pesticides by lc-ms - csic by lc-ms.pdf · logo grupo pesticides determined by lc benzimidazole...

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1SAMA-Food and Environmental Safety Research Group, Facultat de Farmàcia2 Department of Environmental Chemistry, IDAEA-CSIC, Barcelona, Spain3Catalan Institute for Water Research (ICRA), Girona, Spain4 King Saud University, Riyadh, Saudi Arabia

Analysis, fate and risks of organic contaminants in river basins under water scarcityValencia, 7-8 February 2011

Pesticides by LC-MSY. Picó1, M. Farré2, D. Barceló2,3,4

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Monitoring of Pesticide ResiduesGAS CHROMATOGRAPHY

DISADVANTAGES:DISADVANTAGES:

Inappropriate to determine non-volatile, thermolabile and/or polar compounds

ADVANTAGES:ADVANTAGES:High resolutionLow pricesWide variety of selective detectorsEasy coupling to mass spectrometry

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Pesticides determined by GC

Organochlorinep,p’-DDT

OrganophosphorusChlorpyriphos

Pyrethroid insecticidesCypermethrin

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ADVANTAGES:ADVANTAGES:Determine all compounds independently of their physico-chemical characteristics

SPECIFICITYSPECIFICITYSENSITIVITYSENSITIVITY

SELECTIVITYSELECTIVITY

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PESTICIDES DETERMINED BY LC

BenzimidazoleCarbendazim

NitroguanidineImidachloprid

DinitrophenolDinoprop

ConazolePropiconazole

CarbamatePirimicarb

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Requeriments of chromatography

NEUTRAL COMPOUNDS: Reversed phase LC

C18 analytical columns

H2O, volatile buffer solutions, methanol, acetonitrile

Flow rates 0.2-2 ml/min

pH needs carefull optimization

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IONIC COMPOUNDS: Different retention mechanismsIon-exchange chromatographyIon-pair chromatography

Strong salts counter-ions buffers

ADDITIONAL COMPLICATIONS

Requeriments of chromatography

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Ion-pair LC of chlormequat in pears

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Requeriments of mass spectrometry

1.-Interfacing systems

2.-Mass spectrometry data adquisition

3.-The mass spectrometer

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1.-Interfacing systems

Atmospheric pressure ionization (API) sources:

o Electrospray (ESI)o Atmospheric pressure chemical ionization (APCI)o Atmospheric pressure photoionization (APPI)

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APCI vs. ESI

Thurman et al. Anal. Chem. 18 (2000) 2450

N2

N2

N2

Zona deFRAGMENTACIÓN

(CID)

VaporizadorAPCI

N2

Capillary

N2 Nebulizer

N2

Zona deFRAGMENTACIÓN

(CID)

Discharge electrode

APCI

LC

Skimmer

Corone Needle

Octopole High Energy Dinode

Lenses Quadrupole

Electromultiplicator(EM)

IONFOCUSSING

SEPARATION EVAPORATIONAND IONIZATION

DETECTION

IONFOCUSSING

SEPARATION

Drying gasN 2

Capillary

N2Nebulizer

N2

LensesFRAGMENTATION(CID)

Skimmer

N

LC

N2

N2

(CID)

NEBULIZATION AND IONIZATION

Quadrupole

Electromultiplicator(EM)

DETECTION

Ionization source at atmospheric pressure

OctopoleHigh Energy Dinode

Atmospheric Pressure Chemical Ionization (APCI)Electrospray (ESI)

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Carbamates in lettuce

10 15 20

0

50000

100000

150000

200000

250000

min

5 25 30

Res

pons

e ra

nge

min

5 10 15 20 30 35

0

50000

100000

150000

200000

250000

300000

350000

25

Fernández et al. J. Chromatogr. A. 18 (2004) 2450

Res

pons

e ra

nge

APCI ESI≈

thio

benc

arb

thio

benc

arb

feno

xyca

rb

feno

xyca

rb

met

hioc

arb

met

hioc

arb

diet

hofe

ncar

b

Die

thof

enca

rb

+ fe

nobu

carb

feno

buca

rbpi

rimic

arb

pirim

icar

b

isop

roca

rb isop

roca

rb

ethi

ofen

carb

ethi

ofen

carb

carb

aryl ca

rbar

yl

carb

ofur

an carb

ofur

an

prop

oxur

prop

oxur

met

olca

rb

met

olca

rb

oxam

yl

oxam

yl

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Different ESI designsAgilent technologies ZsprayTM by Water/Micromass

Turbo VTM by ABSCIEX

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Comparison of ESI and APCI

Lutz & Alder. J. AOAC Int. 86 (2003) 1015

Relative Response Factor (RRF )Analyte ESI 400ºC APCI 325 ºC APCI 375 ºC APCI 425 ºCFlazasulfuronFlorasulamHaloxyforp-methylHexaflumuronImazalil (reference)ImidaclopridIndoxacarbLinuronMCPAMecoprop-PMetalaxylMetramitronMethamidophosMethiocarb

