European Reference Laboratory - FV - Pesticides · European Reference Laboratory - FV Chlorpyrifos...

European Reference Laboratory - FV


LC-HRAMS

WHY ?BENEFITS ?


Development of control programs for pesticides in Europe

Source: EFSA


DE

IT

FR

NL

UK

RO

HU

DKGR

FI

AT

BE

PL

SE

NO

ESSIIE

CZ

PT

BGSK

CY EELT

ISMT

LU

LV0

100

200

300

400

500

600

700

800

0 100 200 300 400 500 600 700 800 900

Development of control programs for pesticides in EuropeDEITFRNLUKROHUDKGRFIATBEPLSENOESSIIECZPTBGSKCYEELTISMTLULVLI

Source: EFSA


PESTICIDES Nº of reportedresults

Nº of NA (not sought)

False negatives

% of NA results from the total 125

Acetamiprid 56 67 2 54Carbaryl 101 24 0 19Cyprodinil 99 24 2 19Diazinon 123 2 0 2Dimethoate 119 2 4 2Fenhexamid 89 36 0 2Fludioxonil 85 36 4 29Imidacloprid 64 60 1 48Iprodione 113 8 4 6Kresoxim-methyl 104 20 1 16Methomyl 71 45 9 36Monocrotophos 89 30 5 24Procymidone 121 4 0 3Pyrimethanil 98 25 2 20Tetraconazole 70 49 6 39Thiabendazole 104 17 3 14

EUPT-FV07 (2005)Target list: 62 pesticides


ChlorpyrifosDichlofluanidDichlofluanid

EndosulfanMethidathionMyclobutanilMyclobutanil

Vinclozolin

EUPT-7 116EUPT-7 097EUPT-7 122

EUPT-7 125EUPT-7 126EUPT-7 049EUPT-7 125

EUPT-7 097

TestTestTest

TestTestTestTest

Test

0.00160.0130.103

0.1060.1760.260.005

0.022

0.010.050.006

0.0034-

0.010.0006

0.05

Pesticide Lab Code Sample Concentration(mg/kg)

MPRL(mg/kg)

0.050.05

0.05

0.050.020.020.02

0.05

Laboratories that reported false positives

RL(mg/kg)

EUPT-FV07 (2005)


LC‐QqQ‐MSCommon Transitions

226.3‐>142.3

226.3‐>100.1

Prometon

226.0‐>142.3

226.0‐>100.1 226.0‐>100.3

10.54 minSecbumeton10.54 min

226.0‐>142.3

Terbumeton 10.54 min

10.54 min 10.54 min 10.54 minPrometon Secbumeton Terbumeton

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts


0

5

10

15

20

25

30

Citr

us fr

uits

Tree

nut

sPo

me

fruits

Ston

e fru

itsBe

rries

and

sm

all f

ruits

Mis

cella

neou

s fru

its w

ith…

Mis

cella

neou

s fru

its w

ith…

Mis

cella

neou

s fru

its w

ith…

Roo

t and

tube

r veg

etab

les

Bras

sica

veg

etab

les

Leaf

veg

etab

les,

her

bs a

nd…

Legu

me

vege

tabl

esSt

em v

eget

able

sFu

ngi,

mos

ses

and

liche

nsAl

gae

and

prok

aryo

tes…

Puls

esO

ilsee

dsO

il fru

itsC

erea

lsTe

asC

offe

e be

ans

Her

bal i

nfus

ions

from

Coc

oa b

eans

Car

obs/

Sain

t Joh

n's

brea

dsH

ops

Seed

spi

ces

Frui

t spi

ces

Bark

spi

ces

Roo

t and

rhiz

ome

spic

esBu

d sp

ices

Flow

er p

istil

spi

ces

Aril

spic

esSu

gar P

lant

s

Mai

n pr

oduc

tsPlant Origin Matrix Groups

Total number: 250 matrices


Mass

Data base components‐ orangematrix compounds (1 g/mL)

8017 Matrix compounds

m/z: 100‐700Rt: 4‐8 min

m/z: 100‐400Rt: 7‐13 min

DB: 750 components

Orange: 2743 matrix compounds Miner 3D Enterprise


2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

+ESI MRM Frag=380.0V CF=0.000 DF=0.…

* 10.322

2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


* 10.322

Counts vs. Acquisition Time (min)10 10.2 10.4 10.6

2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


* 10.322

2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


* 10.322


2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5+ESI MRM Frag=380.0V CF=0.000 DF=0.…

