Method Validation for Qualitative and Quantitative Analysis of Pesticide Residues in Tomato with GC-MS/MS (TQD) for Food Safety TestingSreenivasaRaoCherukuri*,ShashiBhushanV,HarinathaReddyA,RavindranathD,ArunaM,SwarupaRaniS,RameshBandHymavathyM

All India Network Project on Pesticide Residues, PJTS Agricultural University, Rajendranagar, Hyderabad-500030, Telangana State, India

*Corresponding author: cherukurisrao@yahoo.com

Paper No. 337 Received: 24 June 2014 Accepted: 21 May 2015 Published: 29 June 2015

Abstract

Pesticideresiduesanalysisinfruitsandfreshvegetablesisachallengeforfoodsafetyasthegapbetweenpesticide spraysandharvests isvery less invegetables.Amulti residuemethodwasdeveloped forqualitative and quantitative analysis of 64 pesticides (insecticides, fungicides and herbicides) usingQuEChERS extraction method and GC-MS/MS (Triple Quadrupole) for analysis. Eight differentconcentrationsofcertifiedreferencematerialsfrom0.05ppmto0.30ppmwereinjectedinGC-MS/MSwithMRMmethod,insixreplications,andR2rangedfrom0.990-0.999withRSDof0.55to11.24.Thesamplepreparation approach is through adoptionofQuEChERSmethod,untreated control tomatosampleswerefortifiedwithmixtureofpesticidesat0.05,0.25,0.5mg/kg,eachisfivereplications,andtherecoveryofpesticidesisintherangeof80-95%,andhencemethodcanbeusedforqualitativeandquantitativeanalysisof64pesticidesin/ontomatoformonitoringstudies.

Highlights

• ThesamplepreparationandanalyticalmethodvalidatedinthisstudyformultiplepesticideresiduesanalysisinTomatoisusefulforqualitativeandquantitativeanalysisofresiduesat0.05ppmlevelfor64pesticides

Keywords: Methodvalidation,pesticideresidues,tomato,GC-MS/MS(TQD)

Vegetablesaretheimportantingredientofthehumandietforthemaintenanceofthehealthandpreventionofdiseases.Tomato(Lycopersicon esculentumMill.)iswidelyconsumedvegetable in Indiausually in theformofcurry,andalsoinrawformassalad,home-cooked,orprocessedasjuice,paste,orsauce.AspertheNationalSamplesurveyconductedduring2011-2012 in India, per capita consumption of tomatoin rural and urban area is 586 and 806 grams per

month, respectively (Anonymous, 2014), and thetotal Indian meal constitutes about 150-250 g ofvegetablesperday(MukherjeeandGopal,2003).Awiderangeofpesticidesareusedforcropprotectionagainst pest infection during the cultivation ofvegetables (Agnihotri 1999; Kalra, 2003), and theliteraturerevealsthatvegetablescontaintheresiduesof pesticides above their respective maximumresidue limit (Taneja, 2005;Ashutosh K Srivatsava

AGRICULTURE CHEMICALS

International Journal of Agriculture, Environment and BiotechnologyCitation: IJAEB: 8(2): 457-466 June 2015DOI Number: 10.5958/2230-732X.2015.00053.4©2015 New Delhi Publishers. All rights reserved

458

Cherukuri et al.Ta

ble

1: M

RM

par

amet

ers f

or q

ualit

ativ

e an

d qu

antit

ativ

e an

alys

is o

f pes

ticid

es o

n G

C-M

S/M

S (T

QD

)

Nam

e of

the

Pest

icid

eR

eten

tion

Tim

e (m

in)

Mol

ecul

ar

Wei

ght

Mon

itori

ng Io

nsPr

ecur

sor

Ion

Qua

lifier

Ion

Qua

ntifi

er Io

n

Met

ham

idop

hos

8.54

141.

3414

1, 9

414

114

1>64

, 141

>79,

141

>95

141>

95D

ichl

orvo

s8.

6222

0.98

237,

235

185

185>

63, 1

85>9

3, 1

85>1

0918

5>93

Mon

ocro

toph

os15

.45

223

192,

127

, 164

127

127>

109,

127

>95,

127

>79

127>

109

Phor

ate

15.7

127

626

0, 2

31, 1

2126

026

0>17

5, 2

60>2

31, 1

21>9

312

1>93

Alp

ha H

CH

15.8

429

0.82

219,

181

, 183

219,

181

219>

183,

219

>147

, 181

>145

181>

145

Dim

etho

ate

16.4

522

9.28

125,

229

, 93,

87

125,

229

125>

79, 1

25>9

3, 1

25>1

25, 1

25>8

712

5>12

5B

eta

HC

H17

.00

290.

