Quantitative Evaluation of Pesticide Residues in Fruit Juices by Atmospheric Pressure...

Quantitative Evaluation of Pesticide Residues in Fruit

Juices by Atmospheric Pressure Photoionization High Resolution

Mass SpectrometryPragney Deme

Senior Research Fellow - CSIRNational Centre for Mass SpectrometryIndian Institute of Chemical Tecnology

Tarnaka, Hyderabad – 500007India

Pesticide Residues in Foods Major health hazard

Anticholine esterase activity Hepatotoxic Carcinogenic Thyroidism

Reduces quality of food Affects food exports Monitoring the quantity of residues

Important for regulatory affairs Treatment of health hazards Control of pesticide use

Pesticide Residues

More than 800 listed pesticides Polar Semi-polar Non-polar Volatile Semi-volatile Non Volatile

GC-Based techniques

GC-FID, GC-ECD, GC-NPD, GC-FPD and GC-MS11

Pesticide Monitoring

Polarity of pesticides Non-polar

Polar

Volatility

Volatile

Non-volatile

Liquid-chromatography – Atmospheric Pressure Chemical Ionization

Liquid Chromatography – Electrospray Ionization

Liquid-chromatography – Atmospheric pressure photoionization

Atmospheric Pressure Photoionization(APPI)

Anal. Chem., 2004, 76, 2842

Analysis of Pesticides LC- MS

Polar pesticides Organophosphorus Carbamates Triazines Phenyl acids

Semi-polar pesticides Synthetic pyrethroids

Non-polar pesticides Organochlorines ?

Electrospray ionizationAtmospheric pressure chemical ionization

APCI

Is it possible to develop a multi residue method including organochlorines using LC-MS ?

Objective of the work To develop a UPLC-MS based multiresidue method

for simultaneous determination of

organophosphorus, carbamates, synthetic

pyrethroids and organochlorine pesticides using

APPI

Matrix – Fruit juice (Grape and Mango)

Extraction method – Dispersive solidphase

extraction (Multiwalled carbon

nanotubes)

Analysis Liquid chromatography – APPI- High

resolution mass spectrometry

UPLC-HRMS analysis

Instrument: Thermo Exactive Orbitrap with Accela 600 UPLC system

Column: Hypersil Gold C18, 50 mm x 2.1 mm x 1.9 µ

Mobile Phase: Water (A) : Methanol with 5% toluene (B)

Mode of analysis: Gradient

Scan mode: Full Scan from 100-650 units in dynamic polarity switching between +/- modes

Resolution at 50000 FWHM

Advantages of Orbitrap

Minimal ion loss, hence maximum sensitivity even in full scanIon selection, hence SIM and SRM are possibleHigh resolution for absolute detectionPossibility of qualitative and quantitative analysis within single runLinear dynamic range – 104 orders for absolute quantification

APPI-HRMS of pesticide residues

Organophosphorus pesticides, carbamates and Synthetic pyrethroids Intense [M+H]+ ions when toluene (1%) was used as dopant

Intensity is independent of concentration of dopant

Strong signals in MeOH Weak signals in CH3CN Presence of water in solvent is necessary for intense signals

100 150 200

250m/z

020406080100

%

224.0676

193.0450

127.0148 246.04

86

MonocrotophosDimethoate

150

200

250m/z

020406080100

%

230.0060

198.9635

251.9887

Dichlorvos

100

150 200 250 300m/z

020406080100

%

220.9523

222.9518

224.9456

141.0304

150 200 250m/z

20406080100

%210.1119

168.0654

Propoxur

0

150 200 250 300m/z

20406080100 222.111

9165.0900

123.0438

CarbofuranCarbandezim

100 150 200 250 300m/z020406080100

%

192.0762

134.0706 193.0787

200 250 300 350m/z

20406080100

%

303.1947

257.1894

Allethrin

350 400 450 500m/z

20406080100

416.0808

376.0944

α -Cypermethrin

400 500 600m/z

20406080100 376.156

8

478.9794317.1186

Deltamethrin

505.9792

APPI-HRMS of pesticide residues Organochlorines

Most of the analytes showed signals in negative mode

Showed (M+Cl)-, (M-HCl)- ions

Few analytes DDD, DDE, DDT and Methoxychlor showed signals in positive mode as M+• and [M-CCl2(3)]+• ions