* 10.334

2x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


* 10.322


2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


* 10.334

2x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


* 10.322


2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


* 10.334

2x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


* 10.322


Std in solvent at 0.01 mg/kg

Ethyl Acetate method

Haloxyfop at 0.01 mg/kg in Aubergine. LC-MS/MS

NL method Citrate QuEChERSmethod

Ion Ratio: 0.34Diff.: 11%




Ion Ratio: 0.38

Citrate QuEChERSwithout clean-up

method



0.00

1 D

a

1 D

a


Pyrimethanil[M+H+] m/z 200.11820.01 mg/ kg in onionRT = 7.05 minDilution 5x

0 1 2 3 4 5 6 7 8 9 10 11 12Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

9.51

7.397.38

4.90 6.74 6.754.49

5.64 6.559.806.50 7.52

6.365.83 9.864.22 5.47 7.60 8.894.14 9.468.392.16 10.233.092.27 11.5110.74 11.732.11 3.750.22 1.000.71 1.34

200.1182 ± 1 Da

0 1 2 3 4 5 6 7 8 9 10 11 12Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

7.05

200.1182 ± 0.001 Da

Pyrimethanil at 0.01 mg/kg


EUPT Sample: coriander

LinuronStandard in solventIon ratio: 1.8

Linuron (0.125 mg/kg)Real sample of corianderIon ratio: 2.4

FALSE NEGATIVE


EUPT Sample of coriander

Linuron

Matrix interference


BUT…..


0102030405060708090

100

20 ppb 50 ppb 100 ppb

Com

poun

ds (%

)

0102030405060708090

100

20 ppb 50 ppb 100 ppbC

ompo

unds

(%)

0102030405060708090

100


Com

poun

ds (%

) ,

0102030405060708090

100


Com

poun

ds (%

) ,

0102030405060708090

100


Com

poun

ds (%

) ,

0102030405060708090

100


Com

poun

ds (%

) ,

Solvent

Tomato

Pepper

Zucchini

Orange

Leek

Automatic Detection20 µg kg-1

20 µg kg-1

20 µg kg-1

50 µg kg-1

50 µg kg-1

50 µg kg-1

100 µg kg-1

100 µg kg-1

100 µg kg-1

20 µg kg-1 50 µg kg-1 100 µg kg-1

20 µg kg-1 50 µg kg-1 100 µg kg-1

20 µg kg-1 50 µg kg-1 100 µg kg-1

Manual Detection

Not detected


C20H22N2O +H: C20 H23 N2 O1 pa Chrg 1

307.0 307.5 308.0 308.5 309.0 309.5 310.0 310.5 311.0 311.5 312.0 312.5 313.0m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

307.1805

308.1838

309.1872310.1906 311.1939 312.1948 313.1982

Monoisotopic ionC20H23N2O

Ion with two 13C13C2C18H23N2O

Ion with 13C atom13CC19H23N2O

OUT OF LINEAR RANGE

BELOW LOQ

OUT OF LINEAR RANGE BELOW LOQ

0.005 mg/kg

0.010 mg/kg

0.020 mg/kg

0.050 mg/kg

0.100 mg/kg

0.250 mg/kg

0.500 mg/kg

0.750 mg/kg

1.000 mg/kg

Fenazaquin in tomato extract, calibration with monoisotopic ion, with ion containing one13C atom and with ion containing two 13C atom. Quantitation with monoisotopic ionpossible up to 0.100 mg/kg. Higher concentration levels have to be quantified with ioncontaining two 13C atoms