8221

9, 1

81, 1

8318

1, 2

1918

1>14

5, 2

19>1

8318

1>14

5A

trazi

ne17

.09

215.

6821

5, 2

0021

521

5>20

0, 2

15>1

72, 2

15>1

3821

5>20

0Li

ndan

e17

.36

290.

818

1, 2

19, 1

8318

1, 2

1918

1>14

5, 2

19>1

8318

1>14

5C

hlor

thal

anil

18.1

426

5.91

266

266

266>

133,

266

>168

, 266

>231

266>

231

Dia

zino

n18

.15

304.

330

4, 7

79, 1

7930

4, 1

7930

4>13

7,

304>

164,

30

4>17

9,

179>

137

179>

137,

304

>137

Del

ta H

CH

18.8

029

0.82

219,

183,

181

181,

219

181>

145,

219

>183

181>

145

Phop

hom

idon

20.0

429

926

4, 1

2726

426

4>72

, 264

>127

, 264

>193

264>

127

Chl

orpy

rifos

met

hyl

20.3

532

2.53

286,

125

286

286>

208,

286

>241

286>

241

Met

hyl p

arat

hion

20.7

126

3.21

263,

223

, 125

263

263>

109,

263

>127

, 263

>246

263>

109

Ala

chlo

r20

.81

269.

7618

8, 3

69, 2

38, 2

4018

8, 2

6918

8>16

0,

188>

130,

26

9>16

0,

269>

188

188>

160,

269

>160

Hep

tach

lor

20.9

737

3.32

337,

274

, 272

272

272>

237,

272

>141

, 272

>117

272>

237

Met

alax

yl21

.25

279

206

206

206>

132,

206

>162

, 206

>206

206>

206

Dem

eton

-S-m

ethy

l sul

fone

21.7

029

0.34

142,

109

, 169

169

169>

109,

169

>125

169>

125

Feni

troth

ion

22.1

127

727

7, 2

6026

0,27

726

0>10

9,

260>

125,

26

0>15

1,

277>

109,

277

>260

260>

109,

277

>109

Mal

athi

on22

.79

330.

3617

3, 1

27, 1

2517

317

3>99

, 173

>117

, 173

>127

173>

99A

ldrin

22.8

336

4.91

263,

286,

314

, 293

263

263>

193,

263

>228

263>

193

Chl

orpy

rifos

22.9

935

0.62

314,

286

, 197

314,

286

314>

166,

31

4>25

8,

314>

286,

28

6>93

, 286

>271

314>

258

Fent

hion

23.2

427

827

8, 1

6927

827

8>10

9, 2

78>1

25, 2

78>2

4527

8>10

9Pa

rath

ion

23.4

329

1.3

291,

261

, 235

291

291>

109,

291

>137

291>

109

Dic

ofol

23.7

137

0.48

250,

251

, 759

251

251>

139,

251

>111

251>

139

Die

ldrin

23.7

138

0.9

277,

263

277,

263

277>

241,

277>

206,

277>

170,

263>

193

, 263

>228

263>

193

Method Validation for Qualitative and Quantitative Analysis of Pesticide Residues in Tomato

459

Fipr

onil

25.2

743

7.15

367,

369

, 351

, 213

367

367>

178,

367

>213

, 367

>255

367>

213

Chl

orfe

nvin

phos

25.5

135

9.57

323,

267

267,

323

267>

159,

323

>267

323>

267

Qui

nolp

hos

25.7

629

829

8, 1

46, 1

57, 1

1829

8, 1

46, 1

5729

8>12

9,

298>

156,

29

8>19

0,

146>

118,

157

>129

146>

118

Alle

thrin

-a26

.00

302.

4112

5,13

5,16

9,10

712

512

5>81

, 123

>95

125>

81A

lleth

rin-b

26.0

034

6.42

125,

135,

169,

107

125

125>

81, 1

23>9

512

5>81

2,4

DD

E26

.70

318.

0323

7, 2

3524

6,31

8,16

3,

226

246>

176,

318>

318,

318>

246,

16

3>12

7, 2

26>2

0624

6>17

6

Alp

ha e

ndos

ulfa

n27

.05

406.

9324

1, 2

65, 2

77, 2

4324

1, 2

6524

1>20

6,

241>

170,

26

5>22

9,

265>

195,

265

>193

241>

206

But

achl

or27

.21

311.

