determination of pesticide residue in food grains, milk, vegetables, milk products

2. DETERMINATION OF PESTICIDE RESIDUES
IN
• GRAINS
• FRUITS
• VEGETABLES
• MILK & ITS PRODUCTS
CONT. WITH PESTICIDE ANALYSIS

3. WHAT ISPESTICIDE RESIDUES?
When a crop istreated with a pesticide, a very small amountof the
pesticide, or its metabolites or degradation products, can remain in
thecrop until after it is harvested.

4. WHY DO THEY TURN UP IN OUR
FOOD?
• To control the growth of weeds usedby farmers or
prevent crop damage by insects,rodents and molds.
• usedonfood crops after harvest to prolong their
storagelife.
• usedonanimal farms to control insect pests

5. HARMFULEFFECTSOFPESTICIDE
RESIDUESTOTHEENVIRONMENT
 water systems
 Pollute the air.
 Harming beneficial
• insect species
• Soil
• microorganisms worms
 Weakening plant root systemsand immune systems.

6. DOPROCESSEDFOODSHAVELESS PESTICIDES
THANFRESHFOODS?
 Generally, yes.
 Reason:
Growers : don’t need to ensure that their foods are
cosmetically perfect
Processors: consumerdemand for foods with minimal
pesticide residues
Processing : reduce pesticide residues

7. METHODSUSEDTOREMOVE
PESTICIDERESIDUES.
• Washing
• Peeling
• Hulling
• Trimming
• Cooking + Stir-frying
• Canning
• Freezing
• Drying
• Infusion
• Parboiling
• Storage
• Chemical solution
• Neutral solutions

8. washing
Peeling and coocking
freezing
drying
peeling hulling

9. trimming
perboilig
infusion

10. EFFECTOF CHEMICAL SOLUTIONS ON PESTICIDE
RESIDUES
Acidic solutions
ascorbic acid
acetic acid
hydrogen peroxide
citric acid
 At a concentration of 5 and 10 percent for 10 minutes reduction of pesticide residues.

11. ALKALINE SOLUTION
 Dipping of fruits in NaOH solutionremoves 50 to 60 percent surface residuesof pyrethroids
compared to 40 to 50 percent removed by hydrolytic degradation with NaOH and a detergent
solutionremoved 50 to 60 percent residues,along with thissoap are usedas decontaminating agents
 Solutions of NaOH, acetic acid, potassium dichromate

12. OZONATION
 Ozone because of its powerful oxidizing property iseffectively applied in
drinking water and waste water treatment
 Tapwater treatment + ozonated water treatments significantly reduced
the pesticide residues onvegetables, ascompared to no-wash treatment
pesticide residues

13. NEUTRAL SOLUTIONS
 Sodiumchloride (NaCl)solution
28 to 93% organochlorines
100 % organophoshates
NaCl concentration pesticide residues
5 and 10 % NaCl solution
for 15 minutes rubbed by
hand & water was decanted

14. WHAT CANYOUDO?
 Grow someof your food usingorganicmethods.
 Buyorganicfood.
 Washand scruball fresh fruits, vegetables
 Eatlessor no meat.
 Trimthe fat frommeat.

15. MULTI RESIDUE METHOD FOR
DETERMINATION OF
ORGANOPHOSPHORUS PESTICIDES

16. Apparatus
A.
A gas chromatograph equipped with flame
photometric detector operated in phosphorus
mode (FPD-P), 526 mm filter.
i. Operating conditions:
 Injector temperature 220°C
 Baseline noise should be < 2%
ii. Columns :
 SPB-5 (Supelco, Bellefonte, PA) or
any equivalent 30m x 0.53mm
 N2 carrier gas

17. 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.

18. D.High speed blender.
E. Rotary vacuum evaporator.

19. 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.

20. 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
 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).

21. 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.
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).

22. Partition
• For all groups ( 1 & 2 ) half the volume of food extract equivalent to 25g of sample is placed
in a separating 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.
 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 organic layers are collected and concentrated just to dryness in rotary vacuum evaporator.

23. 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.

24. 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.

25. 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.

26. MULTI RESIDUE METHOD FOR
DETERMINATION OF
ORGANOCHLORINE PESTICIDES

27. Apparatus
i. Refrigerated centrifuge; able to rotate at
3000rpm at 15°C.
ii. SPE (Solid phase extraction)
automatic
iii. Rotary vacuum evaporator.
iii. Gas chromatograph system with
injection device and electron capture
detector.

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

29. 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

30. 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.

31. 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 another 50ml of n-hexane.
 The two extracts are kept together.
 Solvent evaporated at 35°C to 1ml.
 Evaporation is finished with a gentle stream of nitrogen.
`

32. 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.

33. 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.

34. REFERENCES:
 www.google.com
 Slideshares.in
 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.

36. 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)