Presence of water in mobile phase showed a significant effect

Percentage and type of dopant is critical

250 300 350 400m/z

20406080100 324.8260

288.8499

328.8251

HCH

%

A-Endosulfan

150 200 250 300 350 400 450 500m/z

020406080100

%

304.8682

306.8651

268.8916266.894

6340.8046

344.8087 404.806

7

B-Endosulfan

200

250

300

350

400

450

500

m/z020

40

60

80

100

%

304.8682

306.8652268.8713228.9463 340.8047

344.8088404.8059

350 400m/z

20406080100 379.8672

340.95026

381.8698

Dieldrin

%

380 400 420 440 460m/z

20406080100

414.8370

418.8299412.8311

Endrin

200 250 300m/z

20406080100 235.0074

281.9762200.0387

p, p’ - DDD

p,p’- DDT

200 300 400m/z020406080100

%

235.0074

237.0041

317.9338

400 420 440 460 480 500m/z

20406080

100444.7603

446.7683442.7673γ - Chlordane

250 300 350m/z

20406080

100 265.9046

334.8343299.8653

236.8418

336.8349

Heptachlor

Effect of Water on Signal intensity

0 5 10 15 20 25 30 35 40 450

200000

400000

600000

800000

1000000

1200000

1400000

1600000

α-HCHp,p'-DDDα-EndosulfanEndrin

% Water

Signal I

ntensi

ty

Selection of Dopant

deltamethrin

lindane

p, p'-DDD

α-endosulfan

endrin

0200000400000600000800000

1000000120000014000001600000

Toluene

Acetone

Anisole

pesticides

sign

al i

nten

sity

Dopant effect

0 1 2 3 4 5 6 7 8 90

500000

1000000

1500000

2000000

2500000

3000000

α-HCH p,p'-DDDα-EndosulfanEndrin

Percentage of Toluene in mobile phase

Abundance

UPLC-HRMS Chromatograms

D:\Sam ple - ppb

RT: 0.00 - 10.00

0 2 4 6 8 10 Tim e (m in)

0

50

100

1.23 1.21

1.18 1.10

NL: 4.69E7 m /z= 202.07-202.09 NL: 4.29E7 m /z= 222.10-222.12 NL: 8.30E6 m /z= 210.10-210.12 NL: 7.53E6 m /z= 192.06-192.08 NL: 1.96E7 m /z= 229.98-230.01 NL: 3.66E7 m /z= 224.05-224.07 NL: 1.96E7 m /z= 184.00-184.02

0 2 4 6 8 10 Tim e (m in)

0

4.63

4.26

1.33


0 2 4 6 8 10 Tim e (m in)

0

5.56

5.54 5.25

5.10 4.93


0 2 4 6 8 10 Tim e (m in)

0

50

100

Relative Abundance

7.08 6.52

6.50

6.39

NL: 6.64E6 m /z = 227.09-228.01 NL: 1.46E7 m /z= 368.78-368.80 NL: 7.80E6 m /z = 303.18-303.20 NL: 2.00E7 m /z= 404.79-404.81 NL: 3.66E7

m /z= 414.82-414.84

NL: 3.66E7 m /z= 372.93-372.95

0 2 4 6 8 10 Tim e (m in)

0

7.67

7.63

7.45

0 2 4 6 8 10 Tim e (m in)

0

7.90

7.72

NL: 4.69E7 m /z= 510.09-510.11 NL: 6.64E6 m /z= 317.92-317.94 NL: 7.53E6

NL: 1.96E7

NL: 1.96E7

0.73 0.75

0.84

1.40 3.26

4.15

4.51 5.58

5.78 7.51

7.28

7.31 7.42

7.51 7.56

7.60

7.70 NL: 6.64E6 m /z=434.06-434.08 NL: 1.46E7 m /z= 444.75-444.77 NL: 7.80E6 m /z= 349.92-349.94 NL: 2.00E7 m /z= 234.99-235.01 NL: 3.66E7

m /z= 367.98-368.00 NL: 3.66E7 m /z= 379.85-379.87

7.74

7.77

8.10

8.29

NL: 3.66E7 m /z= 416.07-416.09

m /z= 334.82-334.84

m /z= 505.96-505.98

m /z= 230.86-230.88

S.N

o.pesticides

elemental

composition

observed

ion

exact

mass

Observed

mass

erro

r

ppm

R.T.LODm

grape

(ppb)