923

7, 3

23, 2

40, 2

6623

7, 3

2323

7>16

0,

237>

188,

17

6>13

4,

176>

146,

188

>130

176>

146

Hex

acon

azol

e28

.01

314.

2121

4, 1

7521

421

4>12

4, 2

14>1

52, 2

14>1

7221

4>17

2Fe

nam

ipho

s28

.47

303.

330

3,28

8, 1

5430

330

3>13

9, 3

03>1

54, 3

03>1

8030

3>15

4

Prof

enop

hos

28.4

737

233

9, 1

39, 5

59, 7

5933

9, 1

3933

9>18

8,

339>

251,

33

9>26

9,

139>

9713

9>97

4,4

DD

E28

.61

318.

0331

8, 2

4631

8, 2

4631

8>17

6,

318>

246,

24

6>17

6,

318>

318

318>

318

2,4

DD

D28

.91

320.

0523

7, 2

3523

523

5>16

5, 2

35>2

00, 2

35>1

3923

5>16

5

Endr

in29

.72

380.

9328

1, 2

63, 3

17, 2

4528

1, 2

6328

1>17

3,

281>

209,

28

1>24

5,

263>

193,

263

>228

263>

193

Bet

a en

dosu

lfan

30.4

240

6.93

241,

195

195,

241

195>

159,

241

>206

195>

159

4,4

DD

D31

.02

320.

0523

7, 2

3523

523

5>16

5, 2

35>1

99, 2

35>2

0023

5>16

52,

4DD

T31

.02

354.

4923

7, 2

3523

5, 1

4123

5>20

0, 2

35>2

35, 1

41>9

514

1>95

Ethi

on31

.25

384.

4823

1, 3

84, 2

57, 1

5323

123

1>12

9, 2

31>1

75, 2

31>2

0323

1>12

9Tr

iazo

phos

32.1

531

325

7, 1

6125

725

7>11

9, 2

57>1

34, 2

57>1

6225

7>16

2

Endo

sulfa

nsul

phat

e32

.67

422.

9227

4, 2

72, 3

8727

2, 3

8727

2>14

1,

272>

165,

27

2>23

7,

387>

253

272>

237

4,4

DD

T33

.18

354.

4923

7, 2

3523

523

5>16

5,

235>

199,

23

5>20

0,

235>

235,

235

>199

235>

165

Trifl

oxys

trobi

n33

.33

408.

3722

2, 1

16, 1

9022

2, 1

16, 1

9022

2>19

0,

222>

162,

22

2>13

0,

116>

89, 1

90>1

3011

6>89

Tebu

cona

zole

34.2

030

7.8

250,

125

250

250>

125,

250

>153

, 250

>163

250>

125

Bife

nith

rin36

.71

422.

8718

1, 1

65, 1

6618

1, 1

6518

1>11

5,

181>

165,

18

1>16

6,

165>

115

181>

166

Met

hoxy

chlo

r36

.83

345.

722

8, 2

2722

722

7>16

9, 2

27>1

8422

7>16

9

460

Cherukuri et al.Fe

npro

path

rin37

.30

349

265,

165

, 181

,125

265,

165,

181

265>

210,

26

5>18

1,

165>

153,

18

1>15

218

1>15

2

Phos

alon

e38

.66

367

367,

182

367,

182

367>

111,

36

7>13

8,

367>

182,

18

2>13

8, 1

82>1

1136

7>11

1, 1

82>1

11

Lam

bda

cyha

loth

rin40

.97

449.

918

1, 7

9718

1, 7

9718

1>12

7, 1

81>1

5218

1>15

2

Azi

npho

s eth

yl41

.28

345.

416

0, 1

34, 1

55, 1

2716

0,13

4,15

5,

127

160>

102,

160

>105

, 160

>132

160>

132

Perm

ethr

in-I

42.9

390

183,

163

163,

183

163>

127,

183

>153

183>

153

Perm

etrin

-II

43.2

139

018

3, 1

6316

3, 1

8316

3>12

7, 1

83>1

5316

3>12

7

Cyfl

uthr

in44

.48

434.

322

6, 2

06, 1

6320

6, 1

63, 2

2620

6>15

1,20

6>17

7,20

6>17

9,

163>

127,

226

>206

206>

177

Cyp

erm

ethr

in44

.64

416.

3216

3, 1

81, 1

65, 1

2716

3, 1

8116

3>12

7, 1

81>1

5216

3>12

7

Alp

ha c

yper

met

hrin

44.9

240

6.93

241,

265

, 277

, 243

241,

265

241>

206,

241>

170,

265>

229,

26

5>19

5, 2

65>1

9324

1>20

6

Fenv

alar

ate

46.0

441

922

5, 1

6722

522

5>91

, 225

>119

, 225

>147

225>

119

Fluv

alin

ate-

I46

.30

502.

