Comparison of UPLC- and HPLC-MS/MS for the Analysis of ... · PDF fileComparison of UPLC- and...
Transcript of Comparison of UPLC- and HPLC-MS/MS for the Analysis of ... · PDF fileComparison of UPLC- and...
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Comparison of UPLC- and HPLC-MS/MS for the Analysis of Multiple Pesticide Residues
in Food Produce:“ Extension of Scope vs. Shorter Run Times”
Food and Consumer Product Safety Authority
Chemistry Laboratory, R&D - Pesticide Analysis Group ,
National Reference Laboratory (NRL) – Pesticides/Myc otoxines
Amsterdam, The Netherlands
André de Kok,André de Kok, Peter Rensen and Maurice Hiemstra Peter Rensen and Maurice Hiemstra
- VWA
e-mail: [email protected] on Pesticide Residues, Stuttgart, Germany, 6-7 Dec. 2006
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VWA - Food and Consumer Product Safety Authority
Amsterdam
Groningen
ZutphenWageningen
Eindhoven
Zwijndrecht /Rotterdam
VWA - Amsterdam- Primary Agricultural Products
- NRL
• Wageningen
Airport
Harbor
National Reference Laboratory
for:
Pesticides - Plant products- Cereals and Feed
- SRMs
+
Mycotoxines
+
GMO’s
Northwest
Pesticides - Animal products
VWA – East+ RIKILT
- Animal Products- NRL
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OUTLINE
� Status of Multiresidue Methods in Pesticide Analysis
� LC-MS/MS Applications
� Fruits and Vegetables
� Soybean samples
� Expansion of Scope: Quattro Ultima���� Premier XE
� Faster Run Times: HPLC-MS/MS ���� UPLC-MS/MS
� Conclusion
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Official Food Control Lab
Pesticides Monitoring/Enforcement Requirements:
� Accreditation of laboratory (ISO 17025) – EU Food and Feed Law
� Validated and Accredited methods
(Collaborative Studies ���� In-house Validation)
� Quality Assurance / Quality Control (QA/QC)�(Calibration) Standards stability check
�Calibration curve check
�Recovery check
�Detector Response check (RL, drift)
� Proficiency Tests (EU-PTs)
VWA - Food and Consumer Product Safety Authority
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VWA - Food and Consumer Product Safety Authority
Official Food Control Lab Pesticides Monitoring/Enforcement Requirements:
• Up to 1000 pesticides to measure – wide scope ����priorities
• Low MRL’s (Baby food Directive, Organically-grown
products, EU-forbidden pesticides)
����Low Reporting Limits (0.01 mg/kg)
• High Sample Throughput (2 weeks ���� 24-72 hr throughput time)
���� Low-cost, efficient, robust methods
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SPEED
ROBUSTNESS
SENSITIVITY
SELECTIVITY
Extra
ction Cleanup
Detection
}
}
Selection Criteria
Multi Residue MethodsSchemes and Strategies
����COST-EFFICIENCY
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Multi Residue MethodsSchemes and Strategies
����COST-EFFICIENCY
2006
3500 Samples / Year
• Fruits and Vegetables
• Cereals
• Processed foods
• Juices and wines
• Baby foods
2007 (Planning):
4500 Samples / Year
~ 7 FTE’s
~ € 200.- / sampleGC-MS + LC-MS/MS + GC-ECD
30% GC-MS-Dithiocarbamates
(15% 2nd analyses ~ > MRL)
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GCGC--ECDECD
Quantitation + Identification /Confirmation
GCGC--MS (MS (ITD, fullITD, full--scanscan) )
Scheme in Routine
GCGC--MS (SIM)MS (SIM)GCGC--MS/MSMS/MS
LCLC--MS/MSMS/MS
N =172N > 400N =10
N =52
Status MRM’s in Pesticide Analysis
2006
(Captan, etc.)
(OC,PYR, etc.)