Monoisotopic ion

Ion with 13C atom

Ion with two 13C atom


Influence of resolution on detection

317.00 317.05 317.10 317.15 317.20 317.25 317.30 317.35m/z

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

317.1657

317.2014

317.1728

317.1966

317.1544

Exact mass of bupirimate: 317.1642

Matrix ion. Mass error 27.1 ppm

Matrix ion. Mass error -30.1 ppm

Resolution 17500

Resolution 70000

Two non resolved matrix ions. Mass error 4.7 ppm False detect of bupirimate

317.1728

317.1657

317.2014

317.1966

317.1544



LOQ

0%10%20%30%40%50%60%70%80%90%

100%

0.01 mg/kg 0.02 mg/kg 0.05 mg/kg

ddMS2vDIAAIF

0%10%20%30%40%50%60%70%80%90%

100%

0.01 mg/kg 0.02 mg/kg 0.05 mg/kg

ddMS2vDIAAIF

Tomato Apple

0%10%20%30%40%50%60%70%80%90%

100%

0.01 mg/kg 0.02 mg/kg 0.05 mg/kg

ddMS2vDIAAIF

Orange

0%10%20%30%40%50%60%70%80%90%

100%

0.01 mg/kg 0.02 mg/kg 0.05 mg/kg

ddMS2vDIAAIF

Onion

% o

f com

poun

ds

% o

f com

poun

ds

% o

f com

poun

ds

% o

f com

poun

ds

Target Analysis

Non Target MS2 (DIA)Non Target MS2 (DIA-VMW)

Target Analysis


Target Analysis


Target Analysis



Linearity LC-HRAMS

0,0E+00

2,0E+08

4,0E+08

6,0E+08

8,0E+08

1,0E+09

1,2E+09

1,4E+09

1,6E+09

1,8E+09

2,0E+09

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6

Concentration [mg/kg]

Pirimiphos-methyl

Ethoprophos

Flusilazole

Area

Spinach

0.07 0.14 0.21 0.28 0.35 0.42 0.49 0.56

Azoxystrobin

Imidacloprid

Zoxamide


1.3‐1.1

‐1.0

‐0.7

‐0.5‐0.5

‐0.4 ‐0.2‐1.2

‐0.5‐0.7

‐1.0‐0.50.7 ‐1.5

Azoxystrobin 0.01 mg/kg in tomatoDilution 5x (mass errors in ppm)

6.92 6.93 6.94 6.95 6.96 6.97 6.98 6.99 7.00 7.01 7.02 7.03 7.04 7.05Time (min)

0.20.00.00.0

‐0.20.0

0.0‐0.2

0.0 0.00.0

0.0 0.00.0

‐0.20.0‐0.2 ‐0.2

0.2 0.7 ‐1.2 ‐0.7 0.7

QTOF

Orbitrap


Ethirimol 0.1 mg/kg in tomato, MS2 spectrum


0

20

40

60

80

0.1 0.01 0.005 0.001 0.0005495 49 25 5 2

(Da)(ppm)

ThiabendazoleC10H7N3S (m/z 202.0433)

C0‐25H0‐50N0‐5O0‐5Cl0‐5Br0‐5SNum

ber o

f possible molecular fo

rmulae

5 ppm

~ 2 000 ~ 20 000 ~ 40 000 ~ 200 000 ~ 400 000 (FWHM)Resolution

Resolution ~ 70 000 FWHM

9 molecular formulae


‐ Cl, Br

~ 70 000~25 000

Absolute mass error relative to Thiabendazole

Resolution ~ 25 000 FWHM



‐ Cl, Br


0

20000

40000

60000

80000

100000

120000

140000

0 200 400 600 800 1000 1200 1400 1600

Res

olut

ion

m/z

QToFOrbitrap

Resolution dependence on m/zExperimental data


Number of Pesticides with fragments using Orbitrap in Full Scan mode

Solvent 0.10 mg/kg%

of p

estic

ides


RT: 6.72 - 10.15

6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0Time (min)

0

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e A

bund

ance

0

20

40

60

80

100 8.458.47

8.47

8.49

8.54

8.48

Pepper spikedat 0.01 mg/kgmetlaxyl

GrapefruitReal sample 1

Metalaxyl (XIC m/z 280.1543 ± 5 ppm). Full scan MS. Resolution 70000

GrapefruitReal sample 2

Analysis of real samples by LC-HRAMS MS2


Metalaxyl (XIC m/z 280.1543 ± 5 ppm). Full scan MS. Resolution 70000

Expected fragments:148.1119160.1119192.1380220.1380

LC-QOrbitrap

GrapefruitReal sample

192.1382

160.1120 220.1330

148.1121165.1148134.09641011 179.1061

191.0603

245.1078

2203.1068132.0525 172.5912.4849

191 0600

RT: 6.72 - 10.15

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

0

20

40

60

80

1008.45

8.47

8.47

8.49

8.54

8 48

FALSE POSITIVE

XIC m/z 280.1543 ± 5 ppm

XIC m/z 280.1543 ± 5 ppm

Standard of Metalaxyl

279.5 280.0 280.5 28

280.1547

280.227623

280.1547

MS spectrum Experimental MS/MS spectrum

Library MS/MS spectrum

192,1382

191,0603


Combining Full Scan MS and MS2 Mode


Pesticide residue method MS2

Full Scan

• Detection/Identification (mass and retention time)• Quantitation

MS/MS (Target and Non target MS/MS)