9325

0, 1

99, 1

5725

025

0>55

, 250

>200

250>

200

Fluv

alin

ate-

II46

.30

502.

9325

0, 1

99, 1

5725

025

0>55

, 250

>200

250>

200

Del

tam

ethr

in47

.38

505.

2425

3, 1

81, 1

7225

3,17

225

3>17

2, 2

53>1

99, 1

72>9

317

2>93

Method Validation for Qualitative and Quantitative Analysis of Pesticide Residues in Tomato

461

Tabl

e 2.

Lin

eari

ty p

aram

eter

s for

diff

eren

t pes

ticid

es o

n G

C-M

S/M

S (T

QD

) with

MR

M m

etho

d

S.N

oN

ame

of th

e Pe

stic

ide

Coe

ffici

ent o

f va

riat

ion

(R2 )

RSD

S.N

oN

ame

of th

e Pe

stic

ide

Coe

ffici

ent o

f va

riat

ion

(R2 )

RSD

1.D

ichl

orvo

s0.

995

2.91

-4.2

136

.H

exac

onaz

ole

0.99

82.

67-4

.98

2.M

etha

mid

opho

s(1p

pm)

0.99

12.

61-4

.18

37.

Fena

mip

hos

0.99

62.

88-5

.01

3M

onoc

roto

phos

(1pp

m)

0.99

3.66

-4.1

938

.Pr

ofen

opho

s0.

997

3.08

-7.0

24

Phor

ate

0.99

21.

99-3

.88

39.

Die

ldrin

0.99

81.

23-5

.10

5.A

lpha

HC

H0.

994

0.89

-2.0

640

.4,

4 D

DE

0.99

71.

31-1

.98

6.D

imet

hoat

e0.

995

1.31

-4.7

741

.2,

4 D

DD

0.99

72.

01-2

.66

7.B

eta

HC

H0.

996

0.85

-2.9

042

.En

drin

0.99

41.

66-3

.78

8.A

trazi

ne0.

997

2.01

-4.8

843

.B

eta

endo

sulfa

n0.

999

0.55

-3.7

19.

Lind

ane

0.99

91.

01-4

.044

.4,

4 D

DD

0.99

81.

77-5

.10

10.

Chl

orth

alan

il0.

998

2.86

-5.1

145

.2,

4 D

DT

0.99

62.

57-4

.05

11.

Dia

zino

n0.

997

1.44

-3.8

946

.Et

hion

0.99

53.

09-4

.42

12.

Del

ta H

CH

0.99

60.

89-4

.44

47.

Tria

zoph

os0.

996

1.11

-4.2

113

.Ph

opho

mid

on0.

998

1.99

-4.8

848

.En

dosu

lfans

ulph

ate

0.99

61.

23-8

.11

14.

Chl

orpy

rifos

met

hyl

0.99

60.

99-2

.07

49.

4,4

DD

T0.

998

1.42

-4.7

015

.M

ethy

l par

athi

on0.

997

2.39

-3.2

450

.Tr

iflox

tstro

bin

0.99

91.

02-4

.89

16.

Ala

chlo

r0.

998

2.02

-4.7

751

.Te

buco

nazo

le (0

.1pp

m)

0.99

30.

97-4

.32

17.

Hep

tach

lor

0.99

91.

05-3

.652

.B

ifeni

thrin

0.99

91.

44-4

.89

18.

Met

alax

yl0.

991

1.20

-5.0

053

.M

etho

xych

lor

0.99

70.

96-3

.88

19.

Dem

eton

-S-m

ethy

l sul

fone

0.99

0.77

-4.8

754

Fenp

ropa

thrin

0.99

62.

29-4

.04

20.

Feni

troth

ion

0.99

23.

09-3

.81

55Ph

osal

one

0.99

81.

86-4

.89

21.

Mal

athi

on0.

993

0.98

-3.8

756

Lam

bda

cyha

loth

rin0.

994

1.34

-7.5

122

.A

ldrin

0.99

44.

01-4

.89

55A

zinp

hos e

thyl

0.99

32.

09-3

.99

23.

Chl

orpy

rifos

0.99

62.

04-4

.70

58Pe

rmet

hrin

–I

0.99

22.

77-4

.89

24.

Fent

hion

0.99

41.

44-4

.99

59Pe

rmet

rin-I

I0.

