Analysis of pesticides in herbs by GC-QqQ and LC-QqQ · • The aim of the experiments was to...

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European Reference Laboratory - FV

Analysis of pesticides in herbs

by GC-QqQ and LC-QqQ

Amadeo R. Fernández-Alba

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Introduction

• In some cultures herbs have been used as a popular beverages for centuries. Increased interest in those beverages has resulted from perceived health benefits that may be associated with their consumption.

• The EFSA annual report of 2008 revealed that:

– 5.8% and 4.5% of the tea and herbs samples, respectively, contained

more than 5 pesticides, one of the highest percentage of the report.

– these matrices had the highest percentage of MRL exceedance rates

(16% herbs and 9% tea).

• In multiresidue methods one of the main problems is the wide range

of chemical properties of the analytes. Tea leaves comprise sugars,

pigments, polyphenols and alkaloids (especially caffeine).


Objective

• The aim of the experiments was to compare and choose the best

method for the extraction of pesticides residues in tea. Three

popular extraction methods were selected for the comparison:

• modified QuEChERS with CaCl2

• ethyl acetate method

• mini-Luke

• Validate the selected method in four different matrices and apply it

to real samples.

– Green tea -Black tea

– Red Tea - Chamomile


Without

dilution Tomato

Orange

Leek

Tomato

Orange

Leek


912 components (parent molecules, MS fragment ions and Cl isotopes)

m/z: 100-400 Rt: 7-13 min

DB: 227 components

Mass

Data base of 300 pesticides


Mass

Co-extracted matrix components of tomato. LC-TOF-MS

m/z: 200-600 Rt: 4-7 min 2408 Matrix compounds

Absolute height ≥ 10000 counts


Mass

Data base components- tomato matrix compounds

m/z: 200-600 Rt: 4-7 min

m/z: 100-400 Rt: 7-13 min

DB: 227 components Tomato: 656 matrix compounds

2408 Matrix compounds



Co-extracted matrix components of green-tea. LC-TOF-MS

m/z: 100-700 Rt: 4-10 min




Data base components- green tea matrix compounds


Absolute height ≥ 10000 counts m/z: 100-700 Rt: 4-10 min

DB: 227 components Green tea: 3533 matrix compounds

m/z: 100-400 Rt: 7-13 min


Co-extracted matrix components of chamomile. LC-TOF-MS






Data base components- chamomile matrix compounds

m/z: 100-400 Rt: 7-13 min

DB: 227 components Chamomile: 3490 matrix compounds


Selected Compounds

Bifenthrinb

Buprofezin a

Carbaryl a

Carbofuran a

Chinomethionat b

Chlorpyrifos Methyl b

Diazinon b

Dichlorvos b

Dimethoate a

Endosulfan I b

Endosulfan II b

Endosulfan sulfate b

Esfenvalerateb

Ethion b

Fenpropathrin b

Fenvalerateb

Isocarbophos b

Malathion b

Methidathion b

Methiocarb sulfonea

Methiocarb

sulfoxidea

Methiocarba

Methomyl a

Mevinphos b

Monocrotophos a

Parathion Ethyl b

Phorate b

Phosmet b

Pirimiphos Methyl a

Profenofos a

Propargite b

Pyridaben b

Quinalphos b

Tetrachlorvinphos b

Triazophos b

Azoxystrobin a

Benalaxyl b

Biphenylb

Boscalid b

Carbendazim a

Cyproconazole b

Cyprodinil b

Dichlofluanid b

Dicloran b

Fenarimol b

Fludioxonil b

Imazalil a

Iprodione b

Kresoxim Methyl

b Metalaxyl a

Myclobutanil a

Omethoate a

Oxadixyl b

Penconazole b

Pyrimethanil b

Triadimefon a

Triadimenol a

Vinclozolin b

2,4 Da

2,4 DPa

Alachlor b

Atrazine a

MCPP a

Metolachlor a

Metribuzin a

Propyzamide b

Simazine a

Terbuthylazine

a Trifluralin b

Acetamiprid a

Chlorpyrifos b

Cypermethrin b

Ethoprophos b

Etofenprox b

Fenitrothion b

Flucythrinate b

Fonofos b

Imidacloprid a

Isofenphos

Methyl b

Lindane b

Parathion

Methyl b

Permethrin b

Pirimicarb a

Pyriproxyfen b

Terbufos b

Thiodicarb a

Acaricide

Fungicide

PGR

Herbicide

Insecticide

86 LCa- and GCb- amenable pesticides


Spiking procedure

• A key aspect to achieve reliable recovery results is the

spiking process.

