Pesticides by LC-MS - CSIC by LC-MS.pdf · Logo grupo PESTICIDES DETERMINED BY LC Benzimidazole...
Transcript of 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
NAR1ASE 12: MRM of 3 Channels ES+ TIC
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
100NAR1ASE 7: MRM of 2 Channels ES+
TIC7.97e3
13.23
NAR1ASE 8: MRM of 2 Channels ES+ TIC
3.07e314.01
13.7413.41
14.41
NAR1ASE 9: MRM of 3 Channels ES+ TIC
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
100NAR1ASE 4: MRM of 2 Channels ES+
TIC5.57e3
9.29
8.61
6.549.65
10.44
NAR1ASE 5: MRM of 5 Channels ES+ TIC
1.71e49.49
NAR2ASE 6: MRM of 2 Channels ES+ TIC
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
100NAR1ASE 1: MRM of 2 Channels ES+
TIC1.10e3
7.65
6.675.11 6.105.477.13
8.38 8.48 8.89
NAR1ASE 2: MRM of 2 Channels ES+ TIC
5.10e38.54
7.975.22 6.83
6.21 7.45
9.239.67 9.98
10.6010.83
NAR1ASE 3: MRM of 3 Channels ES+ TIC
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
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
NAR1ASE 12: MRM of 3 Channels ES+ TIC
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
100NAR1ASE 7: MRM of 2 Channels ES+
TIC7.97e3
13.23
NAR1ASE 8: MRM of 2 Channels ES+ TIC
3.07e314.01
13.7413.41
14.41
NAR1ASE 9: MRM of 3 Channels ES+ TIC
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
100NAR1ASE 4: MRM of 2 Channels ES+
TIC5.57e3
9.29
8.61
6.549.65
10.44
NAR1ASE 5: MRM of 5 Channels ES+ TIC
1.71e49.49
NAR2ASE 6: MRM of 2 Channels ES+ TIC
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
100NAR1ASE 1: MRM of 2 Channels ES+
TIC1.10e3
7.65
6.675.11 6.105.477.13
8.38 8.48 8.89
NAR1ASE 2: MRM of 2 Channels ES+ TIC
5.10e38.54
7.975.22 6.83
6.21 7.45
9.239.67 9.98
10.6010.83
NAR1ASE 3: MRM of 3 Channels ES+ TIC
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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COMPOUNDCOMPOUND Precursor ion
Transitions CV CE IPs reached
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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COMPOUNDCOMPOUND Precursor ion
Transitions CV CE IPs reached
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
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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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
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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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
Identification of non targeted pesticides
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
Identification of non targeted pesticides
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
Identification of non targeted pesticides
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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.
Logo grupoIdentification of non targeted pesticides
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.
Logo grupoIdentification of non targeted pesticides
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.
Logo grupoIdentification of non targeted pesticides
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
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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
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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!