RESIDUAL PESTICIDES ANALYSIS of VERIOUS VEGETABLES by GC-MS Jyotindrakumar J Bhatt1*, H

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications

Original Research Article DOI: 10.26479/2019.0501.06 RESIDUAL PESTICIDES ANALYSIS OF VERIOUS VEGETABLES BY GC-MS Jyotindrakumar J Bhatt1*, H. P. Gajera2, Daya B. Dobariya1, Mrugesh H Trivedi3 1.Department of Chemistry, KSKV Kachchh University, Bhuj, Gujarat, India. 2.Department of Biotechnology, Junagadh Agriculture University, Junagadh, Gujarat, India. 3.Department of Earth & Environmental Science, KSKV Kachchh University, Bhuj, Gujarat, India

ABSTRACT: This reported work describes residual pesticide analysis in different vegetables, during the period first quarter of the year. A Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method (AOAC Official Method 2007.01) was used to extract pesticide residues from vegetable samples. The vegetable, fruits and grain market-yard of Junagadh the district place of Gujarat is the selected place from which the vegetables are collected. In this study we reported the determination of pesticide residues from selected eight vegetables like cauliflower, brinjal, tomato, cabbage, cluster bean, bottle guard, okra, and chili. The samples were prepared by usual and established method and followed by extraction were subjected to analyses. The extracted pesticide residues were analyzed and quantified by standardized Gas Chromatography/Mass Spectrometry (GC-MS) method developed for 35 pesticide standards. The literature survey of the study of various samples, we tempted to do further study for the residue pesticides and residual insecticides analysis with more interest of its penetration and quantification in vegetables in order to understand the adverse effects and toxicity. The results indicate the presence of pesticide residues in some of the samples above the stated MRL (Maximum Residue Limit) value.

KEYWORDS: Pesticides, GC-MS, Vegetables, Human Health.

Corresponding Author: Dr. Jyotindrakumar J Bhatt*Ph.D. Department of Chemistry, KSKV Kachchh University, Bhuj, Gujarat, India. Email Address: [email protected]

1.INTRODUCTION The aim of this undertaken work is to describe the pesticide residue in fruit and vegetable, mainly how they are introduce, dissipated and degraded. Vegetable are important components of human diet

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications since they provide essential nutrients that are required for most of the reactions occurring in the body. A high intake of fruits and vegetables has been encouraged not only to prevent consequences due to vitamin deficiency but also to reduce the incidence of major diseases such as cancer, cardiovascular diseases and obesity. Like other crops, vegetables are attacked by pests and diseases during production and storage leading to damages that residue and quality and the yield. [1, 2]. A pesticide can also be introduce according to their, Chemical substance, application on different Biological agents (such as virus or bacteria), as disinfectant, antimicrobial agent or its using like device. The Pesticide residue refers to the pesticides that may 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. Exposure of the general population to these residues most commonly occurs through consumption of treated food sources, or being in close contact to areas treated with pesticides such as farms or lawns around houses [3].Pesticides are often referred to according to the type of pest they control. Another way to think about pesticides is to consider those that are chemical pesticides or are derived from a common source or production method. Other categories include bio pesticides, antimicrobials, and pest control devices. Once it reaches the target pest, the chemical may act in different way likewise,blocking the cellular processes of target organisms in a purely mechanical way, by this the pesticide physically prevents a basic cellular function even without any chemical reactions. Destroy or alter the pest’s metabolism. Examples include inorganic copper compounds, dithiocarbamate fungicides, phosphono amino acid herbicides and organophosphate insecticides [4].The application of pesticides has a significant effect on biodiversity. These affect the ability of soil to regenerate itself and remain viable for plant and animal life [5].Environment devastated by pesticides may take years to recover. In some cases, it may never recover at all!Maximum residue levels are the highest levels of residues expected to be in the food when the pesticide is used according to authorised agricultural practices. The MRLs are always set far below levels considered to be safe for humans. Safety limits are assessed in comparison with acceptable daily intake (ADI) for short term exposure or acute reference dose (ARfD). MRLs are subject to legal requirements in most of the countries. MRL setting is based on the national registered good agriculture practice (GAP) data combined with the estimated likely residue from the supervised trials mean residue (STMR), ADI and ARfD. MRLs may be exceeded because of pesticide misuse, false positives due to naturally occurring substances, differences in national MRLs, lack of registered pesticides and incorrect pesticide application [6]. In this present study, systematic path way for residual pesticide analysis have been developed. Generally pesticides occur in food in very low concentration, usually at ppm level. Measuring such a small amount of pesticides is the function of pesticide residue analysis. A variety of analytical methods are currently used to detect pesticide residue, and contain certain basic steps, that include, Sampling (Sampling procedure for