0.5080.5891.1660.0171.0000.3610.1450.4400.0440.0592.8760.6570.5145.261

0.0040.0570.0340.0080.0980.0890.0070.0410.0130.0070.0580.0550.4110.053

0.0040.0490.0320.0060.0990.0750.0040.0380.0150.0080.0470.0500.3440.047

0.0030.0480.0260.0060.0910.0640.0020.0370.0180.0080.0380.0450.3070.040

>>

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Mass spectrometers combined with LC

Low resolution (single quadrupole)

High resolution (time-of-flight, TOF)

Tandem mass spectrometry (MS/MSn)Triple quadrupole (QqQ)Ion Trap (QIT)Quadrupole time-of-flight (QqTOF)

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2.- Mass Spectrometric Data Acquisition

a) Full mass spectra (for library search, structure information)

b) Raw data of small mass range (for the determination of isotope patterns or just molecular weight

c) Mass chromatograms of selected ions (for the quantitation of target compounds)

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Identification points (IPs) earned per ion

MS technique IPs earned per ion

Low resolution mass spectrometry (LR)LR-MSn precursor ionLR-MSn product ionHigh resolution mass spectrometry (HR)HRMSn precursor ionHRMSn product ion

11

1.522

2.5Commission Decision (2002/657/EEC) concerning to the Performance of analytical methods for the determination of organic residues and contaminants in live animal and animal products

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Time-of-flight (TOF)

FOCUSING

ORTHOGONAL ACCELERATOR

FLIGHT TUBE

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Time-of-flight (TOF) characteristics

Elevated mass spectral resolutionSelectivity ↑↑↑↑

Exact mass measurementsSpecificity ↑↑↑↑

High sensitivity in full spectral dataMonitoring of non-target analytes

Low dynamic rangeAccuracy for quantification ↓↓↓

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TOF/MS-Application tomultiresidue analysis

carb

enda

zim

thia

bend

azol

e

thia

clop

rid

Spin

osad

A

Ferrer et al. J. Chromatogr. A. 1082 (2005) 81

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TOF/MS-from mass spectrum to elemental composition

HN NH

S

HN

O

O

HN

S O

C11H11N4O3S2

HN NH

S

HN

O

O

HN

S O

HN NH

S

HN

O

O

HN

S O

C11H11N4O3S2

HN NH

S

C7H7N2SHN NH

S

HN NH

S

C7H7N2S Measured Mass = 151.0321Calculated Mass = 151.0324Error was –2.3 ppm

Measured Mass = 311.0267Calculated Mass = 311.0267Error was <0.1 ppm

Thurman et al. J. Chromatogr. A. 1082 (2005) 71

NONNON--TARGET PESTICIDES AND UNKNOWN METABOLITESTARGET PESTICIDES AND UNKNOWN METABOLITES

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Multiple Tandem MassSpectrometry

Triple Quadrupole MS (QqQ MS)

- MRM – high sensitivity quantitation

Quadrupole Ion-Trap MS (QIT MS)

- MSn - fragmentation pathways

Hybrid Quadrupole Time-of-Flight MS (QqTOF MS)- high mass accuracy spectral data useful for confirmation of formulae

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Triple quadrupole (QqQ) sequence of Triple quadrupole (QqQ) sequence of eventsevents

Ion detection

Ion filtering

Ion transport

Ion production

Ion filtering

Ion processing

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QqQ operating modesQqQ operating modes

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Multiple Reaction Monitoring (MRM)

Q1 Q3Collision cell

filter Precursor Ion - filter Product Ion

Scan Q1 and Q3 to perform this operation on multiple target analytes –25 msec per MRMHigh sensitivity and selectivity but no spectrum

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QqQ characteristicsQqQ characteristics

A LARGE NUMBER OF COMPOUNDS (>100) CAN A LARGE NUMBER OF COMPOUNDS (>100) CAN BE SIMULTANEOUSLY DETERMINED BE SIMULTANEOUSLY DETERMINED

Two precursorTwo precursor--product ion transitions met the product ion transitions met the EU criterionEU criterion

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MRM chromatogram of matrixMRM chromatogram of matrix--matched matched standard at 0.05 mg/kgstandard at 0.05 mg/kg

Hetherton et al. Rapid Commun. Mass Spectrom 18 (2004) 2450

5 10 15 20 25 30 35 40Time/min

0

%

100

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Analyte specific transitions obtained by QqQ for several pesticides/metabolites

Compound Precursor ion First transition (m/z) Second transition (m/z)AldicarbAldicarb sulfoxideAldoxycarbAmidosulfuronAtrazineAzoxystrobinBendiocarbBensulfuron-methylBromoxynilButocarboxim Butocarboxim sulfoxideButoxycarboximCarbarylCarbendazimCarbofuran

