In this slides contains deep introduction about pesticides and analysis of pesticide residue in vegetables. Presented by: M. Malarvannan (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
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presented by
M.MALARVANNAN. (20L81S0704).
M.PHARM
Department of Pharmaceutical Analysis.
Under the guidance/Mentorship of
Dr. P. Ramalingam., Ph.D.
Director- R&D Division,
Professor of pharmaceutical analysis
and medicinal chemistry
METHODS FOR ESTIMATION OF
PESTICIDES IN VEGETABLES

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 2
S.NO CONTENT
1 Introduction
2 Colour code for different pesticide
3 Regulation
4 Terminology
5 Work flow for analysis of pesticide residue
6 Sample collection
7 Sample preparation
8 Extraction of vegetable sample
9 Multiresidue method for determination of Organophosphorus
pesticide.
10 Multiresidue method for determination of chlorinated pesticide.
11 Reference
Content Table

3. 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 3
Pest:
• Pest is any Animal, Plant, or Microorganism that causes trouble,
injuries or destruction of plant or crops.
Pesticide:
• The FAO defines pesticide as any substance intended for preventing,
destroying, repelling, attracting any pest including harm.
Main classes of pesticides:
Introduction
Herbicide Fungicide
Insecticide Rodenticide

4. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
Effect of pests and insects on various foods:
 Damage to existing crops.
 Damage the crops during storage.
 Damage the food products.
 Damage the fresh foods like vegetables and fruits.
 Eg: Onion, tomato, apple etc..
 Damage the milk and milk products.
 Eg: Cheese, butter, cream etc..

5. 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 5
Colour code for different pesticide
LABLE LEVEL OF TOXICITY LD50 CHEMICALS
Extremely toxic 1-50
Monocritophos, Zinc
phosphate, Ethyl mercury
acetate.
Highly toxic 51-500
Endosulfan, Carbaryl,
Quinalphos.
Moderately toxic 501-5000
Malathion, Thiram,
Glyphosate.
Slightly toxic More then 5000
mancozeb, Oxyfluorfen,
mosquito repellant oils
and liquids.

6. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
a). Chlorinated pesticides:
b).Organo-Phosphorus pesticides:
Chemical classification of pesticide
ENDOSULFAN HEPTACHLOR
DDT
ACEPHATE MALATHION
ETHION

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
C). Synthetic pyrethroids :
d). Thiazole Fungicide:
Continued…
FLUVALINATE
FENOPROPATHRIN CYHALOTHRIN
HEXACONAZOLE
PROPICONAZOLE MYCLOBUTANIL

8. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
• It was considered that the ultimate goal in fair practice in
international trade depended, among other things, on the reliability
of analytical results.
• Particularly in pesticide residue analysis, depended not only on the
availability of reliable analytical methods.
• But also on the experience of the analyst and on the maintenance of
good practice in the analysis of pesticides.
• It contains three inter-related parts
The Analyst
Basic Resources
The Analysis
Analysis of pesticides

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
• Each country adopts their own agriculture policies like Maximum
Residue Limits (MRL) and Acceptance Daily Intake (ADI).
• Some countries use the international maximum residue limits –
codex Alimentarius to define the residue limits.
• This was established by Food and Agriculture Organisation of the
United Nations (FAO) and Would health organisation (WHO) in 1963
to develop international food standard.
• “Codex India” the national codex contact point (NCCP) for India
Regulation

10. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
• Pesticide residue: Any specific substance in food, commodities or
animal feed resulting from the use of pesticide.
• Maximum residual limit (MRL): The limit beyond which pesticide
levels are not legally permitted.
• Acceptance daily intake (ADI): It is the daily dose of chemical
which during an entire life time, appears to be without appreciable
risk on the basis of all facts known at that time.
• lowest Calibrated level (LCL): The methods with sufficient
sensitivity to determine residues at levels two or more orders of
magnitude lower.
• Persistence: The chemical that remain active for a long period of
time after application.
Terminology

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Central codex committee of food standards
(The prevention of food adulteration Act,1954)
Maximum Residue Limit in India
PESTICIDE FOOD MRL (mg/kg)
Dithiocarbamates Tomatoes 3.0
Benomyl Fruits
Vegetables
1.0
0.50
Captafol Tomato 5.0
Copper oxychloride Fruits and vegetables 20
carbendazim Fruits
Vegetable
5.0
0.50
Profenfos cabbage, onion, tomato. 0.05
chlorothalonil Potato 0.1
captan Fruits and vegetables 15.0

12. RIPER
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PESTICIDE FOOD MRL (mg/kg)
Dodine Apple 5.0
Penconazole Grapes 0.2
Tricyclazole Rice 0.02
Myclobutanil Grapes 1.0
Dimethoate Carrot, onion, cucumber. 0.02
Propiconazole Wheat 0.05
Malathion Tomato, onion. 3.0
Endosulfan Tomato 0.5
Continued….

13. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
Sampling
Sample
Preparation
Sample Analysis
Data
evaluation
and
Reporting
Workflow for analysis of pesticide
Residue

14. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
Sample collection
COMMODITY EXAMPLES MINIMUM QUANTITY
REQUIRED
Small or light products, unit
barries weight upto about 25 g
Peas, olives parsley 1 kg
Medium sized products unit
weight usually between 25 to
250g
apples, oranges, carrots
potatoes
1 kg
(at least 10 units)
Large sized products unit
weight over 250 g
cabbage, melons, cucumber 2 kg
(at least 5 units)
Dairy products Whole milk, cheese
Butter , cream
0.5 kg
Egg (10 unit if whole) meat Poultry, fat, fish and other
fish and animal products
NA
Oils and fats cottonseed oil margarine 0.5 kg
Cereals and cereal products NA 0.5kg- 1 kg
Spices Chilies, cumin, coriander 0.25kg

15. 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 15
• The well-equipped food analysis laboratory should have a variety of
sample preparation equipment including mechanical choppers,
mincers, grinders, blenders and a hammer or similar mill.
• Generally the vegetables are moist food category so it can be
chopped or minced.
Chopper grinder
Sample preparation
Mincer

16. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
Homogenization of sample composite:
• Ideally, the entire sample should be homogenized using
equipment such as a Hobart food cutter.
• Before being placed in the chopper, samples may need to be
either halved or quartered or cut into small (5-10 cm) pieces. After
homogenization, remove a portion for analysis.
• Pesticides may tend to collect in top of vegetables.
• If the homogenized sample is not immediately
analysed, store it in a clean container with a
tight closure and freeze.

17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
• Normally the whole sample is stored depending analysis at <-10 °C.
• Homogenization will increase the rate of enzymatic hydrolysis so
frozen homogenized samples should be analysed within two weeks.
• In vegetables the edible portion is analysed.
• Soil will be removed from root vegetables, by gentle brushing under
a stream of water and Outer leaves of cabbage, cauliflower, etc. will
be removed.
Sample storage
SAMPLE TYPE STORAGE
Dry feed Room temperature in airtight container
Fruits and vegetables Refrigerate the whole sample
Feeds for fumigant analysis seal in plastic bags and freeze

18. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
Storage of primary standards:
• Proper storage of reference standards in order to maximize
preservation of chemical integrity is an essential part of the
analytical process.
• Small sample vessels, air-tight container, moisture-free atmosphere.
large, wide mouth, resealable glass jars loaded with 2 to 4 cm of an
appropriate desiccant.
Solvents:
• Toluene, Iso-octane (2,2,4-trimethylpentane), ethyl acetate and
hexane. All solvents should be pesticide grade and distilled-in glass.
Pesticide standards

19. 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 19
a) Preparation of stock solution 1 mg/mL (1000 ppm)
• Weigh 50 mg of the pesticide of pesticide reference standard and
transfer to 50 mL volumetric flask. Dissolve the pesticide in a suitable
solvent and make to volume.
b) Intermediate solution 10 µg/mL (10 ppm)
• Pipet 1 mL of the stock solution into individual 100 mL volumetric
flask. Dilute to volume with the suitable solvent.
c) Working solution 1 µg/mL (1 ppm)
• Pipet 10 mL of the intermediate solution into 100 mL volumetric
flask. Dilute to volume with the suitable solution.
Preparation of standard solution of pesticides:

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
The vegetables are covered under the Group-Ⅰcategory.
The Group-Ⅱ&Ⅲ are milk and cereals respectively.
Simple extraction method:
 50g of chopped sample is added into high speed blender jar.
 100ml of acetone
 Blended for 2mins at high speed.
 Filter with Buchner funnel.
 Washed with 50ml of acetone.
 Extract brought to 150-200ml volume with acetone: water (2:1).
Extraction of vegetable sample

21. 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 21
a) Extractive method
QuEChERS Method extraction
Marginizing
the
vegetables
materials
Add solvent Shake
vigorously
(vortex)
Add
extraction
salt.
Transfer to
centrifuge
tube

22. RIPER
AUTONOMOUS
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
Continued…
Shake
vigorously
(vortex)
And transfer
to centrifuge
for 2mins

23. RIPER
AUTONOMOUS
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NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
b) Clean up method:
• The removal of interfering co- extractives from extract is called clean
up. By using dispersive solid phase extraction (dSPE).
Continued…
Shake
vigorously
(vortex)
And transfer
to centrifuge
for 2mins
Collect the
supernatant
Add
supernatant
in dSPE
sorbent
Transfer to
sample vial

24. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
Solid phase extraction cartridges:
• Serves the dual purpose of extraction and clean up. Advantages of
SPE device over other conventional solvent extraction and clean up
of pesticides includes better reproducibility.
Recent Trends in Clean up

25. 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 25
GC method:
Multiresidue method for determination
Organophosphorus Pesticide
Column dimensions and type SPB-5 (Supelco, Bellefonte, PA) or any
equivalent, 30 m × 0.53 mm
Carrier gas Nitrogen
Carrier gas pressure 20 psi
Carrier gas Flow 15 mL/min
Injector temperature 220°C
Detector flame photometric detector
Detector temperature 300°C
Initial temperature 140°C, hold 2 min
Final temperature by 140 °C to 240 °C at 5°C/min.

26. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 26
Chromatography clean-up columns:
 Read to use Extrelut-3, needle is fixed at column end as flow regulator.
 Glass column 30cm x 20cm with glass septum (carbon-celite cleanup).
Reagents:
 Reference standard solution: prepared using heptane or n-hexane.
 Solvents: acetone, acetonitrile, benzene, methanol, n-hexane (all HPLC grade).
 Silanized glass wool.
 Active carbon.
 Sodium chloride.
 Cotton wool washed with acetone and n-hexane.
Continued…

27. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 27
Pesticide standard solution:
 Stock solution of 1mg/ml using ethyl acetate.
 Working solution of 0.5µg/ml.
Extraction:
 50g of chopped sample is added into high speed blender jar.
 100ml of acetone
 Blended for 2mins at high speed.
 Filter with Buchner funnel.
 Washed with 50ml of acetone.
 Extract brought to 150-200ml volume with acetone: water (2:1).
Continued…

28. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 28
Partition:
 25g of sample is placed in a separating funnel.
 100ml of dichloromethane, 100ml of acetone and 10gm Nacl is added.
 Shake vigorously till most Nacl is dissolved and layers are allowed to
separate.
To aqueous layer
 The Aqueous layer is transferred to a second separatory funnel.
 200ml of dichloromethane is added to the second separatory funnel.
Organic layer:
 Organic layer is dried through sodium sulfate.
 The organic layer is dried.
 All the organic layers are collected and concentrated just to dryness in rotary
vacuum evaporator
Continued…

29. RIPER
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NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 29
Chromatographic column clean up:
 Glass column is filled with 2g celite and 4g carbon.
 carbon: celite (4:1) is added.
 Topped with glass wool plug
 Column is washed with 20ml benzene.
 Sample is transferred quantitatively with small portion 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 alalyzed by GC.
Determination:
 Residue are quantified by height or are measurement from solution of known
concentration of authentic compounds.
Continued…

30. RIPER
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 30
• Calculation:
• Retention time:
Compound Sample SPB-5
Acephate NA 0.26
Malathione Onion 1.16
Parathione Tomato 1.19
Paraoxon NA 1.06
Ethion NA 1.73

31. RIPER
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 31
GLC determination:
• For good separation of chlorinated pesticides, a non-polar/fused silicated
quartz capillary column and similar are recommended.
Multiresidue method for determination
Chlorinated Pesticide
Column dimensions and type 30 m × 0.25 or 0.32 mm fused silica capillary column
chemically bonded with SE-54
Carrier gas Helium
Carrier gas pressure 16 psi
Injector temperature 225°C
Detector Electron capture detector
Detector temperature 300°C
Initial temperature 100°C, hold 2 min
Temperature program 100 °C to 160 °C at 15°C/min followed
Final temperature by 160 °C to 270 °C at 5°C/min.

32. RIPER
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 32
• Calculation:
• Retention time:
Compound Sample SE-54
Aldrin NA 14.70
Dieldrin NA 18.74
Endosulfan 1 Tomato 17.62
Endosulfan II Beetroot 20.11
Heptachlor Brinjal 13.59

33. RIPER
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Clean up VS Non clean up

34. RIPER
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Pesticides in spinach sample

35. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
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100-Compound Pesticide mixture

36. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
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224-Compound Pesticide mixture

37. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
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300-Compound Pesticide mixture

38. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 38
1. Joint FAO/WHO Food Standard Programme. “Codex Alimentarius Commission”
Report of the thirty fifth session of the Codex Committee on Pesticide Residues,
2003. pg. 46-55.
2. Joan Stevens, “QuEChERS 101 The basic and beyond” agilent tech slides, 2010.
3. Rohan Dasika, Siddharth Tangirala. “Pesticide residue analysis of fruits and
vegetables” jurnal of environmental chemistry and ecotoxicology Vol.4(2), 2012.
pg 19-28.
4. Application notebook. “Agilent food testing solutions” 2012. pg24-27.
5. Ministry of health and family welfare, govt of India “fassai Manual of methods
of analysis of food pesticide residue” Manual 11. 2015, pg.5-70.
6. Restek “comprehensive solution simplify sample prep and analysis” 2020. pg-5.
7. Manish joshi, “Pesticide residue analysis” www.slideshare.net
Reference