• Herbs are dehydrated samples, so they must be fortified

with the mix thoroughly soaking all sample, and dried

again.

Extend the tea on

the crystallizer Add the spiking mix Stir to be sure of the

homogenizer

procedure

Evaporate the

solvent


Add 10 mL acetonitrile and

200 µL TPP (25 ppm)

Shake automatically

in Agitax

Take 3 mL aliquot

Shake 30 second

in Vortex

Evaporate to dryness

and reconstitute

Shake automatically

in Agitax

Acidify with 10 μL formic

acid 5% per mL of extract

Modified QuEChERS

method

Add 4 g MgSO4 + 1 g NaCl + 1 g

NaAc·2 H2O + 0.5 g NaAc·6 H2O

2 g sample + 4 mL H2O

Shake 30 sec, wait 30 min

Centrifugue 5 min.

3700 r.p.m.

Analysis

Add 150 mg CaCl2

anh. + 150 mg PSA

Centrifugue 5 min.

3700 r.p.m.


Add 10 mL acetonitrile and


Shake automatically

in Agitax

Take 3 mL aliquot

Shake 30 second

in Vortex


and reconstitute

Shake automatically

in Agitax

Acidify with 10 μL formic

acid 5% per mL of extract

Modified QuEChERS

method

5 times

dilution

Add 4 g MgSO4 + 1 g NaCl + 1 g

NaAc·2 H2O + 0.5 g NaAc·6 H2O



Centrifugue 5 min.

3700 r.p.m.

Analysis

Add 150 mg CaCl2

anh. + 150 mg PSA

Centrifugue 5 min.

3700 r.p.m.


Add 10 mL ethyl acetate and


Shake automatically

in Agitax 15 min


and reconstitute

Shake 3 s by hand

Ethyl acetate method



Add 210 mg GCB +

4.8 g MgSO4

anhidrous + 0.9 g NaCl

Centrifugue 5 min.

3700 r.p.m.

Analysis

5 times

dilution


Add 10mL of Acetonitrile and


Shake vigorously

30s by Ultra-Turrax

Shake vigorously

30s by Ultra-Turrax

miniLuke

extraction method 2 g sample + 4 mL H2O


Add 20mL Petroleum ether:

Dichloromethane (1:1)

Centrifugue 5 min.

at 4000 r.p.m.

Evaporate to dryness 3 mL

portion of the upper layer

Reconstitute with 1 mL of

adecuate solvent

Analysis

5 times

dilution


• LC analysis:

– Mobile phases: 0.1% FA in H2O and AcN

– Reversed-phase C8 analytical column of 4.6 mm x

150 mm and 5 µm particle size (Agilent Zorbax SB)

– 6410 LC-QQQ-MS/MS (Agilent Technologies)

– Dynamic MRM software features

• GC analysis:

– Ultra Inert GC column, HP-5MS UI 15 m × 0.25 mm

i.d. × 0.25 μm (Agilent)

– 7000 GC-QQQ-MS/MS (Agilent Technologies)

– Retention Time Locking (RTL) and Blackflushing

– 39 segment SRM method

LC and GC analysis


0

5

10

15

20

25

30

35

40

45

50

0-30 30-40

40-50

50-60

60-70

70-80

80-90

90-100

100-120

n.d.

# C

om

po

un

ds

Recovery (%)

QuEChERS

EtOAc

miniLuke

Concentration level: 100 µg/kg (n=5)

Recoveries


Number of compounds

QuEChERS Ethyl Acetate

method miniLuke

70 - 120% 75 31 54

70 - 60% 2 26 26

< 60% 6 22 2

Not detected 3 7 4

Recoveries


Pvap pKow modified QuEChERS

Compound (Pa, 20ºC) (pH 7, 20ºC) Rec (%) RSD(%)