[M+NH4]+[M+H]+

[M+NH4]+[M+H]+[M+H]+[M+H]+[M+H]+[M+H]+[M-H]-

[M+NH4]+[M+H]+

[M+NH4]+[M+H]+[M+H]+[M+H]+

208→89207→89

240→176370→261216→174404→372224→167411→149276→81

208→116207→75

240→106202→145192→160222→165

208→116207→132240→166370→218216→104404→344224→109411→119278→81

208→116207→132240→177202→127192→132222→123

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The ion trap processes-MS

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IonIon--Trap CharacteristicsTrap Characteristics

Multiplies the stages of mass analysis Multiplies the stages of mass analysis umpteenth timesumpteenth times

FRAGMENTATION PATTERNS FRAGMENTATION PATTERNS STRUCTURAL ELUCIDATIONSTRUCTURAL ELUCIDATION

Limited number of compounds can be Limited number of compounds can be determined simultaneouslydetermined simultaneously

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(MS/MS(MS/MSnn) fragmentation for ) fragmentation for triazinestriazines

N N

NCl NH

NH2+

H2C

R1CH3

R2

CH3

N N

NCl NH

NH3+

H2C

CH3

N N

NCl NH2

NH3+

C CH2

R1

R2

MS2

CH2=CH2

N N

N

NH2

C

NH+C

HCl

CN NH2

MS3

MS4

MS4

CH2=CH2

CN NH2

MS4N

Cl

N

H2C

CH3NH+

N

Cl

N NH2+

CN

NC

NH+

H2C

CH3

C

NH

NH2Cl MS3

MS3

m/z 110

m/z 174

m/z 96m/z 146

m/z 104

m/z 132

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Quadrupole Time of Flight (QqTOF)

FocusingFocusing FilteringFiltering CollisionCollision

High Mass accuracy

High degree of sensitivity

Unlimited mass range

Suitable for trace analysis

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Advantages of MS/MSn

Reduction of interferences for Reduction of interferences for coco--extracted food materialsextracted food materials

Enhanced selectivity

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Enhanced specificity-Elimination of isobaric interferences

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Performance of different LC-MS systems to determine

pesticides in food

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ADIs and MRLsADI(mg/kg)

MRLs (Spanish legislation) mg/kg Residue

Orange Strawberry Cherry Peach Apricot Pear

Acrinathrin 0.02 0.02 0.2 0.2 0.2 0.2 0.1 Acrinathrin

Bupirimate 0.3 0.05 0.5 0.5 0.5 0.5 0.5 Bupirimate+Ethirimol

Buprofezin 0.01 0.2 0.01 0.01 0.01 0.01 0.01 Buprofezin

Λ-cyhalothrin 0.005 0.1 0.5 0.1 0.2 0.2 0.1 Λ-cyhalothrin

Cyproconazole 0.01 0.01 0.01 0.01 0.05 0.05 0.05 Mixture of isomers

Fluvalinate 0.005 1 0.01 0.01 1 1 1 Fluvalinate

Hexaflumuron 0.02 0.5 0.05 0.05 0.5 0.05 0.2 Hexaflumuron

Kresoxim methyl 0.4 0.05 1 0.05 0.05 0.05 0.2 Kresoxim methyl

Propanil 0.007 0.1 0.1 0.1 0.1 0.1 0.1 Propanil

Pyrifenox 0.1 0.05 0.2 0.05 0.2 0.2 0.2 Pyrifenox

Pyriproxyfen 0.003 0.5 0.05 0.05 0.05 0.05 0.05 Pyriproxyfen

Tebufenpyrad 0.002 0.5 0.05 0.5 0.5 0.5 0.5 Tebufenpyrad

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Pressurized liquid extraction (PLE)

Extraction solvent Ethyl acetate

Temperature 75 ºCPressure 1500 psiFlush volume 100 %Heating time 2 minPurge time 60 sStatic Extraction 2 cycles, 7 min

Fruit sample 2.5 g 22 ml stainsteel cell+ 20 g of acidic silica

Extract concentrated to 0.25 mL

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Liquid chromatography-mass spectrometry

TQ. Quattro LC from MICROMASS equipped with a LC Alliance 2690 system and a Max Lynx Software

QIT. Esquire 3000 from BRUCKER and an Agilent 1100 series LC System and Daltonic Esquire Control Software

QqTOF.Qstar Pulsar an Agilent 1100 series LC System and controlled with Analyst 1.3. software from APPLIED BIOSYSTEMS,

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Liquid chromatography separation

Analytical Column

Luna C18 (150 x 4.6 mm I.D., 5 μm) preceded by a securityguard cartridge C18 ( Phenomenex)