993

1.98

-4.3

325

.Pa

rath

ion

0.99

81.

28-4

.77

60C

yflut

hrin

0.99

50.

88-4

.69

26.

Dic

ofol

0.99

51.

08-4

.70

61C

yper

met

hrin

0.99

61.

44-4

.89

27.

Die

ldrin

0.99

81.

66-5

.42

62A

lpha

cyp

erm

ethr

in0.

997

3.71

-8.9

928

.Fi

pron

il0.

994

2.04

-4.0

863

Fenv

alar

ate

0.99

62.

78-1

0.54

29.

Chl

orfe

nvin

phos

0.99

60.

66-3

.89

64Fl

uval

inat

e-I

0.99

82.

81-1

1.24

30.

Qui

nalp

hos

0.99

71.

66-2

.63

65Fl

uval

inat

e-II

0.99

83.

77-8

.91

31.

Alle

thrin

-a0.

993

2.67

-5.0

266

Del

tam

ethr

in0.

995

32.

Alle

thrin

-b0.

992

1.89

-4.0

733

.2,

4 D

DE

0.99

71.

33-2

.49

34.

Alp

ha e

ndos

ulfa

n0.

994

1.99

-4.1

035

.B

utac

hlor

0.99

81.

99-4

.10

462

Cherukuri et al.

Figure 1. Chromatogram of 64 pesticides on GC-MS/MS (TQD) in MRM method

Figure 2. Linearity of organochlorines pesticides on GC-MS/MS (TQD) in MRM method

Figure 3. Linearity of organophosphate pesticides on GC-MS/MS (TQD) in MRM method

Figure 4. Linearity of synthetic pyrethroid pesticides on GC-MS/MS (TQD) in MRM method

Figure 5. Linearity of other pesticides on GC-MS/MS (TQD) in MRM method

Method Validation for Qualitative and Quantitative Analysis of Pesticide Residues in Tomato

463

with 1% phenyl-methyl polysiloxane) maintainedthe column temperatures starting from 50°C to290°Cwith four rampswith a total programof 50minutes.Massspectradetector(TripleQuadrupole)withmassrange50to400wasoperatedat250°Coftransferline,220°Cofsource,and40°Cofmanifoldtemperatures.Limitofdetection(LOD),LOQ(Limitof Quantification) and%RSD (Relative standarddeviation)valueswerecalculatedforeachpesticideunderthestandardconditions.

Field samples and extraction methods

Tomatoes (untreated) were collected from thesupervised fields of Student farm, College ofAgriculture.QuEChERS(QuickEasyCheapEffectiveRugged Safe) method for extraction and clean upwasvalidatedasperSANCO/12571/2013guidelines.Tomato fruits (5 kg) collected from control plotswerehomogenizedwithRobotCoupeBlixer, fromwhich15gwastakeninto50mLcentrifugetubes.Therequiredquantitiesof64pesticidesintermediarystandards are added to each 15 g sample to getfortificationlevelsof0.05mgkg-1,0.25mgkg-1,and0.5mgkg-1, in three replications each.Acetonitrile(30±0.1mL)wasadded to tube,homogenized for1-3minusingHeidolphsilentcrusher(lowvolumehomogenizer). Then 3±0.1 g sodium chloride wasadded to tube and mixed by shaking gently, andcentrifugedfor3minat2500-3000xgwithRemiR-238toseparatetheorganiclayer.Thetoporganiclayerofabout16mLwastakenintothe50mLcentrifugetubetowhich9±0.1ganhydroussodiumsulphatewasaddedtoremovethemoisturecontent.Extract(9mL)wastakeninto15mLtubecontaining0.4±0.1gPSAsorbent(fordispersivesolidphased-SPEcleanup) and 1.2 ± 0.01 g anhydrous magnesiumsulphate,and thesample tubewasvortexed for30secfollowedbycentrifugationfor5minat2500-3000xg. The extract of (2mL)was transferred into testtubesandevaporatedtodrynessusingconcentrationworkstation (TurbovapLVofCapiler lifesciences)with nitrogen gas and reconstituted with 1mLn-Hexane:Acetone (9:1) for analysis. The meanrecoveryoftheresidueswascalculatedtojudgethe

et al. 2011)maypose health hazards to consumers(Elliionet al.2000;MukherjeeandGopal,2003).Thedeterminationofpesticideresiduesinvegetablesandfruitsisofgreatconcernforallcountriestostudytherisk analysis and takeup food safetymeasures forbothexportanddomestic tradepurposes.Theaimofthisworkistodevelopproceduresfortheanalysisofmulticlasspesticidesanditsmetabolitesthroughbestextractionmethods,andbygaschromatographymass spectroscopy. The studies focus on samplepreparation,sampleextractionfollowingQuEChERSmethod and instrumental analysis using MRMmethodsinGC-MS/MS.