Ometoathe a 3,3·10-03 -0,74 19,8 15,3

Monocrotophos a 2,9·10-04 -0,22 34,0 12,6

MCPA a 4,0·10-04 -0,81 45,5 19,3

2,4-D a < 1,0·10-05 -0,83 46,6 22,8

Dichlofluanid b* 1,4·10-05 3,70 49,4 8,3

Dicamba a 1,7·10-03 -1,88 51,7 15,9

Carbendazim a 1,5·10-04 1,48 56,9 17,2

Lowest recoveries with modified QuEChERS method

CaCl2

a Ansalysed by LC b Ansalysed by GC

* Thermical degradation into the GC system


Lowest recoveries with ethyl acetate method

GCB

Pvap pKow EtOAc method


Thiodicarb a 2,7·10-03 1,62 51,6 6,6

Metolachlor a 4,2·10-04 3,40 51,9 6,8

Kresoxim Methyl b 2,3·10-06 3,40 52,7 3,5

Fenarimol b 6,5·10-05 3,69 53,1 2,0

Penconazole b 3,7·10-04 3,72 53,1 2,6

Iprodione b 5,0·10-07 3,10 54,8 4,2

Alachlor b 2,0·10-03 3,09 55,5 6,8

Isofenphos Methyl b 4,04 55,9 4,1

Malathion b 4,5·10-04 2,75 56,8 3,5

Fludioxonil b 3,9·10-07 4,12 56,9 3,3

Quinalphos b 3,5·10-04 4,44 57,4 4,0

Fonofos b 2,8·10-02 3,94 57,7 14,5

Benalaxyl b 6,6·10-04 3,54 58,2 4,8

Vinclozolin b 1,3·10-04 3,02 59,0 6,4

Pvap pKow EtOAc method


Cyprodinil b 5,1·10-04 4,00 0,0 0,0

Pirimiphos Methyl a 2,0·10-03 3,90 31,1 9,9

Imazalil a 1,6·10-04 2,56 31,9 7,1

Chinomethionat b 2,6·10-05 3,78 33,1 8,7

Ometoathe a 3,3·10-03 -0,74 37,4 6,3

Terbufos b 3,5·10-02 4,51 37,7 16,4

Boscalid b 7,0·10-07 2,96 39,3 5,3

Terbuthylazine a 1,5·10-04 3,40 44,4 6,7

Diazinon b 1,2·10-02 3,69 46,3 7,6

Azoxystrobin a 1,1·10-10 2,50 47,1 6,7

Pyrimethanil a 1,1·10-03 2,84 48,4 10,3

Myclobutanil a 2,1·10-04 2,89 48,4 6,1

Phorate b 8,5·10-02 3,86 48,7 3,8

a Ansalysed by LC b Ansalysed by GC


Green Tea Blank

LC-QToF-MS Full Scan Total Ion Chromatogram

Mini Luke method

EtOAc method

QuEChERS method


Mini Luke method

EtOAc method

QuEChERS method

Green Tea Blank

GC-Q-MS Full Scan Total Ion Chromatogram


Set-up

Modified QuEChERS Ethyl Acetate method miniLuke method

Validation in 4 different matrices

with the best method

Green tea

Chamomile

Green tea

Red Tea

Black tea


0

5

10

15

20

25

30

35

40

45

50

Co

mp

ou

nd

s (

%)

Recovery (%)

Concentration level: 25 μg/Kg (n=5)

Green Tea

Red Tea

Black Tea

Camomile

Recoveries


Recoveries

0

5

10

15

20

25

30

35

40

45

50

55

60

Co

mp

ou

nd

s (

%)

Recovery (%)

Concentration level: 100 μg/Kg (n=5)

Green Tea

Red Tea

Black Tea

Chamomile


LODs

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5

10

15

20

25

30

35

40

45

0,1-0,25 0,25-0,5 0,5-1 1-5 5-10 10-50 50-100 > 100

Co

mp

ou

nd

s (

%)

LOD (μg/kg)

Green tea

Red Tea

Black Tea

Chamomile

Mevinphos Quinalphos

Propargite

Fonofos

Mevinphos

Benalaxyl

Cypermethrin

Fonofos

Quinalphos

Profenofos


Linearity and repetitivity

• Linearity

– 7 calibration points, 0,5-200 μg/L

– All the compounds had a R2>0.99

• Repetitivity

– Two concentration levels: 5 and 20 μg/L

– RSD<17%


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4

6

8

10

12

14

16

18

< -50% -50% - (-20%)

-20% - 0

0 - 20%

20% - 50%

50% - 100%

100% - 200%

200% - 300%

300% - 400%

400% - 500%

> 500%

Co

mp

ou

nd

s (

%)

Slope Variation (%)

Green tea

Red Tea

Black Tea

Chamomile

Matrix effects


Blank matrices extracted with modified QuEChERS method

LC-QToF-MS Full Scan Total Ion Chromatogram


Real samples 75 real samples of different kind of herbs bought in:

26 samples 26 samples 9 samples

6 samples 3 samples

1 sample 1 sample 1 sample

1 sample 1 sample


Real samples

68.0%

24.0%

8.0%

Results

Under MRL Above MRL Blank

Uncertainty of 50% is considered to

check the MRL exceedance.