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications vegetablesby collection, transport and storage of sample) [7]. Detection is based on Trace Level Analysis of Pesticide Residues [8]. Extraction method for pesticide residue analysis [9]is having special attention of the researchers.The residues of pesticides have been generally analyzed by gas chromatography with different detectors. High performance liquid chromatography (HPLC) [10] has been also employed, particularly when pesticides are thermally instable. Gas chromatography coupled with mass spectrometry (GC-MS) is more often used at present for pesticide analysis from soil [11-17] due to the possibility of confirming pesticide identity. The main objective of this work was to develop a rapid and simple multi residue method for the analysis of pesticides in our samples, based on the QuEChERS extraction method using a low volume of organic solvent and their determination by GC-MS[18]. 2. MATERIALS AND METHODS The present experimental work on collecting samples and extractions have been carried out systemically in order to approach the system which established from reported survey [19-30]. Analyses was done by the Equipments utilized for sample preparation are like, Centrifuge :REMI Research centrifuge, Vortexer : GeNeiTm, Turbovapour: Caliper Turbovap L-6,Analytical Balance:0.1 mg – 5.0 gm (LC-GC Analyticals), Auto-Pipette: volume range Genaxy(GENPET Auto Pipette), 0.2Micron nylon membrane filters from Himedia and finally GCMS System: GC-2010 plus (Shimadzu). Sample preparation for QuEChERS Organic bottle top dispense, Trace HP-5MS Pesticide (length 30m,diameter 0.250mm, fit thickness 0.25 µm),2 mL amber glass vile, 50 ml FEP centrifuge tubes, Clean up tube: 15 mL tubes , Clean up tube: 2 mL tubes have been taken for preparation and preservation of our samples. Reagents and chemicals that used for treating samples are 15 ml of 1% acetic acid, Acetonitrile (v/v) HPLC grade,6 gm MgSO4 (anhydrous) and 1.5 gm sodium acetate(anhydrous),ENVIRO 900 mg MgSO4, 300 mg PSA 150 mg, 150 mg MgSO4, 50 mg PSA (pk of 100). The Standard pesticide stock solutions were prepared by diluting 1.00mg of each standard pesticide in 10.00mL of HPLC grade (Fisher scientific) ethy-l acetate. The final concentration was 1.00 mg/mL or 100 ppm. For each pesticide standard mentioned above, Stock Solutions (SS) of 100 ppm concentration were prepared in ethyl acetate. A pesticide intermediate standard solution Stock Dilution (SD) 10ppm was prepared by transferring 1 mL from each pesticide Stock Solutions (SS) to a 15ml tarsons tube and diluting to volume with ethyl acetate to obtain a concentration of 100µg/mL.The stock dilutions were then further diluted to prepare Working Solutions (WS) of 1 ppm concentration in ethyl acetate. The working solutions were then used to prepare pesticide standard solution of 500 ppb, 200 ppb, 100 ppb, and 50 ppb and 10 ppb concentrations. The pesticide standard solutions of 10 ppm was used to tune the instrument for pesticide residue analysis in the scan mode, while pesticide standard

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications solutions of 1ppm, 500 ppb, 100 ppb, 50 ppb and 10 ppb concentrations were used to tune the instrument in the SIM (Selective Ion Monitoring) mode. Instrument: GC-MS - Model:- QP2010 Plus Table 1: Criteria, parameter with condition GC-Program HP-5MS (Crossbond, 5% diphenyl / 95% dimethylpolysiloxane) Column: 30 m x 0.25 mm I. D. df = 0.5 µm o - Initial temp.120 C hold for 1 min; o o - Temp. ramping 8 C /min, upto 150 C, hold for 1 min; Column Oven o o - Temp. ramping 5 C /min, upto 200 C, hold for 1 min; Temperature Program: o o - Temp. ramping 4 C /min, upto 262 C, hold for 2 min; o o - Temp. ramping 4 C /min, upto 285 C, hold for 2.5 min. Carrier Gas: Helium (99.999% pure) Injection Temperature: 280 ºC Injection Method: Splitless (1 min), high pressure injection @ 250 psi Injection Volume: 1 µL Total Run Time: 42.50 Solvent cut time: 4.0 min Interface Temperature: 290 ºC Ion source Temperature: 230 ºC Ionization Mode: EI (Electron Ionization) Scan Range: 50-500 m/z Table 2:Method development of Pesticide Standards for Pesticide Residue Analysis