Materials and Methods

Chemicals and Reagents

Solvents like n-hexane, acetone, toluene andacetonitrile(HPLCgrade)werepurchasedfromM/SMerck India. Anhydrous sodium sulfate (Na2SO4)and anhydrous magnesium sulfate (MgSO4) werepurified with acetone and baked for 4 h at 600°Cin muffle Furnace to remove possible phthalateimpurities.Primarysecondaryamine(PSA)bondasil40 µmwas purchased fromM/SAgilent. CertifiedReferenceMaterials (CRMs) of high purity (≥98%)wereprocuredfromM/SSigmaAldrich,USA.

Calibration standards and Linearity studies

Primary(500-1000ppm)andintermediarystandards(50 ppm) of pesticides (Table 1) were prepared incalibrated volumetric flasks from the CRMs usingGC PR grade acetone and hexane solvents. Sixcalibration pesticide solutions (working standards)werepreparedintherangeof0.01ppmto0.5ppmincalibratedgraduatedvolumetricflasksusingdistilledn-hexane as solvent. Each concentration level wasinjected(1µL)sixtimesinBrukerScion436GC-MS/MSTripleQuadrupoleDetector(EI)usingMultipleReaction Monitoring (MRM) method (Table 1).Standardswereinjectedinsplitmode(1:10)at260°Cinjector tempat columnflowof 1ml/min (Helium99.99%purity)usingZebronMR2capillarycolumn(30 mm length, 0.25 mm ID, 0.20 µm film coated

464

Cherukuri et al.Ta

ble

3. R

ecov

ery

resu

lts fo

r di

ffer

ent p

estic

ides

in/o

n To

mat

o

S.N

oN

ame

of th

e Pe

stic

ide

% R

ecov

ery

at d

iffer

ent f

ortifi

catio

n le

vels

S.N

oN

ame

of th

e Pe

stic

ide

% R

ecov

ery

at d

iffer

ent f

ortifi

catio

n le

vels

0.05

mg/

kg0.

25 m

g/kg

0.5

mg/

kgL

OD

0.05

mg/

kg0.

25 m

g/kg

0.5

mg/

kgL

OD

1.D

ichl

orvo

s81

.02

84.0

986

.02

0.00

534

.A

lpha

end

osul

fan

87.6

288

.12

89.0

10.

001

2.M

etha

mid

opho

s(1p

pm)

80.0

981

.11

82.6

40.

005

35.

But

achl

or91

.69

92.1

493

.14

0.00

13

Mon

ocro

toph

os(1

ppm

)83

.86

85.1

286

.09

0.00

536

.H

exac

onaz

ole

87.0

288

.14

89.9

20.

001

4Ph

orat

e84

.16

85.6

686

.88

0.00

137

.Fe

nam

ipho

s81

.22

82.0

282

.99

0.00

55.

Alp

ha H

CH

91.0

291

.99

92.0

90.

001

38.

Prof

enop

hos

89.7

788

.66

85.6

20.

005

6.D

imet

hoat

e80

.08

80.1

480

.99

0.00

539

.D

ield

rin80

.12

81.4

882

.01

0.00

57.

Bet

a H

CH

90.6

091

.89

91.2

20.

001

40.

4,4

DD

E90

.62

91.8

490

.09

0.00

18.

Atra

zine

81.1

881

.42

82.6

40.

001

41.

2,4

DD

D91

.44

92.0

091

.89

0.00

19.

Lind

ane

84.6

685

.12

85.9

90.

001

42.

Endr

in83

.12

82.6

281

.69

0.00

110

.C

hlor

thal

anil

85.6

986

.44

87.1

20.

001

43.

Bet

a en

dosu

lfan

91.2

292

.89

94.6

20.

001

11.

Dia

zino

n81

.24

81.6

987

.88

0.00

544

.4,

4 D

DD

87.6

288

.14

89.2

00.

001

12.

Del

ta H

CH

84.6

984

.01

84.1

20.

001

45.

2,4

DD

T86

.42

85.1

984

.68

0.00

113

.Ph

opho

mid

on83

.21

84.1

083

.04

0.00

546

.Et

hion

89.0

490

.68

88.1

40.

005

14.

Chl

orpy

rifos

met

hyl

88.0

289

.14

88.8

90.