Mobile phase

A: Methanol 10 mM ammonium formateB: Water 10 mM ammonium formate

Gradient 70 % to 90% A in 10 minheld to 90 % 10 min

Flow-rate 0.6 ml/min

Injection volumen

20 μl

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Mass spectrometry conditions

ESI in positive ionization modeTune of the instrument according to manufacturer’s

specificationsOptimization of other conditions using standard solutions of

pesticides

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Results and Discussion

TARGETEDMETHOD

QqQ in SRM modeQqQ in SRM modeScreening of several precursor-product ion transitions

QqToF in MRM modeQqToF in MRM modeScreening of the product-ion mass spectra (from

m/z 50 to m/z 1000)

QIT in MRM mode (isolation of parent ions)QIT in MRM mode (isolation of parent ions)Screening of the product-ion mass spectra (from

m/z 50 to m/z 1000)

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Limits of quantification (mg/kg)(calculated according to doc. SANCO/825/00)

COMPOUNDCOMPOUND QqQQqQ QITQIT QqToFQqToFAcrinathrinAcrinathrin 0.010.01 0.010.01 0.020.02BupirimateBupirimate 0.00010.0001 0.0050.005 0.010.01BuprofezinBuprofezin 0.00010.0001 0.010.01 0.050.05λλ--cyhalothrincyhalothrin 0.010.01 0.40.4 0.30.3CyproconazoleCyproconazole 0.0020.002 0.010.01 0.050.05FluvalinateFluvalinate 0.0010.001 0.20.2 0.40.4HexaflumuronHexaflumuron 0.010.01 0.020.02 0.040.04Kresosim methylKresosim methyl 0.00050.0005 0.010.01 0.050.05PropanilPropanil 0.0010.001 0.020.02 0.070.07PyrifenoxPyrifenox 0.00050.0005 0.010.01 0.030.03PyriproxyfenPyriproxyfen 0.0020.002 0.020.02 0.050.05TebufenpyradTebufenpyrad 0.00050.0005 0.010.01 0.050.05

**

**

**

LOQ

s at

leas

t 10

times

low

er

LOQ

s at

leas

t 10

times

low

er

LOQ

s m

eet M

RLs

for 1

0 pe

stic

ides

LOQ

s m

eet M

RLs

for 1

0 pe

stic

ides

LOQ

s m

eet M

RLs

for 8

pes

ticid

es

LOQ

s m

eet M

RLs

for 8

pes

ticid

es

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Typical chromatogram of an orange sample

naranja 1ppm

Time5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100NAR3ASE 10: MRM of 2 Channels ES+

TIC1.76e5

16.33

13.39

15.87 17.44

NAR1ASE 11: MRM of 3 Channels ES+ TIC

3.42e416.13

15.10

18.4019.49


8.86e317.41

16.76

naranja 0.01 ppm

Time5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0


TIC7.97e3

13.23


3.07e314.01

13.7413.41

14.41


2.63e413.59

naranja 0.01 ppm

Time5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0


TIC5.57e3

9.29

8.61

6.549.65

10.44


1.71e49.49


2.87e411.80

12.63

naranja 0.01 ppm

Time5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0


TIC1.10e3

7.65

6.675.11 6.105.477.13

8.38 8.48 8.89


5.10e38.54

7.975.22 6.83

6.21 7.45

9.239.67 9.98

10.6010.83


2.30e39.90

8.455.965.60 7.886.48 7.20 9.19 10.88

PropanilCV 35, CE 25

CyproconazoleCV 35, CE 30

Kresoxim-methylCV 25, CE 30

PyrifenoxCV 30, CE 30

BupirimateCV 30, CE 20

HexaflumuronCV 30, CE 30

TebufenpyradCE 20, CV 25

PyriproxyfenCV 25, CE 20

BuprofezinCV 30, CE 20

AcrinathrinCE 50, CV 5

λ-cyhalothrinCV 40, CE 2

FluvalinateCV 25, CE 20

naranja 1ppm

Time5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0


TIC1.76e5

16.33

13.39

15.87 17.44


3.42e416.13

15.10

18.4019.49


8.86e317.41

16.76

naranja 0.01 ppm

Time5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0


TIC7.97e3

13.23


3.07e314.01

13.7413.41

14.41


2.63e413.59

naranja 0.01 ppm

Time5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0


TIC5.57e3

9.29

8.61

6.549.65

10.44


1.71e49.49


2.87e411.80

12.63

naranja 0.01 ppm

Time5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0


TIC1.10e3

7.65

6.675.11 6.105.477.13

8.38 8.48 8.89


5.10e38.54

7.975.22 6.83

6.21 7.45

9.239.67 9.98

10.6010.83


2.30e39.90

8.455.965.60 7.886.48 7.20 9.19 10.88

PropanilCV 35, CE 25

CyproconazoleCV 35, CE 30

Kresoxim-methylCV 25, CE 30

PyrifenoxCV 30, CE 30

BupirimateCV 30, CE 20

HexaflumuronCV 30, CE 30

TebufenpyradCE 20, CV 25

PyriproxyfenCV 25, CE 20

BuprofezinCV 30, CE 20

AcrinathrinCE 50, CV 5

λ-cyhalothrinCV 40, CE 2

FluvalinateCV 25, CE 20

Concentration of each pesticide in orange 0.001 mg/kg, except for acrinathrin, λ-cyhalothrin and hexaflumuron, the concentration of which is 0.01 mg/kg