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DETECTION
SEPARATION
MRMsMRMs
GC-MS (ITD)
SAMPLING
HOMOGENIZATION
EXTRACTION
NO CLEANUP
GC LC
ECD
GC-MS (/MS)LC-MS/MS
VWA-Amsterdam MRM Scheme
DATA HANDLING
GPC
Fatty foods:
Non-Fatty foods:
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Fast Acetone Extraction / CH2Cl2 + PE Partitioning
(with or without Na2SO4)15 (or 7.5 g) sample in 250-ml centrifuge tube+ 30 ml acetone
- mix 20 sec. (Polytron homogeniser)
+ 30 ml CH2Cl2 + 30 ml PE (+ 7.5 g Na2SO4) (Partitioning)- mix 20 sec. (Polytron homogeniser)
- centrifuge 4 min. at 3500 rpm
- decant supernatant in 100-ml erlenmeyer flask
- take aliquots for multiresidue methods, no cleanup !
e.g. : LC-MS/MS method: take 1- (or 2-)ml aliquot
- evaporate (batchwise!) and redissolve in 1 ml MeOH
EXTRACTION METHOD
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DETECTION
SEPARATION
DATA HANDLING
LC-MS/MS Chromatographic Run Time
1 sample 1 series
(30 samples)
32 min. (ESI+) 24 hours
10 min. (ESI-) 7.5 hours
Status MRM’s in Pesticide Analysis
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0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00Time0
100
%
���� Ret. Time (min)
MRMs: 26 xMRMs: 26 xMRMs: 30 xMRMs: 26 xMRMs: 31 xMRMs: 30 xMRMs: 13 x
25 %
95 %M
obile
pha
se B
% m
etha
nol
( in
5 m
M f
orm
ic a
cid
)
0 15 27.5 32.5
____182 x
TIC
156 Pesticides Mix
Scheme of Chromatographic Run Time –HPLC gradient and MS/MS scan functions (2004)
7
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MS/MS Scan parameters
Dwell time: 20 msec ���� 20
Inter-channel (-MRM) delay time: 20 msec ���� 10
_______
e.g. 32 MRM’s / channels (32x40 or 30) 1280 msec���� 960
Interscan delay time: 20 msec ���� 10
_______
Duty cycle time: 1300 msec ���� 970(2004) ----------- �(2005)
20 (25) data points per HPLC peak
[10 (13) for 2 overlapping scan functions]
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200 ng/m l
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00Tim e0
100
%
MRMs: 28 xMRMs: 24 xMRMs: 24 xMRMs: 29 xMRMs: 26 xMRMs: 26 xMRMs: 29 x
25 %
95 %m
obile
pha
se%
met
hano
l in
(5 m
M N
H4
form
iate
)
0 15 31.0209 x
163 Pesticides Mix
TIC
MRMs: 23 x
���� Ret. Time (min)
Scheme of Chromatographic Run Time –HPLC gradient and MS/MS scan functions (2005)
____
8
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(HP)LC-MS/MS
HPLC-MS/MS: WATERS Quattro Ultima Triple Quad
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Analytical column: Alltima C18, 5 µm, 150 x 3.2 mm
Column temp.: 30 °C
Mobile phase flow: 0.30 mL.min-1
