Methods For Estimation Of Pesticide Residue in Milk

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
A Seminar as a part of curricular requirement
for 1st year M.Pharm I semester
Presented by
G.HIMA BINDU
Regd no.(20L81S0703)
Under the Guidance of/Mentorship of
Dr. Ramalingam, M.Pharm., Ph.D., R&D Director
Methods for estimation of pesticide
residues in milk
1

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Introduction to Pesticide Residues
Steps involved in Pesticide Analysis
Different Methods for Estimation of Pesticide Analysis
Conclusion
CONTENTS
2

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• Pesticide residue: pesticide residue refers to pesticides that may
remain on or in food after they are applied to food crops, or as a
result of environmental contamination or animal feeding on pesticide
treated feed.
• Different types of pesticide residues present in milk are:
• Organochlorines (DDT, HCH, Endosulfan, etc.,)
• Organophosphates
• phenylpyrazole
Pesticide residues in milk
3

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DDT Endosulfan
Hexachlorocyclohexane Dieldrin
4

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
• The maximum residue limit is the maximum allowed concentration of
residue in a food product obtained from an animal that has received a
veterinary medicine or that has been exposed to a biocidal product for
use in animal husbandry.
Example:
Sample – amul milk.
Pesticide found – chlorpyrifos.
Health impact – affects nervous system.
MRL limit – 0.01ppm.
CODEX MRL – 0.02 ppm.
Maximum residue limit

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Pesticide residues in Dairy products

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• Collection of samples
• Preparation of samples
• Extraction of samples
• Clean up method
• Detection of Samples
Steps involved in analysis of
pesticide residues
7

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• Milk samples are collected from local markets of sahiwal.
• These samples are collected in sterilized glass bottles which were
airtight.
• The fresh sample so collected placed in cool boxes which consists of
ice packets.
Collection of samples
8

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• Each sample of raw milk is mixed thoroughly.
• Sub sample of 10ml was measured in small glass containers.
• The container should be closed with screw teflon capped stoppers.
• Kept the bottles in deply frozen at -20°c.
• For pesticide residue analysis.
Preparation of samples
9

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• The extraction of sample was based on kampire et al.
• The milk sample was defrosted, regimed and mixed thoroughly.
• 1gm of fat was taken and mix anhydrous sodium sulphate in 250ml
flask.
• After the mixture got set, then petroleum ether was filtered in to tube
containing sodium sulfate through glass funnel.
• This concentrated material added in 3ml of hexane to clean out.
Extraction of samples
10

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• Glass column of 15cm long plugged with glass wool and washed
with hexane.
• The concentrated fat extract was added to this column, then it was
eluted to extract 100 ml hexane to extract pesticide residues.
• The resultant material was added to250ml round bottom flask.
• Mix the material with anhydrous sodium sulphate.
• The residue was dissolved in 1ml cyclohexane for GC analysis.
Clean up with florisil method
11

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• After clean up the samples are subjected to the detection.
• In GC analysis, a variant gas chromatograph equipped with Ni63
Electron Capture Detector (ECD).
• It is fitted with semi-polar and non polar fused silica capillary
column.
Source of gas:
• Hydrogen gas with purity 99.9% was used as carrier gas
Temperature:
• The injector temperature was 250°c.
• The initial column temperature was 110°c kept for 1 min then raised
at a rate of 20°c/min to 165°c.
Detection of samples
12

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Injector volume:
• The injector volume was kept up to 1 μ/L.
• Calibration of the GC was achieved before the sample analysis using
the standard pesticide.
• Determination of recoveries for each milk sample was carried out by
spiking the fresh milk sample with particular concentration of
pesticides.
13

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• Firstly all the collected samples were extracted by various
procedures.
• Then all the extracts of samples were cleaned up.
• Then further their determination for pesticide residues were carried
out.
Determination of organochlorine
pesticide residues in milk by GC
15

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Extraction procedure
• Milk(15 ml) + n-hexane(40 ml) + Acetone (40 ml)
• Mix in a glass stoppered cylinder and shake vigorously.
• Allow to stand until a clear separation of two layers.
• Collect the upper organic phase and separate the lower phase.
• Re- extract the lower phase twice with 30 ml of n-hexane.
16

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• Centrifuge at 4000 rpm to separate the solvent.
• Transfer this concentrate in 100 ml separating funnel & add 35
ml conc. Sulphuric acid.
• Wash the clear upper phase with distilled water
• Collect the final extract and analyze using GC equipped with
ECD.
17

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Quantitative determination of
organochlorine pesticides
• Preparation of stock standard:-
• Pesticides stock standard were prepared in μ/ml concentration.
• By dissolving 0.1gm of the standard in 100 ml pesticide quality
hexane using volumetric flask.
• The stock solution was transferred to ground glass stoppered
bottles and stored in refrigerator.
18

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Preparation of chromatographic
working standards
• Standard solutions were prepared from the stock solution using
micro syringes.
• A typical concentration (1ng/1ml) was prepared by diluting 0.1
ml of stock solution to 100 ml using hexane.
• The standard solution were transferred to ground glass
stoppered bottles and stored in refrigerator.
19

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Preparation of extracted samples:-
• The extracted samples was diluted with 0.5 ml of nano grade
hexane.
• 5 μl of diluted extract were injected into electron capture gas
liquid chromatography using standard micro syringe.
• Analysis on Gas Chromatography:-
• Then further analysis of samples were done in GC.
20