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Transitions selected using QqQ

COMPOUNDCOMPOUND Precursor ion

Transitions CV CE IPs reached

AcrinathrinAcrinathrin [M+Na]+ 564→564 50 5 1λλ--cyhalothrincyhalothrin [M+Na}+ 472→472 40 2 1HexaflumuronHexaflumuron [M+Na]+

[M+H]+483→483461→ 158

2030

230

3.5

►►Compounds with the highest LOQsCompounds with the highest LOQs

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CyproconazoleCyproconazole [M+H]+ 292→125292→70

35 30 4

FluvalinateFluvalinate [M+H]+ 503→208503→181

25 20 4

Kresosim methylKresosim methyl [M+H]+ 314→131314→116

25 30 4

PropanilPropanil [M+H]+ 218→162218→127

36 25 4

PyrifenoxPyrifenox [M+H]+ 295→227295→92

30 20 4

PyriproxyfenPyriproxyfen [M+H]+ 322→133322→196

25 20 4

TebufenpyradTebufenpyrad [M+H]+ 334→145334→117

20 25 4

►►Compounds with two transitionsCompounds with two transitions

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BuprofezinBuprofezin [M+H]+ 306→201306→116306→106

35 30 5.5

BupirimateBupirimate [M+H]+

[M+Na}+

317→237317→166317→107339→231339→188

30

30

25

20

9.5

►►Compounds with several transitionsCompounds with several transitions

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Mass spectra of cyproconazole in the QIT

100 125 150 175 200 225 250 275 300 325 m/z0.0

0.5

1.0

1.5

2.0

125 150 175 200 225 250 275 300 325 m/z0.0

6x10Intens.

291.8

313.8

[M+H]+

[M+Na]+

100100 125125 150150 175175 200200 225225 250250 275275 300300 325325 m/zm/z00

11

22

33

44x10x10Intens.Intens.

124.8124.8 MS2 (291.8MS2 (291.8))

138.8138.8

164.7164.7218.8218.8

273.2273.2

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Mass spectra of cyproconazole in the QqTOF

m/z, amu

5000,001,00e41,50e42,00e42,50e43,00e43,50e44,00e44,50e45,00e45,50e46,00e46,50e47,00e47,50e48,00e48,50e49,00e49,50e41,00e5

Intensity, counts,

100 150 200 250 300 350 400 450 500 550 600

292.1142

314.7623

[M+H] +

[M+ Na ]+

m/z, amu40 60 80 100 120 140 160 180 200 220 240 260 280 300

2000,04000,06000,08000,01,0e41,2e41,4e41,6e41,8e42,0e42,2e42,4e42,6e42,8e43,0e43,2e43,4e43,6e4

Intensity,

32,3245

69,0704C5H9

60 80 100 120 140 160 180 200 220 240 260 280 300

2000,04000,06000,08000,01,0e41,2e41,4e41,6e41,8e42,0e42,2e42,4e42,6e42,8e43,0e43,2e43,4e4

Intensity, counts,

32,3245

125,0090C7H6Cl125,0090C7H6Cl

C HMS2 (292)

Cl

+

Cl C

OH

CH2

N

CH

N

N

CH3Cl C

OH

CH2

N

CH

N

N

CH3

CH+ CH3

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Recovery and precision of the selected pesticides from oranges

020406080

100120140

Acrin

athrin

Bupir

imate

Bupro

fezin

cyhalot

hrin

Cyproco

nazole

Fluval

inate

Hexaflu

muron

Kresosi

m me

thyl

Propanil

Pyrif

enox

Pyrip

roxyfen

Tebufenpyrad

Reco

very

(%

)

QqQQITQqTOF


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Matrix effects at the LOQ level

0

20

40

60

80

100

120

Acrin

athrin

Bupir

imate

Buprofezin

cyhalot

hrin

Cyproconazole

Fluvalinate

Hexaflu

muron

Kresosim methyl

Propanil

Pyrif

enox

Pyrip

roxyfen

Tebufenpyrad

Reco

very

(%)

QqQQIT

QqTOF


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Interval of concentrations that gives a linear response (r > 0.99)

