Pesticide residue analysis

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

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.

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

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

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

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

washing
Peeling and coocking
freezing
drying
peeling hulling

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.

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

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

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

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

MULTI RESIDUE METHOD FOR
DETERMINATION OF
ORGANOPHOSPHORUS PESTICIDES

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

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.

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

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.

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).

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).

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.

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.

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.

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.

MULTI RESIDUE METHOD FOR
DETERMINATION OF
ORGANOCHLORINE PESTICIDES

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.

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

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

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 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.
`

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.

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.

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.

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)

Pesticide residue analysis

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