Real samples

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

# S

am

ple

s

# Pesticides

Distribution of the pesticides in the samples


Real samples

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

# S

am

ple

s

# Pesticides

Distribution of the pesticides in the samples

Most found: Biphenyl (37), Acetamiprid (36), Carbendazim (36), Chlorpyrifos (34),

Imidacloprid, (31), Bifenthrin (30) and Endosulfan (Sum) (22).


SAMPLE PESTICIDE DETECTED CONCENTRATION (μg/Kg)

Red tea_1

Acetamiprid

Bifenthrin

Biphenyl

Carbendazim

Imidacloprid

20.9

39.2

13.0

15.4

15.9

Chamomille_3

Carbendazim

Carbofuran

Chlorpyrifos

Endosulphan I

Metalaxyl

Etofenprox

Penconazole

220.2

11.8

15.8

6.0

6.0

18.3

7.1

Green tea_5

Acetamiprid

Bifenthrin

Carbendazim

Endosulphan I

Endosulphan II

Endosulphan sulphate

Imidacloprid

Methomyl

106.2

168.5

15.6

19.0

34.1

38.2

38.9

15.4

Black tea_10

Acetamiprid

Bifenthrin

Carbendazim

Endosulphan I

Endosulphan II


Imidacloprid

29.1

136.5

2.6

18.3

43.6

70.6

26.9

APPLICATION TO REAL SAMPLES

In red, values above MRL


SAMPLE DETECTED

PESTICIDE

CONCENTRATION

(μg/Kg)

Green tea_34

Acetamiprid

Bifenthrin

Biphenyl

Carbendazim

Carbofuran

Clorpyrifos

Endosulphan I

Endosulphan II


Fenpropathrin

Imidacloprid

Methomyl

Pyridaben

Triazophos

271.0

328.7

20.0

14.0

7.7

45.9

34.7

112.7

111.1

33.6

124.9

36.5

2.9

21.7

Green tea_35

Acetamiprid

Bifenthrin

Biphenyl

Carbaril

Carbendazim

Clorpyrifos

Endosulphan I

Endosulphan II


Fenpropathrin

Imidacloprid

Methomyl

Triazophos

112.7

235.0

19.2

16.1

19.8

6.6

9.7

40.8

42.2

60.9

116.6

12.7

6.9



SAMPLE DETECTED

PESTICIDE

CONCENTRATION

(μg/Kg)

