PESTICIDES RESIDUES

Progress Report
Submitted to Subharti University
For partial fulfilment of the degree of
Bachelor of Technology
By
TANYA VERMA
B.Tech (Food Technology)
Batch: 2018-22
[1801000021853]
Submitted to
MR. KAPIL KUMAR

DEPARTMENT OF FOOD TECHNOLOGY

SUBHARTI UNIVERSITY, MEEERUT
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 ACKNOWLEDGEMENTS

It is always a pleasure to remind the fine people in the SWAMI VIVEKANNAND

SUBHARTI UNIVERSITY for their sincere guidance I received to upload my practical as well

laboratory skills in “INSTRUMENTATION AND PESTICIEDS”. I would like to express my

special thanks of gratitude to my teacher MR.KAPIL KUMAR, who gave me the golden

opportunity to do this wonderful project of PESTICIDES RESDIDUES on “NATIONAL

COLLATERAL MANAGEMENT LIMITID COMPANY”, who also helped me in completing my

project. I came to know about so many new things and I am really thankful to them. I received from

their almost for all the work I did there.

Secondly I would also like to thank my parents and friends who helped me a lot in finalizing this

project within the limited time frame.

Finally I apologize all other unnamed who helped me in various ways to have a good training.

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 INDEX

S. NO. CONTENT PAGE NO.

1. INTRODUCTION

2. PESTICIDES
i) Classification Of Pesticides
ii) Pesticide Residues
iii) Health Effects of Pesticides
iv) Brief description of Pesticides
analyzed

3. INSTRUMENTATION

4. MATERIALS AND METHODOLOGY

i) Requirements
ii) Sample
5. RESULT AND DISCUSSION

6. CONCLUSION

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 INTRODUCTION

1. PESTICIDES

Pesticides are the chemical products used for plant protection. According to FAO (1989) a pesticide
is any substance or mixture of substances intended for preventing, destroying, or controlling any
pest including vectors of human or animal diseases, unwanted species of plants or animals causing
harm during, or otherwise interfering with, the production, processing, storage, or marketing of
food, agricultural commodities, wood and wood products, or animal feedstuffs, or which may be
administered to animals for the control of insects, arachnids or other pests in or on their bodies. The
term includes chemicals used as growth regulators, defoliants, desiccants, fruit thinning agents, or
agents for preventing the premature fall of fruits, and substances applied to crops either before or
after harvest to prevent deterioration during storage or transport.

The term, however excludes such chemicals used as fertilizers, plant and animal nutrients, food
additives and animal drugs. The term pesticide is also defined by FAO in collaboration with UNEP
(1990) as chemicals designed to combat the attacks of various pests and vectors on agricultural
crops, domestic animals and human beings. The definitions above imply that, pesticides are toxic
chemical agents (mainly organic compounds) that are deliberately released into the environment to
combat crop pests and disease vectors. They include Insecticides, Fungicides, Herbicides and plant
growth regulators.

1.1 Classification Of Pesticides

The word "pesticide" is an umbrella term for all insecticides, herbicides, fungicides, and
rodenticides, wood preservatives for termites, garden chemicals and household disinfectants that
maybe used to kill pests. There are three most popular ways of classifying pesticides which are:
a) classification based on the mode of action
b) classification based on the targeted pest species
c) classification based on the chemical composition of the pesticide

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2.1.2 Classification Of Pesticides Based On The Targeted Pest Species
In this type of classification, pesticides are named after the name of the corresponding pest in
target
Table 1: Classification of Pesticides based on Targeted Pest species

Type of pesticide Target organism/pest

Herbicides Weeds
Insecticides Insects
Rodenticides Rodents
Fungicides Fungi
Bactericides Bacteria

2.1.3 Classification Of Pesticides Based On The Chemical Composition

Under chemical classification, pesticides are categorized according to the chemical nature of the
active ingredients. The chemical classification of pesticides is by far the most useful
classification as this kind of classification that gives the clue of the efficacy, physical and
chemical properties of the respective pesticides, the knowledge of which is very important in the
mode of application, precautions that need to be taken during application and the application
rates. Based on chemical classification, pesticides are classified into four main groups namely;
organochlorines, organophosphorus, carbamates and pyrethrin & pyrethroids (Buchel, 1983).