Injection volume: 5 µL (in 100% MeOH)
Solvent A: 5 mM HCOONH4 in water
Solvent B: Methanol
Gradient profile: 0 min 75% A 25% B
15 min 5% A 95% B
25 min 5% A 95% B
25.1 min 75% A 25% B
Acquisition time: 31.0 min
Total run time: 32.5 min (incl. Equilibration)
HPLC conditions
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OUTLINE
� Status of Multiresidue Methods in Pesticide Analysis
� LC-MS/MS Applications
� Fruits and Vegetables
� Soy bean samples
� Expansion of Scope: Quattro Ultima ���� Premier XE
� Faster Run Times: HPLC-MS/MS ���� UPLC-MS/MS
� Conclusion
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pepers
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00Time2
100
%
11
100
%
4
100
%
7
100
%
18
100
%
7
100
%
3
100
%
47649315 7: MRM of 16 Channels ES+ TIC
8.98e520.87
20.3221.41 26.8926.33
23.4923.1423.98 27.39
47649315 6: MRM of 30 Channels ES+ TIC
1.09e619.84
19.64
18.7618.32
20.25
20.64
47649315 5: MRM of 30 Channels ES+ TIC
4.32e519.82
17.6817.23
17.02
18.78
47649315 4: MRM of 26 Channels ES+ TIC
1.05e615.06
16.90
16.8017.5517.96
47649315 3: MRM of 30 Channels ES+ TIC
3.00e615.19
14.6013.8013.12
47649315 2: MRM of 25 Channels ES+ TIC
3.77e58.56
8.15 12.438.9710.729.59 12.10 12.72
47649315 1: MRM of 26 Channels ES+ TIC
2.72e64.06
2.051.78 2.82
8.244.15
7.895.31
Pesticide Residues (conc. in mg/kg)
in Red hot chili pepper Sample TICs of 7 Scan Functions
methamidophos 0.26azoxystrobin 0.01carbaryl 0.21carbendazim <0.01carbofuran 0.01methomyl 0.01monocrotophos 0.01profenofos 0.07propiconazole 0.02
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pepers
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50Time3
100
%
0
100
%
0
100
%
3
100
%
3
100
%
4
100
%
1
100
%
47649315 Sm (Mn, 2x3) 1: MRM of 26 Channels ES+ 192 > 160
1.06e68.24
3.933.73
47649315 Sm (Mn, 2x3) 1: MRM of 26 Channels ES+ 192 > 132
2.80e58.24
2.07
47649315 Sm (Mn, 2x3) 1: MRM of 26 Channels ES+ 163 > 106
5.10e47.91
47649315 Sm (Mn, 2x3) 1: MRM of 26 Channels ES+ 163 > 88
5.30e47.91
47649315 Sm (Mn, 2x3) 1: MRM of 26 Channels ES+ 142 > 112
1.21e64.06
47649315 Sm (Mn, 2x3) 1: MRM of 26 Channels ES+ 142 > 94
1.04e64.06
47649315 Sm (Mn, 2x3) 1: MRM of 26 Channels ES+ TIC
2.37e64.06
2.071.79 2.82
8.24
7.955.10
methamidophos
methomyl
carbendazim
Carbendazim< 0.01 mg/kg
Methamidophos0.26 mg/kg
Red hot chili pepper Sample
TIC
Methomyl0.01 mg/kg
MRM’s
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•••• Validation of methods
Comparison of 3 extraction methods for 2 crops, 3 levels (n=6):
Acetone / CH2Cl2 + PE
Acetone / CH2Cl2 + PE + Na2SO4
Acetonitrile (QuEChERS)