Mobile Phase Retention Time Ionization Pesticide Standard (A) 0.1 % Formic Acid (B) Acetonitrile (min) Mode in MilliQ

Imidacloprid 90% 10% 0.34 ESI +

Thiamethoxam (50%) (50%) 0.33 ESI +

Thiodicarb (90%) (10%) 0.33 ESI +

CartapHydrochloride (90%) (10%) 0.34 ESI +

Diafenthiuron (10%) (90%) 0.55 ESI +

Imazethepyr (90%) (10%) 0.49 ESI + © 2019 Life Science Informatics Publication All rights reserved Peer review under responsibility of Life Science Informatics Publications 2019 Jan – Feb RJLBPCS 5(1) Page No.56

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications LC-MS-MS method: Model: Waters Acquity UPLC-PDA, TQD mass detector. Extraction Method& Quantification of Pesticide Residues from vegetables: Vegetables Sampling:Vegetable sampling was done from the Junagadh District of Gujarat. 8 different type of vegetables namely, Cabbage (V1), Cauliflower (V2), Cluster Bean (V3), Bottle guard (V4), Okra (V5), Brinjal (V6), Chilli (V7), and Tomato (V8) were collected from the Junagadh Vegetable Marketing Yard. Procedure for sampling: 1 Kg of each vegetable was purchased from two different vendors. They were then mixed and stored in polyethylene bags and labeled properly. They were stored in the laboratory at 2°- 8°C until analyzed.

V1- Cabbage V2-Cauliflower V3-Clusterbean

V4 - Bottle gourd V5- Okra V6- Brinjal

V7– Chilli V8– Tomato

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications QuEChERS sample preparation The determination of pesticides in fruits and vegetables has been simpliﬁed by a new sample preparation method, QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), and published recently as AOAC Method 2007.011 The sample preparation is shortened by using a single step buffered acetonitrile (MeCN) extraction and liquid-liquid partitioning from water in the sample by salting out with sodium acetate and magnesium sulphate (MgSO4). The MeCN extract is solvent exchanged to hexane/acetone for split less injection with detection by electron ionization and selected ion monitoring (SIM). Experimental condition During the method validation, several experiments were performed to determine the effect of minor modiﬁcation to the QuEChERS method which may impact the performance of the analysis in the laboratory. The recommended consumables required for sample preparation and analysis were austerely tested (Table 3). The sections were evaluated for sample preparation are,Sample Extraction and Clean Up, Solvent Exchange, Injection, Separation, Detection 3. RESULTS AND DISCUSSION

Different vegetables viz. Cabbage (V1), Cauliflower (V2), Cluster Bean (V3), Bottle guard (V4),

Okra (V5), Brinjal (V6), Chilli (V7), and Tomato (V8) were collected from market yard of Junagadh (Gujarat) and studied. The residue concentrations were calculated using reference standards. Out of 35pesticides, total 18 pesticide residues like Trifluralin, Cypermethrin, Propiconazole, Quizalofop- Ethyl, Pendimethalin, Triazophos, Fenpropathrin, Propiconazole, Carbofuran,alpha-Lindane, beta- Lindane, gamma-Lindane, Endosulfan-2, delta-Lindane, Methyl-Parathion, Alachlor, Butachlor and Ethion were detected among 8 vegetables. The maximum residues (Nine pesticides) were detected in Okra followed by Brinjal (Six pesticides). However, least residue (One-Propiconazol) was Bottle guard. In cabbage, three residues were detected among which the concentration of Triazophos was found higher. Cauliflower has two pesticide residues with Quizalofop-Ethyl as higher, while Pendimethalin was detected higher in cluster bean. Okra has five pesticide residues among which two pesticides Lindane and Cypermethrin have their isomers (three for each). Thus, total nine residues including their isomers were detected in Okra. Brinjal has 6 residues with Carbofuran as highest one, while Chilli sample had four pesticide residues among which Butachlor was found higher. Methyl-Parathion was detected higher in tomato in addition to two other pesticide residues. Among all the pesticide residues detected in all the vegetable samples, the concentration of Butachlor was highest, while that of Endosulfan-2 was lowest, whose structures are as below.