005

47.

Tria

zoph

os86

.62

85.1

987

.19

0.00

515

.M

ethy

l par

athi

on90

.09

91.2

492

.66

0.00

548

.En

dosu

lfans

ulph

ate

88.1

485

.02

86.8

80.

001

16.

Ala

chlo

r91

.22

91.3

992

.02

0.00

149

.4,

4 D

DT

82.6

283

.14

84.8

20.

001

17.

Hep

tach

lor

92.0

494

.62

93.8

20.

001

50.

Trifl

oxts

trobi

n87

.88

86.9

288

.14

0.00

5

18.

Met

alax

yl88

.62

88.9

187

.62

0.00

151

.Te

buco

nazo

le

(0.1

ppm

)81

.62

82.1

283

.09

0.00

5

19.

Dem

eton

-S-m

ethy

l su

lfone

79.9

180

.42

79.8

60.

005

52.

Bife

nith

rin91

.02

92..0

893

.62

0.00

1

20.

Feni

troth

ion

82.4

283

.64

84.0

10.

005

53.

Met

hoxy

chlo

r86

.12

85.0

484

.88

0.00

121

.M

alat

hion

84.8

285

.21

85.9

90.

005

54Fe

npro

path

rin81

.92

82.1

283

.04

0.00

522

.A

ldrin

81.9

682

.04

82.9

20.

001

55Ph

osal

one

85.4

484

.46

83.2

20.

005

23.

Chl

orpy

rifos

87.6

286

.14

87.9

90.

005

56La

mbd

a cy

halo

thrin

81.8

682

.22

83.0

90.

005

24.

Fent

hion

90.6

691

.88

89.5

10.

005

55A

zinp

hos e

thyl

79.9

679

.02

80.0

40.

005

25.

Para

thio

n86

.42

81.4

482

.99

0.00

558

Perm

ethr

in –

I81

.02

82.0

484

.99

0.00

526

.D

icof

ol89

.08

89.9

291

.46

0.00

159

Perm

etrin

-II

84.6

485

.02

86.4

20.

005

27.

Die

ldrin

80.4

481

.24

82.1

40.

005

60C

yflut

hrin

81.8

682

.62

84.0

90.

005

28.

Fipr

onil

91.9

492

.01

93.8

20.

005

61C

yper

met

hrin

82.1

280

.86

81.8

90.

005

29.

Chl

orfe

nvin

phos

90.0

191

.62

90.8

90.

005

62A

lpha

cyp

erm

ethr

in83

.24

84.6

286

.12

0.00

530

.Q

uina

lpho

s89

.88

89.0

288

.61

0.00

563

Fenv

alar

ate

84.1

185

.69

86.8

20.

005

31.

Alle

thrin

-a80

.62

81.8

881

.22

0.00

564

Fluv

alin

ate-

I81

.80

82.1

484

.14

0.00

532

.A

lleth

rin-b

80.6

281

.88

81.2

20.

005

65Fl

uval

inat

e-II

85.4

286

.12

87.7

00.

005

33.

2,4

DD

E89

.16

88.1

787

.12

0.00

166

Del

tam

ethr

in88

.62

87.2

988

.24

0.00

5

Method Validation for Qualitative and Quantitative Analysis of Pesticide Residues in Tomato

465

efficiencyofthemethodforqualitativeandanalysisof selected pesticides in/on tomato for nationalmonitoringstudies.