[Ref.: S. Lehotay, A. de Kok, M. Hiemstra and P. van Bodegraven,
J. AOAC Int., 88 (2005) 595-614.]
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recovery RSD
114% 4,8%
99% 6,1%
100% 4,9%
106% 3,8%
100% 1,6%
70% 8,4%
91% 5,4%
108% 7,5%
101% 2,6%
103% 3,0%
99% 8,4%
85% 10,8%
98% 2,3%
99% 3,5%
107% 1,8%
92% 3,8%
101% 12,0%
10 ppb levelrecovery RSD
37% 9.7%
105% 4.7%
109% 3.7%
72% 3.0%
28% 4.2%
67% 7.0%
100% 3.3%
101% 3.0%
101% 2.4%
104% 3.4%
98% 14.9%
92% 6.6%
91% 2.8%
104% 1.5%
88% 1.7%
23% 7.9%
102% 7.5%
10 ppb level
recovery RSD
62% 11,4%
95% 8,0%
99% 6,0%
68% 9,0%
101% 2,9%
50% 8,5%
94% 1,2%
88% 9,3%
61% 2,4%
105% 3,2%
92% 15,2%
37% 5,9%
96% 2,4%
93% 1,6%
87% 3,4%
90% 10,5%
87% 11,1%
10 ppb level
acephate
acetamiprid
aldicarb
aldicarb sulfone
aldicarb sulfoxide
asulam
azaconazole
azamethiphos
azinphos-methyl
azoxystrobin
bitertanol
bromuconazole
buprofezin
butocarboxim
butocarboxim sulfone
butocarboxim sulfoxide
carbaryl
Recoveries for Orange spiked at 10 ppb-level: 3 Extraction methodsAcetone extraction Acetone + Na2SO4 QuEChERS
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orange lettuce cabbage apple
-1% -1% -2% -2%
-1% 5% 0% 4%
-2% 0% -4% 3%
-1% 0% -1% 2%
-2% -2% -5% -4%
-2% -1% -7% -3%
0% 6% -2% 0%
-2% 0% -5% -5%
-25% -51% -7% -4%
-1% 8% 1% -2%
-7% 0% -2% -9%
-40% 2% 0% 0%
-7% -2% -6% -4%
0% 3% -5% 5%
0% 1% -2% -2%
-1% -1% -4% -2%
-8% 1% -4% -4%
matrix effect
Pesticide
acephate
acetamiprid
aldicarb
aldicarb sulfone
aldicarb sulfoxide
asulam
azaconazole
azamethiphos
azinphos-methyl
azoxystrobin
bitertanol
bromuconazole
buprofezin
butocarboxim
butocarboxim sulfone
butocarboxim sulfoxide
carbaryl
Matrix effects, Fruits & Vegetables, 3 Extraction methodsAcetone extraction Acetone + Na2SO4 QuEChERS
orange lettuce5% 2%
5% 1%
2% 0%
-25% 2%
2% 0%
-1% 3%
-2% 3%
-9% 1%
-29% -4%
-5% 2%
-1% -4%
-59% 1%
-4% -2%
1% 0%
-14% 0%
2% 1%
-10% -4%
matrix effectorange lettuce
0% -5%
6% 2%
-3% -1%
-3% 3%
-1% -2%
-3% -1%
0% 4%
-5% 1%
-30% -47%
2% 17%
-12% -4%
-55% 0%
-7% -4%
-8% -1%
-4% -1%
-2% -1%
-10% -1%
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Total ion chromatograms (LC-MS/MS, ESI +) of Soybean Sample , spiked with 155 pesticides at 10, 50 and 100 ppb level
Time5.00 10.00 15.00 20.00 25.00
%
0
100
Time 5.00 10.00 15.00 20.00 25.00
%
0
100
Time5.00 10.00 15.00 20.00 25.00
%
0
100
Time5.00 10.00 15.00 20.00 25.00
%
0
100
Soybean(acetone extract)blank
Soybean(acetone extract)50 ppb
Soybean(acetone extract)10 ppb
Soybean(acetone extract)100 ppb
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Total ion chromatogram (LC-MS/MS, ESI -) of Soybean Sample (acetone extract) , spiked with 14 pesticides at 100 ppb level
Time2.00 4.00 6.00 8.00
%
0
100
���� Total Run time: 10 min.
Routine LC-MS/MS Method on Quattro Ultima
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OUTLINE
� Status of Multiresidue Methods in Pesticide Analysis
� LC-MS/MS Applications
� Fruits and Vegetables
� Soy bean samples
� Expansion of Scope: Quattro Ultima���� Premier XE
� Faster Run Times: HPLC-MS/MS ���� UPLC-MS/MS
� Conclusion
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Limitations:
Acquisition Time: ~ 32 min.
Maximum of 32 “channels”(MRMs) / Scan function (time-window)
����Total number of MS/MS transitions limit: ~ 220
Optional Improvements:
1. Quattro Ultima���� Premier XE:
Dwell time: 20 msec ���� 10 (���� 5 ?)
Inter-channel delay time: 10 msec ���� 5 ?