COMPOUNDCOMPOUND QqQ QIT QqTOFAcrinathrinAcrinathrin 0.010.01--1010 0.010.01--11 0.020.02--11BupirimateBupirimate 0.00010.0001--0.10.1 0.0050.005--22 0.010.01--11BuprofezinBuprofezin 0.00010.0001--0.10.1 0.010.01--77 0.050.05--55λλ--cyhalothrincyhalothrin 0.010.01--1010 0.40.4--4040 0.30.3--2020CyproconazoleCyproconazole 0.0020.002--22 0.010.01--33 0.050.05--55FluvalinateFluvalinate 0.0010.001--11 0.20.2--2020 0.40.4--4040HexaflumuronHexaflumuron 0.010.01--1010 0.020.02--22 0.040.04--11Kresosim methylKresosim methyl 0.00050.0005--0.50.5 0.010.01--66 0.050.05--55PropanilPropanil 0.0010.001--11 0.020.02--55 0.070.07--77PyrifenoxPyrifenox 0.00050.0005--0.50.5 0.010.01--33 0.030.03--33PyriproxyfenPyriproxyfen 0.0020.002--22 0.020.02--77 0.050.05--55TebufenpyradTebufenpyrad 0.00050.0005--0.50.5 0.010.01--33 0.050.05--55

Mor

e th

an th

ree

orde

r of m

agni

tude

Mor

e th

an th

ree

orde

r of m

agni

tude

Mor

e th

an tw

o or

der o

f mag

nitu

de

Mor

e th

an tw

o or

der o

f mag

nitu

de

Betw

een

one

and

two

orde

r of m

agni

tude

Betw

een

one

and

two

orde

r of m

agni

tude

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Scheme to perform the identification of non-target pesticides

SCREENING STEP IN THE FULL MASS SPECTRA SCREENING STEP IN THE FULL MASS SPECTRA LOOKING FORLOOKING FOR THE POSSIBLE THE POSSIBLE PROTONATED PROTONATED MOLECULEMOLECULE OF ANY OF ANY SUSPECTED PESTICIDES SUSPECTED PESTICIDES AND METABOLITESAND METABOLITES

ESTABLISHMENT OF THE IDENTITYESTABLISHMENT OF THE IDENTITY BY THE BY THE STRUCTURAL INFORMATION OBTAINED IN STRUCTURAL INFORMATION OBTAINED IN THE THE PRODUCT ION SPECTRUMPRODUCT ION SPECTRUM

CONFIRMATION AND QUANTIFICATIONCONFIRMATION AND QUANTIFICATIONWITH ANALYTICAL WITH ANALYTICAL STANDARDSTANDARD

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time, min

0.01000.0

5000.0

1.0e4

1.5e4

2.0e4

Inte

nsity

, cps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time, min

0.0

1000.0

5000.0

Inte

nsity

, cps

2000.0

3000.0

4000.0

m/z 317

m/z 209

Identification of non targeted pesticides

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Accurate mass product ion spectrum of bupirimate


40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z, amu

1,0e42,0e43,0e44,0e45,0e46,0e47,0e4

8,0e49,0e41,0e51,1e51,2e51,3e51,4e51,5e51,6e51,7e5

Intensity, counts

237.5294

54,6 98,1 120,980,3 151,641,2

194,2

272,1147209,1527

166.1

178,9

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Identification using QqTOF


40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z, amu

2000,0

4000,0

6000,0

8000,0

1,0e4

1,2e4

1,4e4

1,6e4

1,8e4

2,0e4

2,2e4

2,4e4

2,6e4

2,8e4

3,0e4

3,2e4

3,4e4

3,6e4

3,8e4

4,0e4

4,2e4

4,4e4

Intensity, counts

194.1215

179,1059

164,2197

164,2197

150.0213

136.0092

109.2712

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z, amu

2000,0

4000,0

6000,0

8000,0

1,0e4

1,2e4

1,4e4

1,6e4

1,8e4

2,0e4

2,2e4

2,4e4

2,6e4

2,8e4

3,0e4

3,2e4

3,4e4

3,6e4

3,8e4

4,0e4

4,2e4

4,4e4

Intensity, counts

194.1215

179,1059

164,2197

164,2197

150.0213

136.0092

109.2712

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0 2 4 6 8 10 12 14 16 18 Time [min]0

2

4

6

84x10Intens.

0 2 4 6 8 10 12 14 16 18 Time [min]0

0.2

0.4

0.6

0.84x10Intens.

m/z 210

m/z 317


Logo grupoIdentification of non targeted pesticides

317.1

339.1

150 200 250 300 350 400 450 m/z0

1

2

3

5x10Intens.

[M+H]+

[M+Na]+

N

N

O

NH3C

CH2

CH2

CH2H3C

SO

NO CH3

CH3

H

CH2

CH3

Full mass spectrum (MS) of peak at 9.95 minProduct-ion mass spectrum (MS2 317→)

149.9

165.9

179.9191.9

209.9

237.0

253.0

272.0

140 160 180 200 220 240 260 280 300 m/z0

2

4

6

84x10

Intens.