2. INSTRUMENTATION

2.1 Liquid Chromatography:

Liquid chromatography is a technique used to separate a sample into its individual parts. This
separation occurs based on the interactions of the sample with the mobile and stationary phases.
Because there are many stationary/mobile phase combinations that can be employed when
separating a mixture, there are several different types of chromatography that are classified based on
the physical states of those phases. Liquid-solid column chromatography, the most popular
chromatography technique and features a liquid mobile phase which slowly filters down through the
solid stationary phase, bringing the separated components with it. The first known chromatography

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is traditionally attributed to Russian botanist Mikhail Tswett who used columns of calcium
carbonate to separate plant compounds during his research of chlorophyll. This happened in the 20 th
century (1901). Further development of chromatography occurred when the Nobel Prize was
awarded to Archer John Porter Martin and Richard Laurence Millington Synge in 1952. They were
able to establish the basics of partition chromatography, and also develop Plate theory.

4.1. Chemicals
a) Formic Acid
b) Acetonitrile
c) Ammonium Formate
d) Milli Q
e) Magnesium Sulfate (MgSO4)
f) Sodium Chloride (NaCl)
g) Calcium chloride (CaCl2)
h) Tri-sodium citrate dihydrate
i) Disodium hydrogen citrate sesquihydrate
j) Primary Secondary Amine (PSA)
k) Graphitised carbon black (GCB)

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PROCEDURE:
CEREALS/PULSES/NUTS/FOOD ADDITIVE:
Sample preparation of pesticides residues in cereals,pulses,nuts,food additives and their
produts by LC and GC-MS/MS.
 Weigh 5g sample for cerals and weight 2g for nuts, in 50ml centrifuge tube.
 Add 10ml of water and shake 30 seconds.
 Add 10ml CAN contain 1% acetic acid.
 Add 4gm Mgso4,1gNacl,1g trisodium citrate dihydrate, 0.5g Disodium
hydrogencitrate sesquihydrate.
 Vortex for 1 minute.
 Trasfer 6 ml of organic layer in 15 ml centrifuge tube contain 150 mg PSA and 900
mg magnesium sulphate and vortex. For 3sec.
 Centrifuge at 4000 rpm for 5 minutes
 Transfer 1 ml organic layer in amber vial and inject
 Transfer 2 ml layer from 15 ml centrifuge tube and drying in turbo evaportator at 40
C temperature
 Reconstitute 1 ml with ethylacetate and water.
 Filter 1 ml and injected into G.C.

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Oil [pesticides parameter]

 Weight 2gm of sample oil

 Add 10ml of 100% ACN
 Vortex for 1 min.
 Centrifuge for 10 minute at 4000 rpm.
 Transfer 7-8ml of organic layer in 15ml tube.CC
 Add 900 mg Mgso4, 150g PSA, 350g C18, 50mg GCB.
 LC-2ML in test tube and evaporated and reconstitute.
 GC-2ML in test tube and evaporated and reconstitute.

PESTICIDES IN FRUITS AND VEGETABLES:

 Weight 10gm of sample in 50ml tube.

 Add 10 ml ethyl acetate.
 Vortex for 1 minute.
 Add 10gm sodium sulphate anhydrous, and 1gm NaCl.
 Vortex for 1 minute.
 Put it into in centrifuge for 10 minute.
 To make cleanup , we take15 ml tube in which add 750mg magnesium sulphate, 125mg PSA, and
20mg GCB.
 Centrifuge for 10 minutes.
 LC-2ml and add 200ml 10% DEG in methanol.
 Evaporation and reconstitute 1ml methanol and 1ml water containing 0.1% acetic acid.
 Filter and transfer the layer.
 GC- Direct transfer filter.

MELAMINE [LC]:
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  Weight 1ml sample in 50ml test tube.
 Add 10ml H2O and ACN (100%) in sample.
 Vortex for 1 minute.
 Centrifuge 10 minutes at 4000rpm.
 Add C18(150mg) in 15ml test tube.
 And extract in vials with filtration.

FAME [FATTY ACID PROFILING]

 Take 100mg [15ml centrifuge tube].

 1ml Toluene.
 2ml BF3 [BORONTRICHLOROFULORIDE COMPLES].
 Vortex for 5 minute.
 Water bath 75 degree Celsius for 1 hour.
 Kept out cool room temperature.
 Add water 3ml.
 Sodium sulphate 50mg, layer separate.
 1ml n-hexane
 Vortex for 2 minute.
 Centrifuge for 10 minutes at 4000rpm.
 Direct upper layer, in white vials

SPICES/HERBES/CONDIMENTS:

Sample preparation of pesticides residue in spices, herbs, and condiments by LC and GC-MS/MS.

 Weight 2gm sample for spices 50 ml in centrifuge tube.