-------------------------------
Sum: 30 ���� 20 (or 15)?
Number of MS/MS transitions: 220���� ~ 330 (or 440)
3 or 4 overlapping scan functions possible
Expansion of Scope: Quattro Ultima���� Premier XE
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172 Pesticides Mix
Scheme of Chromatographic Run Time –HPLC gradient and MS/MS scan functions (2006)
9
Quattro Ultima ���� Premier XE
15
Number of transitions (1st) : 172 172
Number of transitions (2nd) : 72 172
Total : 244 344
>220 Pesticides
15
Number of transitions (1st) : 220
Number of transitions (2nd) : 220
Total : 440
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Routine HPLC Method on Premier XE
Column: Alltima C 18; 150mm x 3.2mm; 5µm
Flow rate: 0.3 ml/min
Temperature: 30°C
Dwell time: 20ms
Interchannel delay: 10ms
Nr. of Transitions: 242
Nr. of Pesticides: 172
Pesticide RSDr (%)
methamidophos 1.5
azinphos-methyl 6.7
azoxystrobin 2.7
carbendazim 4.9
cyproconazole 3.0
dimethoate 3.2
omethoate 1.8
dimethomorph 3.6
fenarimol 11.7
fenhexamid 5.2
fenthion sulfoxide 4.7
imazalil 3.5
imidacloprid 11.0
indoxacarb 11.1
linuron 4.1
methomyl 3.9
methoxyfenozide 4.7
monocrotophos 5.6
oxamyl 3.5
prochloraz 3.6
spinosad A/D *
thiabendazole 3.4
tolylfluanid 4.3
triforine 6.5
hexythiazox 5.3
Repeatability
Level 10 ng/ml
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HPLC ���� UPLC
UPLC-MS/MS:
WATERS Acquity UPLC - Quattro Premier Triple Quad
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New Stationary Phase for UPLC
HPLC ���� UPLC
1.7 µm particle size
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Van Deemter Equation
U = Linear Velocity dp = Particle Size
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Objectives
� 1 Extention of the scope of the routine
LC-MS/MS multiresidue method
� 2 At least two transitions per pesticide
(quantitation/confirmation)
� 3 Reduction of analytical run time by a factor of two
HPLC ���� UPLCQuattro Ultima���� Premier XE
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Optimisation of MS/MS parameters for 235 pesticides- 3 MS/MS transitions / pesticide - Cone-voltage and Collision energy - QuanOptimize feature of Masslynx software - Individual pesticides: new autotune wizard
Cone voltage Collision Energy
Optimisation
Cone Voltage / V
%
0
100
5.0 13.0 21.0 29.0 37.0 45.0
1.435e+008m/z 351.99
25
Collision Energy / eV
%
0
100
5.0 14.0 23.0 32.0 41.0 50.0
3.557e+007m/z 351.99-> m/z 124.9
17
MRM 352.0 > 124.9
Tuning Quattro Premier XE
example : Chlorpyrifos
Quattro Premier XE
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Mass spectrum
Optimisation Parent ion
Optimisation Daughter ion
325.0 330.0 335.0 340.0 345.0 350.0 355.0 360.0 365.0 370.0 375.0
%
0
1001.600e+007
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
%
0
1004.597e+006
Quattro Premier XETuning
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Influence of Dwell time and Inter-channel delay time on Repeatability (% RSD r)
Quattro Ultima
Pesticide 5 / 10 10 / 10 10 / 5 20 / 10
% RSDr % RSDr % RSDr % RSDr