[M+H-SO2N(CH3)2]+

Product-ion mass spectrum (MS3 317→237)

136.9

150.9

165.9 179.9

194.9

209.9

100 120 140 160 180 200 220 240m/z0.0

0.2

0.4

0.6

0.8

1.0

4x10Intens.

100 120 140 160 180 200 220 240m/z0.0

0.2

0.4

0.6

0.8

1.0

4x10Intens.


Full mass spectrum of peak at 6.07 min

141.9

149.9165.9

179.9

191.9

209.9

140 160 180 200 220 240 260 280 300 m/z0

2

4

6

85x10Intens.

232.0

[M+H]+

[M+Na]+

N

N

CH2

CH2

H3C

OH

HN

CH2

CH3H3C

Product-ion mass spectrum (MS2 210)

136.9

150.9

165.9 179.9

194.9

100 120 140 160 180 200 220 240m/z0.0

0.2

0.4

0.6

0.8

1.0

4x10Intens.

100 120 140 160 180 200 220 240m/z0.0

0.2

0.4

0.6

0.8

1.0

4x10Intens.


Product-ion mass spectrum (MS3 317→237)

136.9

150.9

165.9 179.9

194.9

209.9

100 120 140 160 180 200 220 240m/z0.0

0.2

0.4

0.6

0.8

1.0

4x10Intens.

100 120 140 160 180 200 220 240m/z0.0

0.2

0.4

0.6

0.8

1.0

4x10Intens.


Product-ion mass spectrum (MS2 210)

136.9

150.9

165.9 179.9

194.9

100 120 140 160 180 200 220 240m/z0.0

0.2

0.4

0.6

0.8

1.0

4x10Intens.

100 120 140 160 180 200 220 240m/z0.0

0.2

0.4

0.6

0.8

1.0

4x10Intens.

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Application to calculate EDIApplication to calculate EDI

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TEN SAMPLES OF EACH

COMMODITY

Sampling from agricultural cooperativesSampling from agricultural cooperatives

STRAWBERRY

STRAWBERRY

ORANGEORANGE

PEARPEAR

PEACHPEACH

CHERRYCHERRY

APRICOTAPRICOT

Application to calculate EDI

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43%

45%12%

Without residues 1 pesticide 2 pesticides

Pesticides found Pesticides found

Application to calculate EDI

Bupirimate

Λ-cyhalothrin

Fluvalinate

Hexaflumuron

Pyrifenox

Pyriproxyfen

Tebufenpyrad

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Samples with pesticide residuesSamples with pesticide residues

Nº of samples that contain residues Orange Strawberry Cherry Peach Apricot Pear

Bupirimate 0 6 5 3 1 5 Λ-cyhalothrin 0 2 0 0 0 0Fluvalinate 0 0 0 0 2 0

Hexaflumuron 3 0 0 2 0 0Pyrifenox 0 1 0 0 0 0Pyriproxyfen 4 0 0 0 0 0Tebufenpyrad 4 0 0 3 0 0


Orange Strawberry Cherry Peach Apricot PearDaily intake (g/person/day)

58.69 3.83 4.29 13.77 2.31 22.65

Bupirimate 0 115* 14* 19* 16* 97

Λ-cyhalothrin 0 55 0 0 0 0Fluvalinate 0 0 0 0 84 0Hexaflumuron 52 0 0 46 0 0

Pyrifenox 0 14 0 0 0 0

Pyriproxyfen 44 0 0 0 0 0Tebufenpyrad 130 0 0 23 0 0

Average level of pesticides found in the Average level of pesticides found in the different commodities (different commodities (μμg/kg)g/kg)

97194 78 39 151

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where Σc is the sum of the concentrations of each pesticide (µg kg-1 of food); C is the mean food daily intake per person (kg /person/day; N is the number of food samples; and K is the mean body weight considered as 60 kg

ESTIMATED DAILY INTAKE (EDI)ESTIMATED DAILY INTAKE (EDI)

EDI =∑c x C

N x K


EDI (ng/kg/day)Orange Strawberry Cherry Peach Apricot Pear

Bupirimate 0 7* 1 * 4* 0.6* 37*

Λ-cyhalothrin 0 3 0 0 0 0Fluvalinate 0 0 0 0 3 0Hexaflumuron 50 0 0 11 0 0

Pyrifenox 0 0.9 0 0 0 0

Pyriproxyfen 40 0 0 0 0 0Tebufenpyrad 100 0 0 5 0 0

EDIs and ADIsEDIs and ADIs

713 18 1.5 57

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Comparison of EDIs and ADIsComparison of EDIs and ADIs

EDI (ng/kg/day)

ADI(ng/kg/day)

Bupirimate 49.6 300000

Λ-cyhalothrin 3 5000Fluvalinate 3 5000Hexaflumuron 61 20000

Pyrifenox 0.9 100000

Pyriproxyfen 40 3000Tebufenpyrad 105 2000

97.5

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ConclusionsConclusionsThe The target analysistarget analysis is is more reliablemore reliable, , rapidrapid and and feasiblefeasible to to check that MRLs are not exceeded.check that MRLs are not exceeded.

