Evaluation of simultaneous MS and MS2 workflows of LC/Q … · 2016. 6. 6. · 0% 10% 20% 30% 40%...
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0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% <2 ppm 2-3 ppm 3-5 ppm ddMS2 vDIA (5 segments/35K) AIF Evaluation of simultaneous MS and MS 2 workflows of LC/Q-Orbitrap for analysis of pesticides in fruits and vegetables Łukasz Rajski, María del Mar Gómez Ramos, Víctor Cutillas, Amadeo R. Fernández-Alba European Union Reference Laboratory for Pesticide Residues in Fruit & Vegetables. University of Almeria, Agrifood Campus of International Excellence (ceiA3). Spain. e-mail: [email protected] INTRODUCTION Acknowledgements: The authors acknowledge funding support from the European Commission, DG SANTE (Specific Agreement No. 7 to Framework Partnership Agreement No. SANCO/2005/FOOD SAFETY/0025-Pesticides in fruit and vegetables). EXPERIMENTAL RESULTS During last decade high resolution accurate mass spectrometers have improved qualitative (resolution, mass accuracy) as well as quantitative (sensitivity, linear range) aspects. HARMS instruments are well known for their high selectivity in full MS mode. Nevertheless even spectrometers which have very high resolution may produce false positive results. Isotopic pattern and alternative adducts not always can correctly discard false positives. A solution to reduce number of false positive results is application of simultaneous full MS and MS 2 . The objective of this work was to compare three workflows of simultaneous MS and MS 2 : All Ion Fragmentation (no precursor ion selection, ions from entire mass range fragmented at the same time), variable Data Independent Acquisition (no precursor ion selection, mass range divided into smaller segments before fragmentation) and data dependent MS 2 (selection of precursor ion). Acatonitrile extracts (blanks and spiked with 166 pesticides) of 11 fruits and vegetables were used for the evaluation. Blank extracts were used to evaluate potential false positives (considering retention time window of 0.2 min) whereas spiked extracts (at 0.01 mg/kg and 0.1 mg/kg) to evaluate the false negatives. Samples were analysed with Q Exactive Focus working with resolution of 70,000 (at m/z 200) in full scan MS and 17,500 or 35,000 in MS 2 mode. Chromatography Mobil phase: - A: 98% H 2 O 2% MeOH 5mM HCOONH 4 0.1% HCOOH - B: 98% MeOH 2% H 2 O 5mM HCOONH 4 0.1% HCOOH Flow: 0.35 mL/min Gradient time: 14 min + 3 min reequilibration Column: Accucore™ aQ C18, 100 mm x 2.1 mm x 2.6 µm Column temperature: 30°C Ijnection volume: 10 µL Acquisition mode: full scan MS -resolution 70,000 -AGC target 1e6 -max IT auto -scan range 120-1000 Da Acquisition mode: MS 2 -dd MS 2 resolution 17,500 -vDIA resolution 17,500 or 35,000, 3 or 5 mass segments - AIF resolution 70,000 CONCLUSIONS Matrices 11.25 11.30 11.35 11.40 11.45 11.50 11.55 11.60 Time (min) ddMS 2 cycle time 0.27s AIF cycle time 0.62s vDIA (3segm x 17,500) cycle time 0.61s vDIA (5segm x 17,500) cycle time 0.79 s vDIA (3segm x 35,000) cycle time 0.83 s vDIA (5segm x 35,000) cycle time 1.28 s 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% RSD 0-5% RSD 0-10% RSD 0-20% 3 segments/ R=35000 5 segments/ R=35000 3 segments/ R=17500 5 segments/ R=17500 AIF dd MS2 Tomato 0.01 mg/kg Peak area repeatability In workflows with shorter cycle times more points per chromatographic peak is obtained, by that peak area repeatability is better. Area Linearity 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] 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 dd-MS 2 vDIA (5 semgents/35,000) Independently of the workflow selected the detector response is characterised by very good linear range and no saturation is observed. Percentage of identified pesticides 80% 85% 90% 95% 100% garlic onion spinach green bean lettuce apple orange cucumber parsley leek tomato 0.1 mg/kg 0.01 mg/kg dd-MS 2 80% 85% 90% 95% 100% garlic onion spinach green bean lettuce apple orange cucumber parsley leek tomato 0.1 mg/kg 0.01 mg/kg 0.1 mg/kg without ion ratio 0.01 mg/kg without ion ratio