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Chromatogram of organochlorine
pesticides analysed by GC-ECD
Name Retention Time
DDT 16.05 mins
DDE 15.4 mins
Dieldrin 16.13 mins
HCH 10.87 mins
Alpha endosulphan 11.96 mins
Beta endosulphan 12.5 mins
Endosulphan sulphate 13.9 mins
Detector Temperature 300℃
Injection Volume up to 1ս/L

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Chromatogram of pesticides
22

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Estimation of pesticide residues by
quechers extraction method
• To improve the efficiency of traditional methods a new sample
preparation approach was introduced by “Anastassiades”.
• This procedure know as “QuEChERS” is an anagram for
Quick, Easy, Cheap, Effective, Rugged, and Safe.
• This procedure uses a single extraction in acetonitrile and
requires a very small sample.
• It is a type of solid phase extraction technique.
23

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Quechers extraction procedure
• Transfer 15 ml of whole milk in to 50 ml centrifuge tube.
• Add 30 μl of 50 ppm triphenyl phosphate (TPP) internal
standard solutions to all samples.
• Add 15 ml of MeCN with 1% Ac. Cap and shake 1min at 1000
strokes/min using a Spex Geno-Grinder.
• Add salts, 6 g of MgSO4 and 1.5 g NaOAc from pouch and
vortex for 10 sec to break up salt agglomerates.
26

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• Shake 1min at 1000 strokes/min using Spex Geno- Grinder.
• Centrifuge at 3830 rcf for 5 min.
• dSPE clean up:-
• Transfer 1 mL of the supernatant to 3 mL dSPE tube.
• Shake 2 min at 1000 strokes/min using spex Geno-Grinder.
• Centrifuge at 15,300 rcf for 5 min.
• Transfer 0.3 mL of the cleaned extract into 2 mL auto- sampler
vial, add 0.3 mL of reagent water and vortex for 30 sec.
28

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• The samples are ready for LC-MS/Ms analysis.
29

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HPLC: Thermo Scientific Dionex UltiMate 3000® LC System
Column: Thermo Scientific, Accucore aQ® , 100×2.1mm, 2.6սm
Guard Column: Thermo Scientific, Accucore aQ®, 10×2.1mm, 2.6սm
Column Temperature: 40℃
Column Flow Rate: 0.200mL/min
Auto-sampler Temperature: 10℃
Injection Volume: 10 μl
Gradient Program:
Mobile Phase A: 0.3% formic acid and 0.1% ammonia formate in water
Mobile Phase B: 0.1% formic acid in MeOH
30

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Estimation of pesticides in milk
by TLC
• In this monitory study milk samples are collected from animals
were analysed for:
• The adulteration by different chemicals.
• For the presence of pesticide residues.
• Different steps involved in estimation are:
• Sampling
• Sample extraction
• Results.
31

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Sampling:
• All milk samples were collected from different dairies and street
vendors.
• The samples were collected in 100 ml screw capped sterilized plastic
bottles.
• Formalin was used for preserving milk samples.
• All the possible precautions were taken to avoid external
contamination at the time of collection and processing of milk.
• After collection, all the samples were stored at 4°c until the
extraction was done.
32

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Sample Extraction
• In 16 ml falcon tube add 4 ml milk + 4 ml Hexane + 4 ml
Acetone then centrifuge at 14000 rpm for 10 min.
• Temperature should be kept at 29°C .
• Then this solution is taken in test tube, upper layer should be
removed.
• Then add 4 ml Hexane + 0.25 ml conc. Sulphuric acid.
33

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• Upper layer of Sample taken in another test tube and put on
water bath evaporation.
• Then the sample reconstitutes with Hexane, this solute used for
spotting on TLC.
34

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Results:
• As per the observation of thin layer chromatography, after spraying
the zinc chloride and DPA (diphenylamine) reagent there was no
immediate colour development.
• The plate was then put in the oven for 10 min at 100°C.
• Pale yellow spots developed after sometime which was for
“chlorpyrifos”.
• Deep bluish colour indicates” DDVP”.
35

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• Two solvent systems were used for better separation.
• Hexane and Acetone (8:2) showed better separation than
Hexane: Acetone (7:3).
36

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• Deep bluish colour spot indicates the presence of DDVP.
37

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Conclusion
Presence of pesticides food and especially in milk is
a growing concern not only in India but also in other
countries as they are widely consumed by all age group of
people especially children. One reason being the fodder
consumed by cattle contain high content of pesticides. The
method TLC can be used for identification and separation of
pesticide residues in milk samples. The findings of the study
might help in extending awareness in dairy farmers and local
people about pesticides and their hazardous effects on human.

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References
1) Astha Pandey et al: “Detection of Pesticides in Buffalo Milk Collected from
Different Areas of Gandhinagar and Ahmedabad by Thin Layer Chromatography”
International Journal of Chem Tech Research, 2017,10(1):231-238.
2) Z.Ishaq & M.A. Nawaz: “Analysis of contaminated milk with organochloride
pesticide residues using gas chromatography” International Journal of Food
Properties,2018,21(1):879-891. https://doi.org/10.1080/10942912.2018.1460607
3) Cihat Ozdemir et al: “Determination of organochlorine pesticide residues in
pasteurized and sterilized milk using QuEChERS sample preparation followed by
gas chromatography-mass spectrometry” Journal of Food processing and
preservation, 2019;00:e14173. https://doi/10.1111/jfpp.14173

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Methods For Estimation Of Pesticide Residue in Milk

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