction
Spike levels 5,10,20, 50µg/ml -
1
Blank matrix
10mL acetonitrile with 1% acetic acid
3g anhydrous magnesium sulfate ,
1.7g anhydrous sodium acetate, 1g sodium citrate
GC-MS (NCI-SIM) analysis.
Handly and vigorously shake for 1 min
4mL of upper layer transferred to another tube
With 600mg anhydrous MgSo4 and 500mg C18
Centrifugation 8mins at 4000rpm
Handly and vigorously shake for 1 min
Centrifugation 8mins at 4000rpm
Handly and vigorously shake for 1 min
10g slurry

19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
Chromatogram of wheat sample

20. RIPER
AUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
References
• D. S. Chormey, C. Buyukpinar, F. Turak, O. T. Komesli, and S.
Bakırdere, pesticides in water medium at trace levels by GC-
MS after dispersive liquid-liquid
microextraction,” Environmental Monitoring and Assessment,
vol. 189, no. 6, p. 277, 2017.View at: Publisher Site | Google
Scholar.
• D. Harshit, K. Charmy, and P. Nrupesh, “Organophosphorus
pesticides determination by novel HPLC and
spectrophotometric method,” Food Chemistry, vol. 230, pp.
448–453, 2017.View at: Publisher Site | Google Scholar.
• I. Timofeeva, A. Shishov, D. Kanashina, D. Dzema, and A.
Bulatov, “On-line in-syringe sugaring-out liquid-liquid extraction
coupled with HPLC-MS/MS for the determination of pesticides
in fruit and berry juices,” Talanta, vol. 167, pp. 761–767, 2017.

21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
T