LOQm

grape

(ppb)

LODm

mango

(ppb)

LOQm

mango

(ppb)

MRLs

EU/FAO

(ppb)

1 acephate C4H10NO3PS [M+H]+184.019

2184.01878

-

2.170.73 0.5 2 0.5 2 10

2 monocrotophos C7H14NO5P [M+H]+224.068

2224.06757

-

2.990.75 0.5 2 1.5 5 10

3 dimethoate C5H12NO3PS2 [M+H]+230.006

9230.006

-

3.910.84 1.5 5 3 10 20

4 carbandezim C9H9N3O2 [M+H]+192.076

8192.0762

-

2.861.1 1.5 5 1.5 5 500

5 propoxur C11H15NO3 [M+H]+210.112

5210.11185

-

2.951.18 1.5 5 1.5 5 50

6 carbofuran C12H15NO3 [M+H]+222.112

5222.11186

-

2.751.21 1.5 5 1.5 5 10

7 carbaryl C12H11NO2 [M+H]+202.086

3202.08563

-

3.121.23 1.5 5 3 10 10

8 dichlorvos C4H7Cl2O4P [M+H]+220.953

2220.95226

-

4.161.33 3 10 3 10 10

9 malaoxon C10H19O7PS [M+H]+315.066

2315.06494

-

3.971.4 3 10 3 10 20

10 methiocarb C11H15NO2S [M+H]+226.089

6226.08894

-

3.053.26 3 10 3 10 100

11 phosmet C11H12NO4PS2 [M+H]+318.001

8318.00064

-

3.684.15 0.5 2 1.5 5 50

12endosulfan

alcoholC9H8Cl6O2 [M+2+Cl]─

394.830

9394.83224 3.39 4.26 3 10 3 10 50

13methyl-

parathionC8H10NO5PS [M-H] -

261.994

5261.99365

-

3.054.51 0.5 2 1.5 5 10

14 malathion C10H19O6PS2 [M+H]+331.043

3331.04263

-

2.144.63 0.5 2 1.5 5 20

15 fenitrothion C9H12NO5PS [M-H] -276.010

1276.00894

-

4.204.93 0.5 2 0.5 2 20

16 endrin aldehyde C12H8Cl6O[M+4+2Cl-

H]-

450.795

2450.79368

-

3.285.1 1.5 5 0.5 2 10

17 quinalphos C12H15N2O3PS [M+H]+299.061

4299.06012

-

4.215.25 0.5 2 0.5 2 50

18 phorate C7H17O2PS3 [M+H]+261.020

1261.01944

-

2.535.54 0.5 2 0.5 2 10

19 lindane C6H6Cl6 [M+2+Cl] -324.825

4324.82603 1.88 5.56 0.5 2 0.5 2 10

20 diazinon C12H21N2O3PS [M+H]+305.108

3305.10796

-

1.215.58 0.5 2 0.5 2 10

21 endrin ketone C12H8Cl6O [M+2+Cl]- 414.836 414.83752 3.69 5.78 1.5 5 3 10 10

22 profenofosC11H15BrClO3P

S[M+H]+

372.942

4372.94103

-

3.736.39 0.5 2 1.5 5 20

23 phosaloneC12H15ClNO4PS

2[M+H]+

367.994

1367.99356

-

1.586.5 1.5 5 3 10 10

24 α-endosulfan C9H6Cl6O3S [M+2-H] -404.806

7404.80656

-

0.226.52 1.5 5 3 10 50

25 β-endosulfan C9H6Cl6O3S [M+2-H]-404.806

7404.80588

-

1.907.08 3 10 1.5 5 50

S.N

o.pesticides

elemental

composition

observed

ion

exact

mass

Observed

mass

erro

r

ppm

R.T.LODm

grape

(ppb)

LOQm

grape

(ppb)

LODm

mango

(ppb)

LOQm

mango

(ppb)