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Butachlor Endosulfan-2 Table 3:Pesticide residues quantified from samples

Name of Pesticide(s) found Concentration MRLs (mg/ kg) the vegetable & (mg/Kg) / (As per std. of 2013) Code No. Codex EU

V1 Trifluralin 0.027 - 0.01 Cabbage Triazophos 0.156 - 0.01 Cypermethrin 0.056 1 1

V2 Propiconazole 0.076 - 0.05 Cauliflower Quizalofop Ethyl 0.088 - 0.04

V3 Pendimethalin 0.598 - 0.05 Cluster bean Triazophos 0.211 - 0.01 Fenpropathrin 0.401 - 0.01

V4 Propiconazole 2.602 - 0.05 Bottle gourd

V5 Carbofuran 0.021 - 0.01 Okra alpha-Lindane 4.304 - 0.01 beta-Lindane 0.133 - 0.01 gamma-Lindane 0.400 - 0.01 Endosulfan-2 0.021 0.5 0.05 Triazophos 0.338 - 0.01 Cypermethrin-1 0.196 0.1 0.05 Cypermethrin-2 0.287 0.1 0.05 Cypermethrin-3 0.736 0.1 0.05

V6 Carbofuran 1.789 - 0.01 Brinjal Trifluralin 0.046 - 0.01 gamma-Lindane 0.475 - 0.01 delta-Lindane 0.442 - 0.01 Methyl-Parathion 0.102 - 0.01 Fenpropathrin 0.389 - 0.01

V7 beta-Lindane 2.126 - 0.01

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications Chilli Alachlor 0.299 - 0.01 Butachlor 13.006 - - Ethion 0.161 - 0.01

V8 Methyl Parathion 0.610 - 0.01 Tomato Alachlor 0.072 - 0.01 Fenpropathrin 0.485 - 0.01

(x10,000,000) 1.6 TIC

1.5

1.4

1.3

1.2 37

1 1.1

1.0 19

0.9 10

0.8 30

0.7 23

0.6 8

6 0.5 17

0.4 42

27 0.3 35

0.2 2

9 0.1

4 0.0

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 Figure 1: Chrometograph-1

Figure 2: Chrometograph-2 © 2019 Life Science Informatics Publication All rights reserved Peer review under responsibility of Life Science Informatics Publications 2019 Jan – Feb RJLBPCS 5(1) Page No.60

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications Development of Calibration Curve of Pesticide Standards 1 μL of mixture of 35 pesticide standards at different working concentration (500 ppb, 100 ppb 50 ppb and10 ppb) were injected in to the developed and SCAN mode and SIM. 2 Table 4:Straight Line Equation and R Value of each pesticide standard in CC Set

