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TRAINING REPORT

ON

EXTRACTION AND ANALYSIS OF PESTICIDE RESIDUE IN FRUITS AND VEGETABLES BY GC-

MS/MS AND LC-MS/MSBy

DUKALE RAHUL PUNDLIK(BTL/14/2010)

Under the Department of

Post-Harvest and Food Biotechnology

Guide Co- Guide

Mr. V. K. Gupta Prof. S.K. Gadhe

TUV India Pvt. Ltd. college of agricultural Biotechnology

Pune Loni

COLLEGE OF AGRICULTURAL BIOTECNOLOGY,LONI2014

Total slides - Training period: 10 Dec 2013 To 31 Mar 2014

GENERAL INTRODUCTION OF COMPANY

Company profile: Type : Private

Industry : Quality Analysis

M.D. : Dr. Korade N. U.

Analysis of: food and food products

Office : TUV-Nord House, Pune

Web : www.tuv@nord.com

TUV India Pvt. Ltd. Pune

Contents

Introduction Objectives Materials and Methods Results and discussion Summary and conclusion Work outcome References

Introduction

Fruits and Vegetables Important supplement to the human diet as they provide the essential minerals, vitamins and fibre required for maintaining health.

In India, the total fruits and vegetable production is about 137 million tonnes per year.

India contributes 8.56% and 13.5% respectively to world’s fruits and vegetables production.

The presence of pesticide residues is a concern for consumers.

Share of India’s fruit and vegetable production in World

Export of fruits and vegetables Mainly exported to Kuwait, Dubai and Saudi

Arbia.

Fresh onions and mangoes are the main commodities entering in export trade.

Fruits juices, fruit pulp and pickles are mainly imported by the USSR, Yemen, and Arab Republic.

Important fruits exported are Melon, Sweet Melon, Grapes, Pomegranate, Sapota, Custard Apple, Orange, Papaya, Pineapple.

Pesticide

Substances meant for preventing, destroying or mitigating any pest.

Protect plants from damaging influences.

A chemical or biological agent that through its effect deters, incapacitates, kills or otherwise discourages pests.

Used in food production to increase food security despite the fact that they can have negative health effects on consumers.

Pesticide Residue

The pesticides remain on or in food after they are applied to food crops.

The levels of these residues in foods are often stipulated by regulatory bodies in many countries.

Pesticides exhibit bioaccumulation which could build up to harmful levels in the body as well as in the environment.

Found in various fruits and vegetables; both raw and processed.

Dirty Dozen Plus

Apples Strawberries Grapes Celery Peaches Spinach Summer squash

Sweet bell peppers Nectarines-

imported Cucumbers Potatoes Cherry tomatoes Hot peppers Leafy greens

Pesticides and Health

Chronic Impacts Increased risk of cancer

Neurological impairment

Organ damage

Intrusion with the human hormone system

Acute Impacts Eyes: tearing, irritation, conjunctivitis

Skin: rash, blistering, burns, sweating, contact dermatitis, jaundice

Gastrointestinal tract: nausea, vomiting, diarrhea, abdominal pain

Maximum residue limits (MRLs)

Currently, more than 800 pesticides (active ingredients) are sold worldwide.

Many of these compounds, legal action levels in food have been established.

MLRs for pesticide residues are set at European Union (EU) level for approximately 150 plant protection products and at member state level for any other unharmonized products.

In general, the MRLs are in the range of 0.01–10 mg/kg.

In India the APEDA give the list of 181 Pesticides

Principle of GC-MS

GC-MS is a technique which perform sample identification and quantification according to mass, charge and it work on the principle that a mixture will separate into individual substance when heated.

GC-MS is analytical Method which combines the features of gas chromatography and mass spectrometry to identify different substance within test samples.

Principle of LC-MS

Liquid chromatography(LC) separates molecules in a liquid mobile phase using a solid stationary phase. Liquid chromatography can be used for analytical or preparative applications.

Objectives

To study the presence of pesticide residue in fruit and vegetables.

To compare the concentration of pesticide with MRLs as given in APEDA list of 181 pesticide molecules.

Materials and method

Materials Grapes, Pomegranates, Okra, Chilli

RequirementReagent / reference materials Methanol (LCMS grade)

Ethyl Acetate

Water (HPLC Grade)

Anhydrous Sodium Sulphate: - pre-heated for 3 hrs. At 5000C.