• Identification (at least one fragment)


Pesticides Full scan QTOF Orbitrap MS2 QTOF OrbitrapAzoxystrobin 404.1241 -1.5 0.0 372.0979 0.6 -0.3Diazinon 305.1083 -1.5 0.3 169.0794 0.9 0.0Dichlorvos 220.9532 0.5 0.5 109.0047 -1.9 1.6Fenamiphos - sulfone 336.1029 -0.2 0.6 266.0246 -1.6 0.4Profenofos 372.9424 1.9 0.5 302.8642 1.0 -0.3

Pesticides Full scan QTOF Orbitrap MS2 QTOF OrbitrapAzoxystrobin 404.1241 0.4 0.0 372.0979 1.4 0.3Diazinon 305.1083 1.2 -0.3 169.0794 -1.1 -0.6Dichlorvos 220.9532 1.9 -0.5 109.0047 -2.3 2.4Fenamiphos - sulfone 336.1029 -0.2 0.0 266.0246 1.5 0.0Profenofos 372.9424 -0.4 0.0 302.8642 1.1 -1.0

Tomato

Orange

0.01 mg/kg

0.01 mg/kg


32k 32 k

32k 32k

32k 32k 32k 32k

32k 32k 32k 32k 32k 32k

32k 32k 32k 32k

TARGET ANALYSIS (DATA DEPENDENT ACQUISITION)

NON TARGET MS2 DIA-VMW (FS/SWATH)

Full scan MS

MS2

0.1 – 0.5 s 0.1 - 0.5 s

32k

NON TARGET MS2 (DATA INDEPENDENT ACQUISITION) FS/AIF-MS2

32k 32k 32k 32k 32k 32k 32k 32k 32k

TOF32k

32k

32k

32k

32k

32k

32 k32k

32k

32k

32k

32k

32k


LC‐QTOF‐MS

Full scan detection

Formetanate Tomato 0.02 mg/kg

Auto MS/MS mode

4x10

0

1

2

3

Counts vs. Mass-to-Charge (m/z)218219220221222223224225226227228229

No MS/MS spectrum

All ions mode

MS/MS spectrum

165.1022

222.1242

120.0444

Formetanate Identified with two fragments ions


Full scan detection IDA mode

No MS/MS spectrum

SWATH mode Propoxur Identified by fragments ions


70k 17,5k

70k 70k

70k 35k 35k 35k 35k 35k

70k 35k 35k 35k

70k 17,5k

17,5k

17,5k

17,5k

17,5k

70k 17,5k

17,5k

17,5k

TARGET ANALYSIS (DATA DEPENDENT ACQUISITION) FS/dd-MS2

FS/AIF-MS2

FS/vDIA-MS2

0.27 s 1.28 s

Orbitrap Full scan MS

MS2


11.25 11.30 11.35 11.40 11.45 11.50 11.55 11.60Time (min)

ddMS2

cycle time 0.27s

AIFcycle time 0.62s

vDIA (3segm x 17,500)cycle time 0.61s

vDIA (5segm x 17,500)cycle time 0.79 s



Fenazaquin0.02 mg/L in solvent


0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

RSD 0-5% RSD 0-10% RSD 0-20%

3 segments/ R=350005 segments/ R=350003 segments/ R=175005 segments/ R=17500AIFdd MS2

Tomato0.01 mg/kg

Repeatability(Five Analyses)

% o

f com

poun

ds


11 Matrices

Cucumber Orange Green bean

Apple

Lettuce

Onion

Tomato

Parsley Leek

Garlic Spinach


Identification

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

dd MS2 vDIA AIF

0.01 mg/kg0.1 mg/kg

166 pesticides x 11 matrices = 1826 results%

of c

ompo

unds


Pesticide Asigned value(mg/kg)