Results and Discussion

The standard chromatogram with 64 pesticides at500 ppb onGC-MS/MSwas presented in Figure 1andthedataonretentiontime(RT),MRMpapmeterswaspresentedinTable1.Eightpointlinearitycurvewasdrawnbyinjectingmixtureofvariouspesticides(Figure 2,3,4,5) and data on regression valuesincluding% RSD from linearity for each pesticidewas given in Table 2. It is seen that the R2 value(CoefficientofDetermination:ameasureofgoodnessof fit of linear regression) ranged from 0.990-0.999andpercentageofRelativeStandardDeviationisinbetween 0.55-8.11 explains that the instrument haswide linearity for quantitation purposes. Limit ofdetectionoforganochlorinepesticidesisintherangeof0.001to0.005mg/kg,withrecoveryof79.91%to92.00%at0.05mg/kg,81.24to94.62%at0.25mg/kgand79.86to94.62%at0.5mg/kglevels(Table3).Limitofdetectionfororganophosphateareintherangeof0.001to0.005mg/kgandrecoveryisintherangeof79.96to90.66%at0.05mg/kg,79.02to91.88%at0.25mg/kgand80.99to93.82%at0.5mg/kgfortificationlevels.Thepercentrecoveryofsyntheticpyrethroidpesticides is in therangeof81.02 to91.02%at0.05mg/kg, 80.86 to 92.08% at 0.25mg/kg and 81.89 to93.62%at0.5mg/kgfortificationlevel.Therecoveryofotherpesticidessuchatherbicidesandfungicidesis in the range of 88.81 to 91.69% at 0.05 mg/kg81.42 to 92.12% at 0.25mg/kg and 82.64 to 93.14%at0.5mg/kgfortification levels.Theextractionandcleanupmethodology followedproved to be rapidand highly effective for extraction of 64 pesticidesfrom tomatowith a recovery of various pesticidesin the range of 80-120% and themethod used forestimation of pesticides using MRMs method inGC-MS/MS(TQD)ishighlyusefulforidentificationofpesticidesatverylowlevels,asthecoefficientofdeterminationisveryhighinthelinearrangeof0.01ppmto0.50ppm,whichisveryusefulformonitoringstudiesaretheMRLs(MaximumResidueLimits)forthe targetedpesticidesare>0.01mg/kg in tomato.

The method developed by Anastassiades M et al.(2003) for fast and easy extraction procedures foranalysis of multiple pesticides in foods has beenfollowedwordwideinvariousmatrices,andduringthe present investigation, it was known that themethodisgoodfortomatomatrixalsoastherewereno matrix interferences. Sample preparation is animportant step for better extraction and cleanupforMSanalysisandinthepresentinvestigationthemethoddescribedbyLehoteySJ(2011)wasfollowedwith somemodifications, and based on the resultsobtainedthepresentstudy,itcanberecommendedtobeusedinthenationalresiduemonitoringprogramsasthemethodisvalidatedaspertheinternationallyfollowed“Guidancedocumentonanalyticalqualitycontrol and validation procedures for pesticideresidues analysis in food and feed” ofHealth andConsumerProtectionDirectorateGeneral,EuropeanCommission(SANCO/12571/2013(2013).

ReferencesAgnihotri NP 1999. Pesticide safety evaluation andmonitoring. All India Coordinated Research Project ofPesticideResidues,IndianAgriculturalResearchInstitute,DivisionofAgriculturalChemicals,NewDelhi,pp146.

AnastassiadesM, Lehotay SJ, StajnbaherD and Schenck FJ2003. Fast and easy multiresidue method employingacetonitrileextraction/partitioningand“dispersive solid-phase extraction” for the determination of pesticideresidues in produce. Journal of AOAC International 86(2):412-431.

Anonymous.2014.Householdconsumptionofvariousgoodsand services in India 2011-12.Ministry of Statistics andProgramme Implementation, Government of India. pp1-1143.

Fillion J, Sauve F and Selwyn J 2000.Multiresiduemethodforthedeterminationofresiduesof251pesticidesinfruitsandvegetablesbygaschromatography/massspectrometryand liquid chromatographywith fluorescence detection.Journal of AOAC International 83(3):698-713.

KalraRL2003.Assessmentofhumanexposureofpesticideresidues through food, and water and other sources inIndia. In:Proceedingsof symposiumon risk assessmentofpesticideresiduesinwaterandfood,ILSIWashingtonDC,ITRCLucknowandICMR,NewDelhi,India,pp.E1-9.

Lehotey SJ 2011. QuEChERS sample preparation approachfor mass spectrometric analysis of pesticide residues infoods.Methods in Molecular Biology 747:65-91.

466

Cherukuri et al.

Mukherjee I and Gopal M 2003. Pesticide Residues inVegetables. In: Proceedings of symposium on riskassessmentofpesticide residues inwater and food, ILSIWashingtonDC, ITRCLucknowand ICMR,NewDelhi,India,pp.A1-8.

SANCO/12571/20132013.Guidancedocumentonanalyticalquality control and validation procedures for pesticideresiduesanalysisinfoodandfeed.HealthandConsumerProtectionDirectorateGeneral,EuropeanCommission.

SrivastavaAshutoshK,TrivediPurushottam,SrivastavaMK,LohaniM, Srivastava Laxman Prasad. 2011. Monitoringof pesticide residues in market basket samples ofvegetablefromLucknowCity,India:QuEChERSmethod.Environmental Monitoring and Assessment 176:465–472.

Taneja A 2005. Monitoring of organochlorine pesticideresidues in vegetables fromAgra, India – a case study.Environmental Monitoring and Assessment 110:341-346.