Acetic Acid Glacial (HPLC Grade)

Primary Secondary Amine (PSA)

Reference standards of pesticides as per Annexure I (GCMSMS) & II (LCMSMS)

Apparatus Centrifuge Tube (FEP) 50 ml

Micro centrifuge tube- 2 ml

Micropipette- 1 ml

Vials -2 ml

Volumetric Pipette- 10 ml

Equipment Blender

Homogenizer

Centrifuge

Balance

GCMS-MS Triple Quadra pole

LCMS-MS Triple Quadra pole

Multi-residue Method

Weigh 10g of crushed sample + 10 ml D/W + 10 ml Ethyl Acetate

Add 1.5g sodium acetate + 10g activated sodium sulphate

Homogenize for 5 minutes

Centrifuge at 7000 rpm for 5 minutes at 40 c

For GCMS/MS

Take 50 mg PSA in eppendrof tube

Add 2ml of extract

Vortex Centrifuge at 13000 rpm

Filter through 0.2µ membrane filter

Inject into GCMS/MS

For LCMS/MS

Take 50mg PSA in test tube

Add 5ml of extract

Vortex allow PSA to settle

Take above 1ml in another TT + 0.2ml of 10% DEG in methanol

Evaporate to dryness

PGR Method

Weigh 10gm homogenized sample in centrifuge tube

Add 20ml 0.1% formic acid in methanol

Homogenize for 5 minutes

Centrifuge at 7000 rpm for 5 minutes at 4oc

Take 2ml of supernatant in eppendorf tube (containing 50mg C18 powder)

Centrifuge at 13000 rpm for 5 minutes at 4oc

Filter through 0.2µ nylon membrane in vial

Inject in LC-MS/MS

Results and discussion

Multi-residue Method Analysis of Pesticide Residue in Grapes

by GC-MS/MS

Fig.No.1

Quantitation Results

Compound RT Response Final conc.

(mg/kg)

MRLs(mg/kg)

4-Bromo 2-Chlorophen

ol

5.312 110 0.1141 0.01

Lambda-Cyhalothrin

e

14.885 92118 0.1987 0.2

Permethrin II

15.712 14826 0.0419 0.5

Phorate-Sulphone

9.843 18650 0.01737 0.01

Table No. 1

Analysis of Pesticide Residue in Pomegranates by LC-MS/MS

Fig.No.2

Quantitation Results

 Compound  

RT

 Respons

e

 Final Conc.

(mg/kg)

MRLs

(mg/kg)

ACETAMIPRID 

6.388 

546060 

0.011 

0.5

IMIDACLOPRID

 

5.472 

105233 

0.013 

1

DIFENCONAZOLE

 

14.610 

51456 

0.45  0.5

Table No.2

PGR Method

Analysis of Pesticide Residue in Grapes by LC-MS/MS

Fig.No.3

Quantitation Results

 Compound  

RT

 Response

 Final Conc.

(mg/kg)

MRLs

(mg/kg)

2, 4 - D 

4.342 

65 

0.0148 

0.05

CHLORAMQUAT CHLORIDE 

1.339 

9062 

0.03174 

0.05

6 BA 

4.367 

73 

0.01 

0.01

Table No.3

The pesticide residues present in the samples were identified and quantified with reference to standard of pesticides molecules.

Pesticide molecules are frequently found- 4-bromo,2 chlorophenol, Lambda Cyhalothrin, Chlormequat-chloride, Imidacloprid and Acetamiprid.

Pesticide molecule like 4-bromo, 2-chlorophenol found to be maximum concentration than MRL in Table No.1.

Summary and conclusion The analysis of pesticide residue has been

detected by GC-MS/MS and LC-MS/MS.

The extraction is done by the Standard Operating Procedures which are set by the laboratory.

Two major important SOPs i.e. Multi-residue Method and Method for PGR are studied during work.

The molecules with excess concentration than MRLs are failed to achieve the export requirements.

Work Outcome

In present work protocol for “Studies on Extraction and Analysis of Pesticide Residue in Fruits and Vegetables” was successfully completed.

Protocol for the study of Pesticide Residue from Fruits and Vegetables was learnt.

Good laboratory practices were learnt.

Proper instrument handling was learnt.

Gained knowledge of thesis writing and presentation skills.

REFERENCES FAO. Agricultural data. FAOSTAT (2005).

EU. Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. Off J L230:1–32.Brussels. (1991).

USA. Food Quality Protection Act (Public Law 104–170, Aug, 1996).disposable at http://www.epa.gov/ (1996).

EU. MLRs sorted by commodity updated 04/11/2004, Disposable at http://www.europa.eu.int/comm/food/plant/protection/pesticides/index_en.htm, (2005b).

TUV/03/SOP/001

Ganry, J. ‘Fruits and Vegetables for Healthy diet in Developing Countries’. ActaHort 2005; 744:55- 60.

Gurevitch, J., Hedges, L.V., 1999. Statistical issues in ecological meta-analyses. Ecology 80 (4), 1142–1149 (Available from: ISI: 000081368500005).

Hayes T.B., Falso P., Gallipeau S., and Stice M. “The cause of global amphibian declines: a developmental endocrinologist’s perspective”. The Journal of Experimental Biology 2010; 213:921-933. doi:10.1242/jeb.040865.

Thank You