Obtained value (Difference)AIF dd MS2 vDIA

Bupirimate 0,165 (±50%) 0,157 (-5%) 0,152 (-8%) 0,160 (-3%)

Carbendazim 0,512 (±50%) 0,415 (-19%) 0,404 (-21%) 0,404 (-21%)

Diazinon 0,071 (±50%) 0,051 (-28%) 0,051 (-28%) 0,051 (-28%)

Difenoconazol 0,53 (±50%) 0,625 (18%) 0,631 (19%) 0,610 (15%)

Diflubenzuron 0,319 (±50%) 0,367 (15%) 0,332 (4%) 0,354 (11%)

Metoxyfenozide 0,349 (±50%) 0,300 (-14%) 0,304 (-13%) 0,293 (-16%)

Spinosaad 0,051 (±50%) 0,044 (-14%) 0,044 (-14%) 0,044 (-14%)

Thiabendazole 1,90 (±50%) 2,07 (9%) 2,01 (6%) 2,00 (5%)

Trifloxystrobin 0,466 (±50%) 0,592 (27%) 0,592 (27%) 0,559 (20%)

Pendimethalin 0,062 (±50%) 0,070 (13%) 0,059 (-5%) 0,065 5%)

EUPT-FV-17 Broccoli 2015


• “Old” or rarely detected compounds

• “Very expensive” analytical standards

• Compounds “produced” during the analysis

• Complex residue definitionCycloxydim including degradation and reaction products which can be determined as 3-(3-thianyl)glutaric acid S-dioxide (BH 517-TGSO2) and/or3-hydroxy-3-(3-thianyl)glutaric acid S-dioxide (BH 517-5-OH-TGSO2) ormethyl esters thereof, calculated in total as cycloxydim


0

50000

100000

150000

200000

250000

0 50 100 150 200 250 300 350

solvent AcN

AcN/H2O

BroccoliAscorbic Acid

ARE

A

min

Diafenthiuron


Standard Broccoli

R1 Broccoli

R2 Broccoli

Diafenthiuron

Urea

Sulfomonoxide

Urea

Sulfomonoxide

Urea

Sulfomonoxide

Diafenthiuron

Diafenthiuron

[M+H]+ =369.2536

[M+H]+ =369.2536

[M+H]+ =369.2536


C23H32N2O2369.2536 C23H30N2O2

351.2430

C23H32N2OS385.2308

C23H32N2O2S401.2257


0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

Difenthiuron Sulfomonoxide Urea

ARE

A

Diafenthiuron in Broccoli

St1St2St3R1_t1R1_t2R1_t3R2_t1R2_t2R2_t3

R2:0%R1:0%

LC-QTOF-MS


0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

Difenthiuron Sulfomonoxide Urea

ARE

A

Diafenthiuron in Orange

St1St2St3R1_t1R1_t2R1_t3R2_t1R2_t2R2_t3

LC-QTOF-MS


Standard Orange

R1 Orange

R2 Orange

Diafenthiuron

Sulfomonoxide

Sulfomonoxide

Sulfomonoxide

Diafenthiuron

Diafenthiuron

Urea



Number of Laboratories analising by HRAMSin the last three EUPTs-FV-SM

No.

of L

abor

ator

ies


% of Laboratories analising by HRAMSin the last three EUPTs-FV-SM

% o

f Lab

orat

orie

s


24 EU/EFTA Countries

AustriaBelgiumBulgaryCroatiaCyprusCzech Republic DenmarkEstoniaFinlandFranceGermanyGreeceHungaryItalyIrelandLatviaNetherlandsNorwayRomaniaSerbiaSloveniaSpainSwedenSwitzerlandUnited Kingdom

Participation EUPT-FV-SM08

AustriaBelgiumBulgaryCroatiaCyprusCzech RepublicDenmarkEstoniaFinlandFranceGermanyGreeceHungaryItalyIrelandLatviaNetherlandsNorwayRomaniaSerbiaSloveniaSpainSwedenSwitzerlandUnited Kingdom

HRAMSCzech Republic

DenmarkFrance

GermanyGreece

ItalyNetherlands

NorwayRomania

SpainSweden

United Kingdom



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