irrational usage of pesticide leads to development of resistance, resurgence and toxic residue problems in our food. ultimately imbalance of environment . so that detection of pesticide residue in all materials of earth especially in our food, milk, meat, water, soil aquatic ecosystem and agriculture land. for the analysis of resiude set of procedure, methods, instruments, skills and laboratory must required. In this seminar would like to enlighten the best, suitable and feasible methods are discussed.

2. Methods of Pesticide Residue Analysis in Fruits
and Vegetables
Ravi Biradar
PHD16AGR6008
Seminar on

3. •Pesticide
•Boon and bane of pesticide
•Pesticide Residues
•Sampling, Extraction and Cleanup
•QuEChERS method
•Enzyme inhibition method
•Spectrophotometric method
•Gas Chromatography
•Liquid chromatography
•Residue control
•Conclusion
Content

4. Any chemical, biological substance or mixture of substances intended for
preventing, destroying, attracting, repelling or controlling any pest and includes
any substance or mixture of substances intended for use as PGR, or defoliant .
WHAT ARE PESTICIDES….. ??

5. Category 1960 1970 1980 1990 2000 2005
Sale* % Sale* % Sale* % Sale* % Sale* % Sale* %
Insecticides 310 36.5 1002 37.1 4025 34.7 7655 29 7559 27.9 7798 25.0
Herbicides 170 20.0 939 34.8 4756 14.0 11625 44.0 12885 47.5 14971 48.0
Fungicides
&
Bactericides
340 40.0 599 22.2 2181 18.8 5545 21 5306 19.6 7486 24.0
Others 30 3.5 159 5.9 638 5.5 1575 6.0 1354 5.0 936 3.0
Total 850 100 2700 100 11600 100 26400 100 27104 100 31191 100
Changes of Pesticides Consumption Worldwide
*Sale: million US dollars
Aktar et al. (2009)
In India: 76% insecticide , 10% herbicide and 13% fungicide

6. Knock down effect
Quantity
Flexibility
Cost effectiveness
Save your Life

7. Reduction of beneficial species
Resistance
Ground water contamination
Drift of sprays and vapors
Residues in food

8. Biomagnifications…

9. “The amount of insecticide left over after a lapse of time.”
Disappearance of pesticide residue take place by two way i.e.
Dissipation : In which the disappearance of the residue is fast
Persistence : In which there is a slow decrease in amount of residue

10. Residues in News…….!

11. What is the need to study pesticide residue?
persistence of the pesticide
High quantity of pesticides (lack of awareness from farmers)
Prolonged use of pesticides ( Kerala incidence)
monitoring the residues in environment
TO fix the MRL, safe waiting period,.

12. NOAEL : No observed adverse effect concentration derived from animal or
human studies
Maximum Residue Levels (MRL): The legal limit of pesticide allowed
(expressed in mg/kg of produce) in food or animal feeds.
Acceptable Daily Intake (ADI): The amount of chemical that can be
consumed (in mg/kg bodyweight/day) every day for an individuals entire
lifetime in the practical certainty, on the basis of all known facts that no
harm will result.
Important Terminologies

14. SAMPLING
Previous history
character of the sample
Application of insecticide
Formulation of insecticide
Storage of samples

15. EXTRACTION
Extraction is the process by which toxicant is transferred from the
treated bulky biological material in to a solvent.
TYPES
Surface rinsing
Soxhlet extraction
Blending extraction
Solvents:
Water, acetonitrile, ethyl
acetate, methanol, acetone and
n-hexane.
•Blending and sonication
•Microwave- assisted extraction (MAE)
•Pressurized liquid extraction (PLE)
•Supercritical fluid extraction (SFE)

16. Franz von Soxhlet
(1848 – 1926)
In the Beginning There Was Soxhlet...

17. CLEAN UP
Isolation of toxicant from interfering substance or solvent.
Conditions to use
When the insecticide is evaluated by direct measurement.
When the insecticide can be measure only after all the interfering
substance are removed or converted to product which shall not interfere
during analysis.
When insecticide cannot be measured in isolated form and its converted
to suitable derivative for measurement.