methamidophos 6.4 3.5 14.6 3.3azinphos-methyl 9.5 6.0 15.6 8.8azoxystrobin 1.6 2.6 5.7 6.3carbendazim 1.7 6.2 14.0 5.5cyproconazole 8.8 7.8 15.8 15.2dimethoate 2.5 3.7 15.2 7.1omethoate 2.3 3.0 16.4 6.4dimethomorph 9.5 4.1 13.1 5.1fenarimol 13.1 8.3 20.6 13.0fenhexamid 14.4 9.6 20.9 9.1fenthion sulfoxide 5.0 2.7 21.9 7.2imazalil 3.8 3.1 15.5 11.7imidacloprid 9.1 6.4 16.3 15.9indoxacarb 21.1 9.5 18.2 10.4linuron 10.3 3.4 10.8 5.1methomyl 3.8 4.3 12.7 9.4methoxyfenozide 4.5 3.3 16.9 11.4monocrotophos 4.8 3.2 18.5 3.6oxamyl 8.9 5.3 28.3 4.4prochloraz 6.0 4.4 8.1 9.5thiabendazole 2.1 8.3 17.6 4.0tolylfluanid 7.2 4.5 28.0 8.6triforine 8.8 13.8 17.3 4.6hexythiazox 7.8 6.8 6.6 15.7
Quattro Premier
dwell-time/interchannel delay (ms)
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Analytical column: Acquity UPLC BEH C18, 1.7 µm, 150 x 2.1 mm
Column temp.: 65 °C
Mobile phase flow: 0.30 mL.min-1
Injection volume: 2 µL (Std. In 100% MeOH)
Solvent A: 5 mM HCOONH4 in water
Solvent B: Methanol
Gradient profile: 0 min 75% A 25% B
15 min 5% A 95% B
25 min 5% A 95% B
25.1 min 75% A 25% B
Acquisition time: 31.0 min
Total run time: 32.5 min
UPLC conditions
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2 transitions / pesticide
Column: Acquity UPLC TM BEH C18, 2.1 x 150mm, 1.7µm
Flow rate: 0.45 ml/min
Temperature: 65°C
Dwell time: 10ms
Interchannel delay: 10ms
Nr. of Transitions: 344
Nr. of Pesticides: 172
UPLC- Method on Premier XE
Pesticide RSDr (%)
methamidophos 5.5
azinphos-methyl 3.8
azoxystrobin 3.8
carbendazim 3.8
cyproconazole 6.4
dimethoate 5.1
omethoate 7.1
dimethomorph 4.9
fenarimol 11.0
fenhexamid 8.7
fenthion sulfoxide 4.7
imazalil 7.2
imidacloprid 4.8
indoxacarb 4.8
linuron 7.9
methomyl 9.8
methoxyfenozide 5.4
monocrotophos 9.3
oxamyl 25.0
prochloraz 5.2
spinosad A/D 2.9
thiabendazole 4.8
tolylfluanid 8.5
triforine 28.4
hexythiazox 8.0
Repeatability
Level 10 ng/ml
Total Run time: 32 min.
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Time-scheduled Multiple Reaction Monitoring
Function Time (min) Transitions Max.
1 1.0 - 4.0 24 32
2 3.0 - 5.5 24 32
3 4.0 - 7.0 24 32
4 6.5 - 10.0 24 32
5 9.5 - 12.0 24 32
6 11.5 - 13.7 24 32
7 13.0 - 15.0 24 32
8 14.5 - 16.0 24 32
9 15.0 - 16.6 24 32
10 15.6 - 17.5 24 32
11 16.5 - 18.0 24 32
12 17.2 - 19.0 24 32
13 17.9 - 20.5 24 32
14 18.5 - 23.0 24 32
15 21.0 - 28.0 8 32
Total Transitions: 344 480Nr. of pesticides: 172 240
Experimental UPLC-Method 28 min
Column: Acquity UPLC BEH C18, 2.1 x 150mm, 1.7µm
Temp. Column: 65°C
Mobile phase: A: 5 mM Ammonium Formiate
B: methanol
Gradient:
Time (min) A % B %
0 80 20
23 5 95
24 80 20
28 80 20
Run time: 32 min
flow rate: 0.45 ml.min-1
injection volume: 2 µl
Detection: Micromass, Quattro Premier XE, ESI positive mode
15 functions, dwell time 10 ms
Capillary voltage: 1 kV
Cone voltage: dependable
Collision energy: 5 - 50 eV
UPLC- Method on Premier XE
Possible Maximum Scope
200-240 with 2 transitions / pesticide
or ~ 400 with 1 transition / pesticide
Recent method:
220 pesticides in 32 min.