However, the However, the identification of unknown compoundsidentification of unknown compounds and and metabolitesmetabolites can not be abandon because can not be abandon because EDIs can be EDIs can be much highermuch higher than that calculated from the target analysis than that calculated from the target analysis of unaltered.of unaltered.

QqQ QqQ provides provides better LODsbetter LODs and compliance with the criteria and compliance with the criteria to to confirm the identityconfirm the identity of the pesticides. QIT and QqTOF of the pesticides. QIT and QqTOF can determine most of these compounds also at levels can determine most of these compounds also at levels below the MRLs. below the MRLs.

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NonNon--target analysis: identification of products coming target analysis: identification of products coming from the postfrom the post--harvest treatment of apples and pearsharvest treatment of apples and pears

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ExtractionExtractionExtract 15 min in an ultrasonic bath

Filter throughNa2SO4

Evaporate to dryness at 40 ºC and 240 mbar

Redissolve in 5 mL of methanol

1 piece of apple or pear

Cover with ethyl acetate

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QqTOF-MS

ESI +

Capillary (V) 3000

Sample cone (V) 30

Desolvation Tª (ºC) 350

Source Tª (ºC) 125

TOF Resolution (FHWM) 5000

Lock mass (m/z) 380.2185

ESI ‐

Capillary (V) 2800

Sample cone (V) 30

Desolvation Tº (ºC) 350

Source Tª (ºC) 125

TOF Resolution (FHWM) 5000

Lock Mass (m/z) 378.2029

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Imazalil

Ethoxyquin

DPA

Thiabendazole

PostPost--harvest treatment of apples and pearsharvest treatment of apples and pears

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N

H

N

H

HO

N

H

H3CO

N

O

Methoxydiphenylamine

Diphenylamine

Hydroxydiphenylamine

N-phenyl-4-quinoneimine

Diphenyamine and ThiabendazoleDiphenyamine and Thiabendazole

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HN

CH3

CH3

CH3

CH3CH2O

N

CH3

CH3

CH3

H3CH2CO

Ethoxyquin-N-oxyl

Ethoxyquin

N

CH3

CH3

CH3

CH3CH2O

O*

N

CH3

CH3

CH3

CH3CH2O

CH3

N

CH3

CH3

CH3

O

N

CH3

CH3

CH3CH2O

NH

CH3

CH3

H

CH3CH2O

NH

CH3

CH3

CH3

CH3CH2O

Methylethoxyquin

2,2,4-Trimethyl-6-quinolone

Demethylethoxyquin

Demethyldehydroethoxyquin

Ethoxyquin and imazalilEthoxyquin and imazalil

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HN

CH3

CH3

CH3

CH3CH2O

N

CH3

CH3

CH3

H3CH2CO

CH3

CH3

CH3CH2O

N

CH3

CH3

CH3

H3CH2CON

CH3

CH3

CH3

O

NCH3H3C

HN

CH3

CH3

CH3

HO

N

CH3

CH3

CH3

HO

N

CH3

CH3

CH3

O

N

CH3

CH3

CH3

HO

MS/MS of the ethoxyquin dimerMS/MS of the ethoxyquin dimer

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LCLC--MS/MS only monitor targeted MS/MS only monitor targeted pesticidespesticides

Disadvantages of MS/MSn

Fails to identify non-targeted pesticides and unknown metabolites

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Features of the different mass spectrometers

Sensitivity in full scan

Selectivity Accuracy Dynamic range Unique features

QqQ Medium High Low. Unit resolution

High Neutral loss

IT MS/MS High High Low. Unit resolution

Medium MSn

TOF/MS High Medium High Low Accurate mass and

sensitivityQ-TOF/MS

Single quadrupole

Medium

Low

High

Medium

High

Low. Unit. resolution

Medium

High

Accurate mass and

selectivity

---

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Conclusions

LC-MS will become an even more important factor in pesticide residue analysis in food than it already is.

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Best choice:

-nature of applications (most important)-types of mass spectrometers already available-infrastructure available-equipment cost (investment)-equipment complexity (maintainance)-skill level of the operatorsThere

is not o

nly one

There is

not only

one

correct a

nswer!

correct a

nswer!

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Thanks for your attention!

pesticides by lc-ms - csic by lc-ms.pdf · logo grupo pesticides determined by lc benzimidazole...

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