Sr.No. Pesticide Standard Straight Line Equation R2 value

1. Carbofuran Y = 9616.542X + 25394.2 0.9985434 2. Trifluralin Y = 2373.789X +9891.049 0.9977985 3. Phorate Y = 7168.044X +27948.38 0.9992948 4. Dimethoate Y = 1967.341X -27528.05 0.9961004 5. Fluchloralin Y = 1840.894X + 3704.859 0.9989157 6. Methyl-Parathion Y = 1826.126X +12287.39 0.9859142 7. Alachlor Y = 3772.375X -28848.37 0.9990137 8. Heptachlor Y = 1929.796X -9616.518 0.9979476 9. Malathion Y = 2102.9X - 75749.91 0.9983016 10. Chlorpyriphos Y = 4353.934X+90131.99 0.9963005 11. Aldrin Y = 3374.273X+15733.85 0.998087 12. Pendimethalin Y = 1299.304X -2768.327 0.9979654 13. Butachlor Y = 5011.607X +26036.97 0.9843095 14. Endosulfan-1 Y = 574.0606X + 16593.61 0.9990655 15. Profenofos Y = 643.1272X -9073.941 0.9741099 16. Endosulfan-2 Y = 1194.472X +15636.13 0.9965006 17. Ethion Y = 3930.323X -68253.03 0.9933582 18. Triazophos Y = 1930.781X -53887.57 0.982844 19. Quizalofop-ethyl Y = 3870.936X -145147.5 0.9901825 20. Cabaril Y = 9616.542X +25394.2 0.9985434 21. a-BHC Y = 94.4106X +94108.07 0.6315221 22. b-BHC Y = 1212.904X +6387.056 0.9995311 23. g-BHC Y = 769.121X + 29049.18 0.9642116 24. d-BHC Y = 756.0549X +57914.01 0.9918748 25. Fluchlolalin Y = 1840.894X +3704.859 0.9989157 26. Fipornil Y = 6936.443X -221473.4 0.9911576 27. Oxyflurofen Y = 831.2414X -29468.57 0.9895674 28. Propiconazol-1 Y = 1832.952X -32373.87 0.9992266

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications 29. Propiconazol-2 Y = 2621.924X -41706.33 0.9990739 30. Bifenthrin Y = 9984.335X -82663.65 0.9991796 31. Fenpropethin Y = 7948.786X -90511.99 0.9993834 32. Cypermethrin Y = 2453.772X +126229.7 0.9720539 33. Fenvelarate Y = 2061.077X +156991.1 0.9785813

Figure: 3. Chromatogram of different vegetables sample (black-V1, pink—V2, blue-V3, brown-V4, green-V5, purpal-V6, light green-V7, black-V8) DISCUSSION 1µL of mixture of 35 pesticide standards at different working concentration (1 ppm, 500 ppb, 100 ppb and 50 ppb) were injected in to the developed and optimized GC-MS method. Depending on the retention time obtained from analysis of each individual pesticide standard and relative reference ions using Total Ion Chromatogram (TIC), the standard analyte in mixture were eluted and confirm by similarity search from the inbuilt Pesticide Library an NIST Library.The peaks of each individual pesticide standard obtained were integrated manually for each working concentration. A calibration plot was developed depending upon Area vs. Concentration. From each individual CC plot of standard, a straight line equation was obtain. From the straight line equation the concentration of residues obtained from the vegetable sample will be quantified in ppb concentration.The chromatogram of the mixture of 35 pesticide standards injected in a single run of GC-MS and the retention time (RT) of each individual pesticide standards and their relative isomers, majored. There was no effect of mixture of pesticide on retention time obtained when individual standards were © 2019 Life Science Informatics Publication All rights reserved Peer review under responsibility of Life Science Informatics Publications 2019 Jan – Feb RJLBPCS 5(1) Page No.62

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications eluted. The peaks obtained show more than 80 % similarity with the inbuilt NIST and Pesticide Library.Figure-shows the relative intensities of chromatogram obtained at different concentration of pesticide mixture.Figure-shows the calibration plot of pesticide standards Phorate, Dimethoate,β- BHC etc. As these pesticides residue were extracted from the collected vegetable samples used in this study Table 5: Retention Time, peak area and identified compounds from samples

VEGETABLE SAMPLE-V1 to V8 Retentio Straight Line Concentration Compound Sr. No. n Time Area Equation from CC of pesticide Identified (min.) Set Residues(ppb) (Cabbage) Y = 1212.904X 11.65 60676 678.879 beta-Lindane V1 +6387.056

Y = 3772.375X - 15.92 14777 758.016 Alachlor 28848.37

Y = 5011.607X 21.08 28205 876.340 Butachlor +26036.97

Y = 3930.323X - 24.21 124481 268.752 Ethion 68253.03 Y = 1832.952X - Cauliflower 25.75 121835 237.637 Propiconazole 32373.87 V2 Y = 3870.936X - Quizalofop 35.51 90473 356.645 145147.5 Ethyl Cluster Y = 1299.304X - 19.19 54303 600.314 Pendimethalin Bean 2768.327

V3 Y = 1930.781X - 24.81 57321 156.656 Triazophos 53887.57 Y = 7948.786X - 28.50 131939 Fenpropathrin 90511.99 640.204 Bottle Guard 25.75 121835 Y = 1832.952X - 237.637 Propiconazole