vDIA (5 semgents/35,000) 80% 85% 90% 95% 100% garlic onion spinach green bean lettuce apple orange cucumber parsley leek tomato 0.1 mg/kg 0.01 mg/kg 0.1 mg/kg without ion ratio 0.01 mg/kg without ion ratio AIF 0.01 mg/kg of Propargite in leek Full scan MS 368.1890 ± 5ppm Full scan MS 368.1890 ± 5ppm MS2 (120 – 1000 m/z) 231.1745 ± 5ppm All Ion Fragmentation vDIA MS2 (295 – 405 m/z) 231.1745 ± 5ppm Resolving power 70,000 Resolving power 35,000 Mass errors in full scan MS 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% <2 ppm 2-3 ppm 3-5 ppm Tomato Orange % of compounds 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% <2 ppm 2-3 ppm 3-5 ppm ddMS2 vDIA (5 segments/35K) AIF Tomato % of compounds Orange % of compounds 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Tomato Apple Orange Onion -50%<ME -21%<ME<-50% -20%<ME<20% 21%<ME<50% 50%<ME Selectivity in AIF and vDIA MS2 151.0326 ± 5 ppm 0.01 mg/kg 0.02 mg/kg 0.05 mg/kg Full Scan MS 343.0529 ± 5 ppm Thiophanate methyl in onion (vDIA, 5 segments, 35K). vDIA provides more selectivity than AIF Quantitation in MS 2 (AIF, vDIA) If full scan MS does not have enough selectivity, quantitation is possible in MS 2 Matrix effects Mass errors in MS 2 LC–Q Exactive Focus MS operated in full scan at the resolution of 70,000 detected close to 100% of the selected pesticides in all matrices with mass errors below 2 ppm and mass errors < 5 ppm in MS 2 . From the point of view of peak area repeatability the most robust workflow was dd MS 2 . Matrix effects were lower than 20% in the majority of the cases facilitating quantification. AIF and vDIA offer the possibility to quantify with accurate MS 2 , this can help with "difficult" matrices. Dd MS 2 had the highest identification rate (96-100%, depending on the matrix). In vDIA it was 86-100% and in AIF 81-100%.
Transcript of Evaluation of simultaneous MS and MS2 workflows of LC/Q … · 2016. 6. 6. · 0% 10% 20% 30% 40%...
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<2 ppm 2-3 ppm 3-5 ppm
ddMS2
vDIA (5 segments/35K) AIF
Evaluation of simultaneous MS and MS2 workflows of LC/Q-Orbitrap for analysis of
pesticides in fruits and vegetables Łukasz Rajski, María del Mar Gómez Ramos, Víctor Cutillas, Amadeo R. Fernández-Alba
European Union Reference Laboratory for Pesticide Residues in Fruit & Vegetables. University of Almeria, Agrifood Campus of International Excellence (ceiA3). Spain. e-mail: [email protected]
INTRODUCTION
Acknowledgements: The authors acknowledge funding support from the European Commission, DG SANTE (Specific Agreement No. 7 to Framework Partnership Agreement No. SANCO/2005/FOOD
SAFETY/0025-Pesticides in fruit and vegetables).
EXPERIMENTAL
RESULTS
During last decade high resolution accurate mass spectrometers have improved qualitative (resolution, mass accuracy) as well as quantitative (sensitivity, linear range) aspects. HARMS instruments are well known for their high selectivity in full MS
mode. Nevertheless even spectrometers which have very high resolution may produce false positive results. Isotopic pattern and alternative adducts not always can correctly discard false positives. A solution to reduce number of false positive results is
application of simultaneous full MS and MS2.
The objective of this work was to compare three workflows of simultaneous MS and MS2: All Ion Fragmentation (no precursor ion selection, ions from entire mass range fragmented at the same time), variable Data Independent Acquisition (no precursor
ion selection, mass range divided into smaller segments before fragmentation) and data dependent MS2 (selection of precursor ion). Acatonitrile extracts (blanks and spiked with 166 pesticides) of 11 fruits and vegetables were used for the evaluation. Blank
extracts were used to evaluate potential false positives (considering retention time window of 0.2 min) whereas spiked extracts (at 0.01 mg/kg and 0.1 mg/kg) to evaluate the false negatives. Samples were analysed with Q Exactive Focus working with
resolution of 70,000 (at m/z 200) in full scan MS and 17,500 or 35,000 in MS2 mode.