 Add 10ml water and vortex for 1 minute.
 Add 10ml ACN contain 1% acetic acid.
 Add 4gm MgSO4, 1gm NaCl, 1g, trisodium citrate dehydrate and 0.5g disodium hydrogen citrate
sesquihydrate.
 Vortex for 1 minute.
 Centrifuge for 4000 rpm for 5 minutes.
 Transfer 7ml of organic layer in 15 ml centrifuge tube contain 1.2g MgSo4, 350mg PSA, 350mg C18
and GCB (if color pigment is present).
 Vortex for 1 minute.
 Centrifuge at 4000rpm for 5minutes.
 LC-MS/MS – transfer 3 ml organic layer and drying in turbo-evaporator.
 Reconstitute with 1ml of methanol : water.
 Filter into amber vial and injected into LC-MS/MS.
 GS-MS/MS transfer 3ml of layer from 15ml centrifuge tube and drying in turbo evaporator.
 Reconstitute 1ml of ethyl acetate.
 Filter into white vial and injected into GC-MS/MS.

HONEY/MILK

PROCEDURE FOR DETERMINATION OF NITROFURAN PARENT COMPONENTS IN MILK AND HONEY.

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  Weight 10gm of sample for honey 50ml in centrifuge tube.
 Add 8ml of water and vortex for 1 minute.
 Add 10ml of 1% acetic acid in ACN and vortex for 1 minute.
 Add 4g Na2SO4, 1g NaCl , 1g sodium citrate dehydrate , 0.5g Na2H citrate sesquidrate and vortex
for 1 minute.
 Centrifuge at 4000 rpm for 5 minutes.
 Transfer 8ml of ACN layer to another tube containing 900 mg MgSO4, 150mf PSA, 150mg C18.
 Vortex for 1 minute.
 Centrifuge at 4000 rpm for 5 minutes.
 Transfer 5ml of supernatant to a test tube.
 Evaporate to dryness by passing nitrogen gas.
 Reconstitute to 1ml wuth 70:30 [5Mm Ammonium acetate: ACN].

PROCEDURE FOR DETERMINATION OF ANTIBIOTIC RESDUES IN MILK, MEAT,

EGG, ANIMAL FEED, FISH, SEA FOOD AND THEIR PRODUCTS BY LC-MS/MS.[CAP]

 Weight 10g sample into a 50ml tube and 10ml water.

 Add 0.1ml of 30 micro/internal standard.[30ppb spike, 100 microliter.
 Vortex for 2 minutes.
 Add 10ml acetonitrile and vortex for 2 minutes.
 Add 4gm MgSO4 and 1gm NaCl.
 Vortex for 30 seconds and centrifuge at 4000 rpm for 10 minutes.
 Dry the ethyl acetate layer under turbo evaporator at 40 degree Celsius.
 Reconstitute the sample to water : methanol(60:40).
 Vortex for 15 seconds.
 Filter through 0.22 PVDF filter and inject the sample into LC-MS/MS.

ACRYLEAMIDE [LC]:

 Weight 2gm of sample in 50ml tube.

 Add 10ml water and 10ml ACN(100%).
 Vortex for 1 minute.
 Transfer organic layer 6ml in 15 ml tube.
 Add MgSo4, 1gm NaCl, 1.5g trisodium, di-sodium(0.5g).
 Vortex and centrifuge.
 Transfer 2ml in the test tube ad evaporate.
 Reconstitute 80 : 20 (H2O : ACN) 1ml in vials.

CHOLESTROL:

 Weight 1gm liquid, 3gm solids in amber flask.

 Add 40 ml of 95% ethanol and 8ml KOH(50%)

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  Magnetic stirrer two plat with condenser for 70 minutes.
 After that, turn of heating plates & add 95% ethanol(60ml) through top of condenser.
 Cooled at 10 minutes.
 Transfer in separating funnel. Add 100 ml toluene.
 Wash with 110ml of 1 molar KOH solution.
 Wash with 40 ml of 0.5 Molar KOH.
 Wash with 40 ml of H2o at least 3 times at a time
 Until toluene layer in crystal clear.
 Filter toluene layer into the conical flask
 25 ml extract in ria vials & evaporate until dryness 40 degree Celsius.
 3 ml acetone & evaporate again.
 Reconstitute with DMF(1ml)
 Transfer 1 ML in 15Ml tube added chlorotrimethyle saline (100micro liter)
 Hexamethyldisilane (200 Ml)
 Kept at room temperature (for 15 Min.)

ORGANIC ACID:

 Weight 2gm of sample.