V4 32373.87 Okara Y = 9616.542X + 4.34 315855 875.456 Carbofuran V5 25394.2 Y = 94.4106X 8.32 138767 875.980 alpha-Lindane +94108.07

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications Y = 1212.904X 11.65 60676 678.879 beta-Lindane +6387.056 Y = 769.121X + gamma- 12.76 44141 245.968 29049.18 Lindane Y = 1194.472X 23.32 126868 875.765 Endosulfan-2 +15636.13 Y = 1930.781X - 24.81 57321 276.572 Triazophos 53887.57 Cypermethrin Y = 2453.772X 35.14 54641 2077.472 -1 +126229.7

Y = 2453.772X Cypermethrin 35.45 72570 25.850 +126229.7 -2 Y = 2453.772X Cypermethrin 35.65 100668 25.850 +126229.7 -3 Brinjal Y = 9616.542X + 27.99 273240 875.456 Carbofuran V6 25394.2 Y = 1930.781X - 11.25 110074 156.656 Trifluralin 53887.57 Y = 1212.904X gamma- 11.65 60676 245.968 +6387.056 Lindane Y = 1212.904X 12.96 88539 245.968 delta-Lindane +6387.056 Y = 1826.126X Methyl- 15.59 155723 355.469 +12287.39 Parathion Y = 7948.786X - 28.50 131939 640.204 Fenpropathrin 90511.99 Chilli Y = 1212.897X beta-HCH 11.65 60567 678.669 V7 +6387.056 Y = 3772.367X – 15.92 14767 758.011 Alachlor 28848.37 Y = 5011.589X 21.08 27901 142.507 Butachlor +26036.97 Y = 3930.318X - 24.21 124468 268.752 Ethion 68253.03

Bhatt et al RJLBPCS 2019 www.rjlbpcs.com Life Science Informatics Publications Tomato Y = 1826.126X Methyl 15.59 155723 355.469 V8 +12287.39 Parathion Y = 3772.375X - 15.92 14777 758.016 Alachlor 28848.37 Y = 7948.786X - 28.50 131939 640.204 Fenpropathrin 90511.99 4. CONCLUSION The results from the above study highlighted the presence of pesticide residues in samples. Some different types of pesticides are present in one type of vegetable indicating the increasing amount of use of pesticides. Hence, it can be absorb in vegetable samples in law to moderate amount. Study has been done with systematic process via collection, preservation followed by extraction. The standard method was applied to treat the collected vegetables for sample preparation. The standards were taken as reference and each of for calibration in order to quantify and compare with known molecules of pesticides. Identification of such type of pesticides has been done with compare to that of known. The main objective was to conduct trace level analysis of pesticide residues from vegetable samples using GC–MS technique. The well established QuEChERS method is used with its physico-chemical parameters for extraction of pesticide residue. The extracts were reconstituted in appropriate solvent such as ethyl-acetate and analyzed by GC–MS. Finally the analyzed samples were quantified using calibration curve of 35 pesticide standards by GC–MS. The entire study is fulfilling the aim to show the non-negligible presence of the pesticides in vegetable samples. Results are indicating the adverse effects and toxicity of these pesticides also harmfulness for human being. ACKNOWLEDGEMENT Authors are thankful to the authorities of Gujarat Agricultural University, Junagadh and the Department of Chemistry, KSKV Kachchh University, Bhuj for providing research facility. CONFLICT OF INTEREST Authors have no any conflict of interest. REFERENCES 1. KeikotlhaileB.M., and SpanogheP., Psticidee residues in fruit and vegetables, Ghent University Belgium, www.interchopen.com 2. http://www.businessdictionary.com/definition/pesticideresidue.html#ixzz2tB941shj 3. Pritam S. & Mukherjee I.,Substitution of Toxicologically Critical Solvents in the Residue Analysis of Acetamiprid: Towards Green Chemistry, Toxicological and Environmental Chemistry,(2010),Vol.92, No.1, pp. 13-19 4. Govindrajan S., http://www.tc.umn.edu/~allch001/1815/pestcide/sim/background 5. Dr. Sharma K. K., Hand book of pesticide analysis manual, ICAR, 2ndaddition: February, 2013,

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