Chromatography Mobil phase:
- A: 98% H2O 2% MeOH 5mM HCOONH4 0.1% HCOOH
- B: 98% MeOH 2% H2O 5mM HCOONH4 0.1% HCOOH
Flow: 0.35 mL/min
Gradient time: 14 min + 3 min reequilibration
Column: Accucore™ aQ C18,
100 mm x 2.1 mm x 2.6 µm
Column temperature: 30°C
Ijnection volume: 10 µL
Acquisition mode: full scan MS -resolution 70,000
-AGC target 1e6
-max IT auto
-scan range 120-1000 Da Acquisition mode: MS2 -dd MS2 resolution 17,500
-vDIA resolution 17,500 or 35,000,
3 or 5 mass segments - AIF resolution 70,000
CONCLUSIONS
Matrices
RT: 11.00 - 11.70
11.00 11.05 11.10 11.15 11.20 11.25 11.30 11.35 11.40 11.45 11.50 11.55 11.60 11.65
Time (min)
0
50
1000
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1000
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1000
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100
Re
lative
Ab
un
da
nce
0
50
1000
50
100 NL: 1.01E8
Base Peak m/z= 307.1790-307.1820
F: FTMS + p ESI Full ms
[120.00-1000.00] MS
Tomate_100ppb_dil5_dd_150pest_R
2
NL: 8.99E7
Base Peak m/z= 307.1790-307.1820
F: FTMS + p ESI Full ms
[120.00-1000.00] MS
Tomate_100ppb_dil5_AIF_R2
NL: 9.66E7
Base Peak m/z= 307.1790-307.1820
F: FTMS + p ESI Full ms
[120.00-1000.00] MS
Tomate_100ppb_dil5_vDIA_17500_3
segm_R2
NL: 9.34E7
Base Peak m/z= 307.1790-307.1820
F: FTMS + p ESI Full ms
[120.00-1000.00] MS
Tomate_100ppb_dil5_vDIA_17500_5
segm_R2
NL: 9.48E7
Base Peak m/z= 307.1790-307.1820
F: FTMS + p ESI Full ms
[120.00-1000.00] MS
Tomate_100ppb_dil5_vDIA_3segm_
R2
NL: 9.28E7
Base Peak m/z= 307.1790-307.1820
F: FTMS + p ESI Full ms
[120.00-1000.00] MS
Tomate_100ppb_dil5_vDIA_5segm_
R2
ddMS2
cycle time 0.27s
AIF
cycle time 0.62s
vDIA (3segm x 17,500)
cycle time 0.61s
vDIA (5segm x 17,500)
cycle time 0.79 s
vDIA (3segm x 35,000)
cycle time 0.83 s
vDIA (5segm x 35,000)
cycle time 1.28 s
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
RSD 0-5% RSD 0-10% RSD 0-20%
3 segments/ R=35000
5 segments/ R=35000
3 segments/ R=17500
5 segments/ R=17500
AIF
dd MS2
Tomato 0.01 mg/kg
Peak area repeatability
In workflows with shorter cycle times more
points per chromatographic peak is obtained,
by that peak area repeatability is better.
Are
a
Linearity
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]
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
dd-MS2 vDIA (5 semgents/35,000)
Independently of the workflow selected the detector response is characterised
by very good linear range and no saturation is observed.
Percentage of identified pesticides
80%
85%
90%
95%
100%
garlic
onion
spinach
green bean
lettuce
apple orange
cucumber
parsley
leek
tomato
0.1 mg/kg
0.01 mg/kg
dd-MS2
80%
85%
90%
95%
100%
garlic
onion
spinach
green bean
lettuce
apple orange
cucumber
parsley
leek
tomato
0.1 mg/kg
0.01 mg/kg
0.1 mg/kg without ion ratio
0.01 mg/kg without ion ratio
vDIA (5 semgents/35,000)
80%
85%
90%
95%
100%
garlic
onion
spinach
green bean
lettuce
apple orange
cucumber
parsley
leek
tomato
0.1 mg/kg
0.01 mg/kg
0.1 mg/kg without ion ratio
0.01 mg/kg without ion ratio
AIF
0.01 mg/kg of Propargite in leek
Full scan MS
368.1890 ± 5ppm
Full scan MS
368.1890 ± 5ppm
MS2 (120 – 1000 m/z)
231.1745 ± 5ppm
All Ion Fragmentation vDIA
MS2 (295 – 405 m/z)
231.1745 ± 5ppm
Resolving power
70,000
Resolving power
35,000
Mass errors in full scan MS
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
<2 ppm 2-3 ppm 3-5 ppm
Tomato
Orange % o
f co
mp
ou
nd
s
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
<2 ppm 2-3 ppm 3-5 ppm
ddMS2
vDIA (5 segments/35K) AIF
Tomato
% o
f co
mp
ou
nd
s Orange
% o
f co
mp
ou
nd
s
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Tomato Apple Orange Onion
-50%<ME
-21%<ME<-50%
-20%<ME<20%
21%<ME<50%
50%<ME
Selectivity in AIF and vDIA
MS2
151.0326 ± 5 ppm
0.01 mg/kg 0.02 mg/kg 0.05 mg/kg
Full Scan MS
343.0529 ± 5 ppm
Thiophanate methyl in onion (vDIA, 5 segments, 35K).
vDIA provides more selectivity than AIF
Quantitation in MS2 (AIF, vDIA)
If full scan MS does not have enough
selectivity, quantitation is possible in MS2
Matrix effects
Mass errors in MS2
LC–Q Exactive Focus MS operated in full scan at the resolution of 70,000 detected close to 100% of the selected pesticides in all matrices with mass errors below 2 ppm and
mass errors < 5 ppm in MS2. From the point of view of peak area repeatability the most robust workflow was dd MS2. Matrix effects were lower than 20% in the majority of the
cases facilitating quantification. AIF and vDIA offer the possibility to quantify with accurate MS2, this can help with "difficult" matrices. Dd MS2 had the highest identification rate
(96-100%, depending on the matrix). In vDIA it was 86-100% and in AIF 81-100%.