MRLs

EU/FAO

(ppb)

26 allethrin C19H26O3 [M+H]+303.195

5303.1947 2.87 7.28 0.5 2 0.5 2 -

27heptachlor-

epoxideC10H5Cl7O

[M+2-H2O-

H]-

368.795

2368.7962 2.71 7.31 1.5 5 1.5 5 10

28 methoxychlor C16H15Cl3O2 [M-CCl3]+227.106

7227.10635

-

1.367.42 0.5 2 0.5 2 10

29 dieldrin C12H8Cl6O [M+2] +●379.868

2379.86723

-

2.637.45 1.5 5 1.5 5 10

30 endrin C12H8Cl6O [M+2+Cl]─414.837

1414.83704

-

0.107.51 1.5 5 3 10 10

31 p,p'-DDD C14H10Cl4 [M-CHCl2] +235.007

6235.00737

-

0.897.56 3 10 3 10 10

32 chlorpyrifos C9H11Cl3NO3PS [M+H]+349.933

6349.93307

-

1.407.6 3 10 3 10 30

33 p,p'-DDT C14H9Cl5 [M-CCl3] +235.007

6235.0074

-

0.777.63 3 10 3 10 50

34 γ-chlordane C10H6Cl8 [M+4+Cl]─444.760

2444.76035 0.38 7.67 3 10 3 10 10

35 β-cyfluthrin C22H18Cl2FNO3 [M+H]+434.072

1434.07255 1.15 7.7 3 10 3 10 20

36 heptachlor C10H5Cl7[M+2-HCl-

H]─

334.834

2334.83434 0.45 7.72 3 10 3 10 10

37 α-cypermethrin C22H19Cl2NO3 [M+H]+416.081

5416.08078

-

1.687.74 3 10 3 10 2000

38 deltamethrin C22H19Br2NO3 [M+2+H]+505.978

4505.97916 1.50 7.77 3 10 3 10 50

ISpentachloro

benzeneC6H1Cl5

[M+2-

Cl+O]─

230.874

7230.8739

-

3.257.90 3 10 1.5 5 -

39 p,p'-DDE C14H8Cl4 [M+2]+●317.934

5317.93382

-

2.178.1 3 10 3 10 10

40 flumethrin C28H22Cl2FNO3 [M+H]+510.103

4510.10199

-

2.678.29 1.5 5 1.5 5 -

Validation Parameters

Limit of Detection - 0.5 to 3 ng/mL

Limit of Quantification - 2 to 10 ng/mL

Method linearity - 2 to 1000 ng/mL

The extraction recoveries - 62 to 109 % (grape

juice)

- 54

to 103 % (mango juice)

Data Variation - less than 15% (RSD)

Analysis of field samples

Grapes and mangoes were collected from various

local markets

Juice was extracted using Philips fruit Juicer

Juice samples were centrifuged at 15000 rpm to

remove suspended fibers

Samples were subjected to DSPE

Analysed on UPLC-APPI-HRMS

DSPE Work flow

10 mL fruit juice

DSPE extraction with 30 mg

MWCNT

Centrifuge,Extract from MWCNT by DCM

MeoH ,UPLC-HRMS analysis

0 10Time (min)

0

50

100

7.53

7.177.66

3.42

1.24

0.70

NL: 1.21E5m/z= 416.07-416.09 MS Mango juice- Batch 5NL: 1.02E5

m/z= 234.95-235.02 MS Grape Juice Batch 3NL: 3.51E5

m/z= 226.07-226.09 NL: 6.72E5

m/z= 202.07-202.09 Grape juice Batch 4NL: 1.97E7

m/z= 184.01-184.03 MS Grape Juice Batch 2

Relative

Abund

ance

Endrin

DDT

Methiocarbcarbaryl

Acephate

Pesticides found in real samples

Acknowledgements

Dr. U. V. R. Vijaya Saradhi Sr. Scientist (research Supervisor)

Dr. R. Srinivas Chief Scientist Head of NCMS

Dr. S. Prabhakar Principal Scientist NCMS

Colleagues of NCMS

CSIR – For sponsoring the projects AARF, FAC-

005

Quantitative Evaluation of Pesticide Residues in Fruit Juices by Atmospheric Pressure...

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