20. Sample Preparation
Homogenize fruit and
vegetables at a low
temperature
Extraction/Partition Place a 10
g sample into a 50 mL Teflon
tube with acetonitrile and
NaSO4. Shake well. Spin for 5
minute in a centrifuge
Transfer the supernatat to a 15 ml
Teflon tube. Add MgSO4 and PSA,
shake well for 20 seconds then
centrifuge for 5 min.
GC/MS/MS, LC/MS/MS Same extract
injected into both LC/MS and GC/MS
systems.

22. Enzyme inhibition method of residual analysis
Cholinesterase inhibiting by the insecticide action
Methods assess the cholinesterase activity which can be used in residue analysis:
Potentiometric Method:
The enzyme is allowed to act on acetylcholine in a buffer solution for definite
period of time (usually 1-2 hrs) at constant temperature. The pH of mixture is
measured at the beginning and at the end. Change in pH due to the release of acetic
acid is measured which indicates the enzymatic activity.
(Michel, 1949)

23. Advantages
This method is particularly suitable when the insecticides
undergo change in the plant to produce metabolites with high
inhibitory activity.
Insecticidal residues in food crops can be analyze rapidly.
In this technique less clean up of extracts is needed.
Disadvantages
It’s limited to organophosphate and carbamate compounds
only.

24. •Three kinds of spectrophotometric methods used in pesticide residue analysis.
•Ultraviolet, visible and infra-red methods
•Measure the conc. Of pesticide in solution by measuring the amount of the light
that is absorbed by the solution.
•Range 180nm to 400nm.
Chlorobenzilate in citrus (Blinn et al. 1954)
2, 4-D in alfalfa (Gordon et al. 1952)
DDT in milk and citrus & diquat in potatoes (Herriot 1946).

25. Chromatography
chromo means “colour” Graphy means “to write”
The chromatography consist in the unique distribution of the components of a
mixture between stationary phase and mobile phase due to different affinity of a single
compounds towards both phase (Stationary phase and mobile phase).
It involves passing a mixture dissolved in a "mobile phase" through a
stationary phase, which separates the analyte to be measured from other molecules in
the mixture based on differential distribution coefficient and different net rate of
migration between the mobile and stationary phases.
Mikhail
Tswett(1872-1919)

27. Various techniques of chromatography
1. Planar Chromatography
Paper chromatography Thin layer chromatography
2. Column chromatography
Gas chromatography Liquid chromatography
3 Affinity chromatography
4 Ion exchange chromatography

28. Planar chromatography is a separation technique in which the stationary
phase is present in or on a plane.
The plane can be a paper, serving as such or impregnated by a substance as the
stationary bed (paper chromatography)
or
a layer of solid particles spread on a support such as a glass plate (thin layer
chromatography).
Planar Chromatography

29. The method has been widely used for analysis of insecticide residue. In
this method samples are spotted on whatman filter paper no.1 (20 X 20 cm).
•Pencil line is drawn at 4-5 cm from one edge of the paper and the samples are
applied on this line via micro pipette.
•The solvent of sample is allowed to evaporate.
•The paper is kept in air tight chamber saturated with vapours of solvent.
•The number of solvents such as carbon tetrachloride, carbon disulfide, diethyl ether
and chloroform are used depending on the polarity.
Paper Chromatography

30. •It is a versatile technique widely used in residual analysis
•the only different in thin layer chromatography and paper
chromatography is that in TLC the glass plates (20X20 cm or
20X10 cm) having a thin film of absorbent used in the place of
filter paper
• Number of absorbents such a silica gel G, silica gel HR, or
alumina G etc. are available commercially.
Thin Layer Chromatography

32. •Stationary phase is packed into glass or metal column.
•The mixture of analytes is then applied and the mobile phase, commonly referred
to as the eluent
•passed through column either gravity feed or by use of pumping system or
applied gas pressure.
•The stationary phase either coated on to discrete small particles (matrix) and
packed into column.
•As the eluent flow through the column analyte separate on the basis of their
distribution coefficient and emerge individually in the eluate as it leaves the
column.
Column Chromatography

33. Gas chromatography (GC)
• Volatility and thermal stability
• Carrier gases: helium, argon and nitrogen , which are chemically inert.
• The stationary phase is adhered to the inside of a small-diameter glass tube
(a capillary column) or a solid matrix inside a larger metal tube (a packed column).
•80% of pesticide can analysed.
•Organochlorines, organoposphates, carbamates