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UPLC- Method on Premier XE
Dwell time: 10 ms 10 ms
Interchannel delay: 10 ms 10 ms
Nr. of Transitions: 344 14
Nr. of Pesticides: 172 14
Nr. of Functions: 15 1
Total Run time: 32 min.
ESI+ ESI-
Inter scan delay time : 20 ms
Pos./Neg. IonisationPos./Neg. Ionisation in 1 Run
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Column: Acquity UPLC TM BEH C18, 2.1 x 150mm, 1.7µm
Flow rate: 0.45 ml/min
Temperature: 65°C
Total Run time: 32 min.
ESI+ ESI-Pos./Neg. Ionisation in 1 Run
Imazalil
297.1 > 69.1
Nr of scans: 8
UPLC- Method on Premier XE
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2 transitions / pesticide
Column: Acquity UPLC TM BEH C18, 2.1 x 150mm, 1.7µm
Flow rate: 0.45 ml/min
Temperature: 65°C
Dwell time: 10ms
Interchannel delay: 10ms
Nr. of Transitions: 344
Nr. of Pesticides: 172
UPLC- Method on Premier XE
Shorter Run times:
Total Run time: 16 min. !!!
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20 %
95 %
Mob
ile p
hase
B%
Met
hano
l(
in 5
mM
NH
4fo
rmat
e)
Scheme of Chromatographic Run Time –HPLC gradient (2006)
Total Run time: 16 min. !!!
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Experimental UPLC-Method, 16 min.-Run time
Column: Acquity UPLC BEH C18, 2.1 x 150mm, 1.7µm
Temp. Column: 65°C
Mobile phase: A: 5 mM Ammonium Formiate
B: Methanol
Gradient:
Time (min) A % B %
0 80 20
11 5 95
12 80 20
16 80 20
Run time: 16 min
Flow rate: 0.45 ml.min-1
Injection volume:2 µl
Detection: Waters, Quattro Premier, ESI positive mode
15 functions, dwell time 10 ms
Capillary voltage:1 kV
Cone voltage: dependable
Collision energy: 5 - 50 eV
Time-scheduled Multiple Reaction Monitoring
Function Time (min) Transitions Max.
1 0.5 - 2.0 24 32
2 1.5 - 2.5 24 32
3 2.1 - 3.5 24 32
4 2.5 - 4.5 24 32
5 4.5 - 5.5 24 32
6 5.5 - 6.5 24 32
7 6.0 - 7.4 24 32
8 7.0 - 7.6 24 32
9 7.3 - 8.1 24 32
10 7.6 - 8.5 24 32
11 8.1 - 8.7 24 32
12 8.5 - 9.2 24 32
13 8.8 - 10.0 24 32
14 9.5 - 11.0 24 32
15 10.5 - 14 8 32
Total Transitions: 344 480
Nr. of pesticides: 172 240
UPLC-MS/MS (Premier XE) conditions
Nr of scans: ~ 8 ~ 6Limit !! Too low !!
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Imazalil
297.1 > 69.1
Nr of scans: 10
Imazalil
297 > 255
Nr of scans: 10
Imazalil
297.1 > 69.1
Nr of scans: 10
Imazalil
297.1 > 69.1
Nr of scans: 8
Routine Method on Premier XE
UPLC-Method on Premier XE
with two transitions/Pesticide
UPLC–Method on Premier XE
Routine Method on Quattro Ultima 2006
Run time 16 min.
Number of data points (scans) / HPLC peak
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CONCLUSIONSUPLC-MS/MS
n Expands scope of Multiresidue Methods
n Increases sensitivity
n Decreases run time, maintaining resolution
n Increases sample throughput
n Implementation in routine: 2007
- VWA