 Makeup sample by 50 ml ultra pure water.
 Vortex for 5 minutes.
 Centrifuge for 5 minutes.
 Filtration of the sample.
 Mobile phase of organic acid.
 1.34gm KH2PO4 [potassium dihydrogen phosphate].
 500 ml distilled water (Ph-2.8).

BETA CAROTENE:

 Weight 1 gm of sample.
 Makeup 15 ml petroleum ether.
 Vortex for 5 minutes.
 Centrifuge 5 minutes.
 Transfer upper layer in another tube.
 Repeat three times at a time.
 Take 5-10 ml in test tube.
 Evaporation.
 Reconstitute 1ml of methanol.
 Filtration done.

HYPORSINE:

 Weight 1gm of sample.

 Makeup 10ml methanol.

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  Vortex for 5 minutes.
 Centrifuge 10 minutes.
 Filtration.

ANTIOXIDENT IN COLD DRINK:

 Weight 2gm of sample.

 Add up to 20 ml ACN.
 Vortex for 5 minutes.
 Centrifuge for 10 minutes at 4000 rpm.
 Filtration.

PRESERVATIVES:

 Weight 2gm of sample.

 Add 1ml zinc acetate and 1ml ferrocynate.
 Vortex for 5 minutes.
 Centrifuge for 5 minutes.
 Filtration.
 Linearity points-1, 2, 5, 10, 20, 50 ppm.

VITAMIN A & E:

 Weight 2gm of sample.

 Add 50mg butylated hydroxytoluene (BHT).
 Add 4ml KOH (50%).
 Makeup 20ml ethanol.
 Reflex 75degree Celsius for 50 minutes.
 Cool for 5 minutes.
 4ml acetic acid mixer.
 Makeup 40ml by THF & ethanol 1:1.
 Vortex for 10 minutes and filter the pure layer.

ABBREVATIONS:

DILUTION

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  0.1% formic acid to makeup in 250ml.
 Instrument use (micro ppt)
 Tip (1ml) blue.
 Tip (200ml)yellow.
 Vail ( brown / white).
 Stock – 100 ppm.
 LC- Methanol + water.
 GC-Ethylacetate(900) + 100ml sample.
 Three types of linearity are aqueous, Matrix match, based linearity.

COMMODITIES:

 CEREALS - 5gm
 FRUITS AND VEGETABLES - 10gm
 SPICES - 2gm
 MEAT AND MEAT PRODUCTS - 10gm
 OIL AND OIL PRODUCTS - 2gm
 SUGAR AND SYRUP - 2gm
 ANTIBIOTICS - 10gm

CLEANUP SETUP:

 CEREALS ---- PSA & MgSO4.

 SPICES, TEA, OIL, F & V, TOBBACCO ----- PSA, MgSO4, C18, GCB.

QUALITY ASSURANCE & QUALITY CONTROL:

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 Quality assurance can be defined as "part of quality management focused on providing
confidence that quality requirements will be fulfilled." The confidence provided by quality
assurance is twofold—internally to management and externally to customers,
government agencies, regulators, certifiers, and third parties. An alternate definition is
"all the planned and systematic activities implemented within the quality system that can
be demonstrated to provide confidence that a product or service will fulfill requirements
for quality."

 Quality control can be defined as "part of quality management focused on

fulfilling quality requirements." While quality assurance relates to how a process is
performed or how a product is made, quality control is more the inspection aspect of
quality management. An alternate definition is "the operational techniques and activities
used to fulfill requirements for quality."

CONCLUSION
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 The “NATIONAL COLLATERAL MANAGEMENT Ltd. was a very good
learning experience as it helped me to learn practical aspects of the food industry like
food processing, quality control, quality assurance, food safety, packaging etc.
Analyzed various criteria of testing the product from raw materials pre to the final
finished product which gives the product its standard quality.

The several criteria of quality control include instrumentation, chemical and

microbiological analysis. There were several tests like pesticides analysis, oil content,
FFA and finished products etc. which not only assures the good taste and shelf life of
the product but also the quality of the product which is popular among consumers for
its assurance that it is safe and healthy.

On analysis I found that the product is good in all parameters of quality and within
the permissible limit which is obtained by utilization of instrumentation, chemical,
microbiological, nutritional and sensory parameters. Quality control and assurance is
an important part of any food industry, it plays a vital role for providing safety to the
consumers.

The guidelines issued by the certified bodies are followed strictly. The efficiency of
effluent treatment plants is such that it does not produce any hazard or harmful effect
to the environment.

A proper dress-code and decorum of the company is maintained. The quality standard
of NCML could be understood by the fact that there is significantly high brand
loyalty for NCML products among the consumer. Market share of NCML is more
than double to its competitors.

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