35. Sharma et al., 2010

36. Pesticides Insect MRLs Sample I
(Dusehri)
Sample II
(Chaunsa)
Sample lll
(Sindhri)
Cypermethrin Tip Borer 0.50 0.25 0.38 0.19
Methamedophos Mango
hopper
1.00 0.16 0.41 0.36
Monocrotophos Fruit fly 1.00 0.46 0.25 0.69
Cyfluthrin Mealy bug 0.50 0.24 0.18 0.39
Dialdrin Termites 2.00 - - 0.56
Methyl Parathion Thrips 1.00 0.15
-
-
Quantitative analysis of pesticides residues in mango (GC)
(-) stands for not detected.
Saqib et al. (2002)Pakistan

37. Residues of endosulfan on brinjal fruits (GLC)
Method of
estimation
Residues* (ppm) days after spray
0 1 5 10 15
Bioassay 2.40 ± 0.44 1.80 ± 0.18 0.76 ± 0.08 0.36 ± 0.04 0.21 ± 0.03
GCL (I) 1.40 ± 0.22 1.11 ± 0.06 0.37 ± 0.04 0.11 ± 0.05 0.08 ± 0.01
GCL (II) 1.03 ± 0.23 0.59 ± 0.05 0.18 ± 0.01 0.09 ± 0.01 0.08 ± 0.01
GCL (ES) 0.19 ± 0.01 0.24 ± 0.01 0.28 ± 0.01 0.18 ± 0.03 0.09 ± 0.01
(I+ II ES) 2.63 ± 0.43 1.93 ± 0.04 0.84 ± 0.02 0.40 ± 0.02 0.23 ± 0.03
Average 2.52 ± 0.41
-
1.86 ± 0.13
(25.8)
0.80 ± 0.07
(68.0)
0.38 ± 0.03
(84.8)
0.22 ± 0.03
(90.9)
*Mean of three replication, GLC= Gas liquid chromatography, I= -endosulfan, II=β-endosulfan, ES= endosulfan
sulphate, Figures following ± signs are standard deviation to the mean.
Sharma and Anil (2010)Palampur

38. Pesticides identified in spinach by GC/MS
Pesticides
Identified
Retention
Time
Elution
Time
Relative
Abundance
Molecular
Weight
Piperonylbutoxide 16.275 17.661 2-3% 338
Crotoxyphos 17.283 17.901 3-4% 325
Glyophosphate 3.175 3.165 2-3% 169
Zaib et.al. (2010)Lahore

39. Pesticides Residue identified in spinach by GC/MS
Lahore Zaib et.al (2010)
Sample without pesticide Sample with pesticides
Sample identified pesticide of Crotoxyphos

40. Sampling
date
(M/D)
Days
after
treatm
ent
Chlorpyrifos Cypermethrin Chlorothalonil
Residue
(mgkg-1) (mean
± SD)
Degrada
tion rate
(%)
Residue
(mgkg-1) (mean
± SD)
Degrada
tion rate
(%)
Residue
(mgkg-1)
(mean ± SD)
Degradat
ion rate
(%)
5/8 0 2.510 ±0.204 0 0.374 ± 0.062 0 15.246 ± 0.628 0
5/10 2 0.961 ±0.109 62 0.164 ±0.045 56 10.614 ±0.617 31
5/12 4 0.372 ± 0.087 85 0.107 ±0.005 71 4.417 ±0.110 71
5/14 6 0.284 ± 0.006 89 0.080 ± 0.008 79 3.899 ± 0.178 74
5/16 8 0.276 ± 0.058 89 0.050 ± 0.002 87 3.620 ±0.041 76
5/18 10 0.196 ±0.016 92 0.020 ± 0.002 95 2.729 ± 0.190 82
The concentrations and degradation rates of pesticides residues in
Cabbage (GC/MS)
China (Zhang et.al 2006)
Half life: Chlorpyrifos: 2.9; Cypermethrin: 2.6; Chlorothalonil: 4.1 days.

41. 6. LIQUID CHROMATOGRAPHY
It is a separation technique in which the mobile
phase is a liquid. Present day liquid chromatography that
generally utilizes very small packing particles and a
relatively high pressure is referred to as high performance
liquid chromatography (HPLC).
In the HPLC technique, the sample is forced
through a column that is packed with irregularly or
spherically shaped particles or a porous monolithic layer
(stationary phase) by a liquid (mobile phase) at high
pressure.