Black tea_41

Acetamiprid

Biphenyl

Carbendazim

Methiocarb sulfone

Pyridaben

15.6

7.2

43.0

21.2

4.9

Black tea_57

Acetamiprid

Bifenthrin

Buprofezin

Chlorpyrifos

Imidacloprid

Methiocarb Sulfone

13.3

81.9

1.3

4.5

7.6

7.6

Black tea_10

Acetamiprid

Bifenthrin

Carbendazim

Endosulphan I

Endosulphan II

Endosulphan

sulphate

Imidacloprid

29.1

136.5

2.6

18.3

43.6

70.6

26.9

Black tea_21

Acetamiprid

Biphenyl

Carbendazim

Chlorpyrifos

Imidacloprid

Methiocarb sulfone

Pyridaben

26.7

13.9

19.3

3.2

61.6

20.5

3.2



Green tea_9

Acetamiprid

Bifenthrin

Carbaril

Carbendazim

Endosulphan I


Imidacloprid

Methomyl

Propargite

20.1

55.2

11.8

6.8

13.9

64.6

35.1

3.9

44.1

Black tea_10

Acetamiprid

Bifenthrin

Carbendazim

Endosulphan I

Endosulphan II


Imidacloprid

29.1

136.5

2.6

18.3

43.6

70.6

26.9

Green tea_75

Acetamiprid

Bifenthrin

Buprofezin

Carbendazim

Carbofuran

Chlorpyrifos

Endosulfan I

Endosulfan II

Endosulfan suphate

Imidacloprid

Fenvalerate/Esfenvalerate RR/SS

Fenvalerate/Esfenvalerate RS/SR

Methomyl

Pyridaben

Triazophos

217.2

478.3

75.3

33.6

1.9

16.1

25.3

100.0

114.0

118.4

56.8

63.5

49.7

10.8

39.0





Rooibos tea_19

Carbendazim

Chlorpyrifos

Etofenprox

Imazalil

Iprodione

Methomyl

5.2

18.7

29.3

41.2

0.6

4.4

Herbs and fruits_23

Biphenyl

Boscalid

Carbendazim

Carbofuran

Chlorpyrifos


Fludioxonil

Iprodione

Pirimetanil

Pirimicarb

Propargite

Pyrimethanil

Pyrimiphos methyl

9.9

32.4

30.0

3.7

73.9

5.0

20.2

64.6

46.5

2.7

278.2

78.7

1.7

Black-green tea_30

Acetamiprid

Bifenthrin

Biphenyl

Carbendazim

Chlorpyrifos

Endosulphan I

Endosulphan II


Imidacloprid

Methomyl

Monocrothophos

Triazophos

45.7

105.0

21.1

10.6

6.6

10.2

37.0

49.5

52.4

3.8

10.3

7.8




Conclusions

• From the three MRM the preferred method for the analysis of

pesticides residues in green tea was QuEChERS with calcium

chloride. This method was the best in terms of recoveries and the

full scan chromatograms were the least influenced with coextracted

matrix compounds.

• The method was validated studying recoveries, limits of detection,

linearity , repeatability and matrix effects, with good results.

• It was applied to real samples from different countries, where 92% of

them were positives, as average they contained 5 pesticides per

sample and 24% exceeded the EU MRL.


Tea infusion


Infusion for LC analysis

50mL of boiled

milliQ water

2 g sample

Stir during 15 min

Take 900µL of

infusion + 100µL of

acetonitrile

Filter

Analysis by

LC-MS/MS

27.8

times

dilution


Transfer (%) of the pesticides from the leaves to the infusion

Water solubility


Ring Test Tea

Results


1

1

1

1

1

2

Participating labs

Number of participating labs in each country 1


Preparation of test material


Compound Lab 1

(France)

Lab 3

(Sweden)

Lab 4

(Germany)

Lab 5

(Austria)

EURL-

FV

EU

Average

(mg/kg)

EU

RSD

(%)

Lab 2

(Sri

Lanka)

Lab 6

(Thailand)

Lab 7

(Thailand)

EU and

3rd

Countries

RSD (%)

Acetamiprid 0.565 0.583 0.517 0.647 0.606 0.584 8.3 0.567 0.582 6.9

Bifenthrin 0.392 0.683 NA 0.711 0.634 0.605 24.1 0.402 0.801 28.0

Buprofezin 0.043 0.116 0.142 0.108 0.102 41.2 0.098 0.078 34.9

Carbofuran 0.022 0.016 0.027 0.022 26.3 Detected 26.3

Chlorpyrifos 0.014 0.013 0.009 0.012 20.8 Detected NA 20.8

Cypermethrine 0.065 NA 0.113 0.089 38.1 Detected 0.067 NA 33.2

Endosulfan Alpha 0.047 0.099 NA 0.087 0.100 0.083 29.8 0.079 1.781 189.8

Endosulfan Beta 0.077 0.183 NA 0.126 0.171 0.139 34.6 0.506 0.704 85.4

Endosulfan Sulfate 0.088 0.072 NA 0.049 0.080 0.072 23.3 NA 0.086 0.072 19.0

Fenpropathrin 0.090 0.193 NA 0.182 0.200 0.166 30.9 0.281 NA 0.219 32.0

Fenvalerate 0.031 0.050 NA 0.059 0.050 0.047 30.6 Detected 0.038 28.0

Imidacloprid 0.115 0.079 0.077 0.136 0.120 0.105 24.9 0.111 NA 22.2

Methomyl 0.231 0.278 0.388 0.345 0.311 22.5 NA NA 0.327 19.4

Pyridaben NA 0.024 0.024 0.095 84.0

Triazophos 0.028 0.042 0.044 0.033 0.037 20.4 0.219 0.015 121.0

Results

Concentration expressed as mg/kg

NA: Not Analised


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0.1

0.2

0.3

0.4

0.5

0.6

0.7

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Ace

tam

iprid

Bife

nth

rin

Bu

pro

fezin

Carb

ofu

ran

Chlo

rpyrifo

s

Cyp

erm

eth

rin

e

En

do

su

lfa

n A

lph

a

En

do

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lfa

n B

eta

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do

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ulfa

te

Fen

pro

pa

thrin

Imid

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prid

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tho

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ab

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zo

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os

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va

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Co

nc

en

tra

tio

n (

mg

/kg

)

Lab 1 (France) Lab 3 (Sweden) Lab 4 (Germany) Lab 5 (Austria) EURL-FV

European labs results


Thank You

for Your Attention

Analysis of pesticides in herbs by GC-QqQ and LC-QqQ · • The aim of the experiments was to...

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