42. High Pressure Liquid Chromatography (HPLC)
A better form of column chromatography. Instead of
draining down through the stationary phase, the solvent is
forced through under high pressure.
Stationary phase :Silica
Mobile phase :Suitable solvent

44. Insecticide Insect
Insecticide residues in Brinjal fruit (ppm)
Sampling Periods
0 Day 3 Days 7 Days
Carbofuran Fruit and Stem borer 0.084 0.039 0.026
Chlorpyriphos Fruit/stem
Borer/Sucking Insect
0.095 0.049 0.035
Imidachloprid Sucking Insect 0.097 0.051 0.039
Dichlorovos Fruit/stem
Borer/Sucking Insect
0.070 0.049 0.034
Acephate Fruit and Stem borer 0.071 0.046 0.028
Quantitative analysis of residues in Brinjal fruit (HPLC)
Iqbal et.al (2007)Faisalabad
*Values are mean of 5 samples from supervised trail.

45. Sampling site No. of
Samples
Analysed
Pesticides detected Frequencies Residue level
(mg kg')
MRL
(mg kg-1)
Bada Ber 5 Cypermethrin 1 0.112 0.1
Methomyl 1 0.087 0.2
Endosulfan 1 0.100 0.2
Zangli 10 Cypermethrin I 0.176 0.1
Pishthakhera 3 N.D - - -
Urmar 8 Chlorpyriphos 2 0.08, 0.120 0.01
A,-Cyhalothrin 1 0.165 b
Hussain Abad 5 N.D - - -
Mewra 7 Methamidophos 4 1.610, 0.150,
0.101,0.75
0.5
Cypermethrin 2 0.130, 0.106 0.1
Methomyl 3 0.150, .087,
.095
0.2
Nahqi 3 Fenvalerate 1 1.21 b
Pesticide residue in onion sample through HPLC
Khan (2005)Pakistan
b: No MRL available

47. LC-MS Analysis
GC-MS Analysis
Sample Prep: QuEChERS
Data Processing/Analysis
Employed a combination of GC–MS and LC–MS/MS for the analysis of 446 pesticides in
fruits and vegetables and 450 pesticides in honey, fruit juice and wine
Pang et al., (2006)

49. If you are willing to change a few habits in your life,
you can dramatically reduce your exposure to pesticides.
Buy organic produce when you can.
Wash your produce
with warm or running
tap water cleans it better
than dipping it, or
rubbing produce with
your hand.

50. Trim the outer part of fruits and vegetables. Take off
the outer leaves of cabbage and lettuce. Peel fruits and
vegetables when practical.
Cook fruits and vegetable thoroughly. Cooking can
break down some pesticide residue because they tend to
be heat sensitive.
Follow the instruction on pesticide container labels
when using .

51. Green grapes
S/N Pesticide Max residue
limit (mg/kg)
No wash Lukewarm water
wash
Salted lukewarm water
wash
1 Imazalil 5 8.6 6.9 5.4
2 Thiabendazole 10 21.4 17 14.7
3 Phosmet 10 11.8 8.9 7.6
4 Decamethrin 0.05 0.03 ND ND
Black grapes
S/N Pesticide Max residue
limit (mg/kg)
No wash Lukewarm water
wash
Salted lukewarm water
wash
1 Imazalil 5 9.3 8.2 6.6
2 Thiabendazole 10 32 27.4 21
3 Phosmet 10 16.7 11.3 8.9
4 Decamethrin 0.05 0.05 0.04 0.02
PESTICIDE RESIDUES IN GRAPES
Dasika et.al (2012)Hyderabad

52. Decontamination of carbaryl and isoprocarb residues in grapes
Processing procedure
Carbaryl 0.15% Isooprocarb 0.2%
Residues*
(mg/kg)
Per cent
removal
Residues*
(mg/kg)
Per cent
removal
Initial residues 0.465 -- 0.214 --
Water wash 0.007 98.56 0.003 98.74
5% tamarind solution dip
(30 min.)followed by water
wash
0.004 99.01 0.058 73.09
2% salt solution dip (30
min.) followed by water
wash
0.003 99.46 0.001 99.16
1% acetic acid solution dip
(30 min.) followed by water
wash
0.004 99.18 0.043 79.83
*Average of three replications.
Reddy and Rao (2003)Hyderabad