Fast, Selective, and Sensitive Screening for More Than 1000 Targeted Pesticides with Identification Using Automated Library Searching André Schreiber1, Tania Sasaki2, Doina Caraiman1 1 AB SCIEX, Concord, Ontario, Canada; 2 AB SCIEX, Foster City, California, USA
Introduction MRM
Recent regulations on food and environmental analysis require screening for pesticides using confirmatory techniques, such as GC/MS and LC/MS/MS. More than 1000 pesticides are used worldwide and, along with their metabolites and degradation products, are present in food and the environment. Thus, there is a demand for powerful and rapid analytical methods that can detect very low concentrations of pesticides. Alder et al. compared the use of GC/MS and LC/MS/MS for multi residue pesticide analysis and concluded “…the benefits of LC-MS/MS in terms of wider scope, increased sensitivity, and better selectivity are obvious.”1 The big challenges for pesticide residue laboratories at the moment are the requests to test for larger number of compounds, in a wider range of commodities, in shorter time, all without sacrificing data quality. The novel AB SCIEX QTRAP® 5500 LC/MS/MS System uses ® advanced eQ™ electronics, the new Qurved LINAC collision cell, and the patented Linear Accelerator™ Trap to address the above challenges. The developed method demonstrates that the unmatched speed allows screening for more targeted pesticides using fast LC, fast MRM, and identification of detected compounds by Enhanced Product Ion scanning and library searching of the spectra.
intensity threshold
Threshold
EPI 297 159
69
173 81
109
141
176
255 201
115
Library Search
Figure 1. Setup of an Information Dependent Acquisition (IDA) experiment: A survey in Multiple Reaction Monitoring (MRM) screens for a multitude of targeted analytes with highest selectivity and sensitivity. The chromatographic signal above a specified threshold triggers automatically the Enhanced Product Ion (EPI) scanning. Acquired EPI spectra are searched against a mass spectral library for identification.
Method Details • QuEChERS extraction of food samples and 50 times dilution to minimize possible matrix effects • Direct injection of water samples • Ultra High Pressure Liquid Chromatography using a Shimadzu UFLCXR system with a Phenomenex Synergi Fusion-RP (2.5 μm) column and a fast gradient of water and methanol with ammonium formate buffer • Total run time of less than 10 min ®
• AB SCIEX QTRAP 5500 System with Turbo V™ Source and ESI probe • Detection of 1064 Scheduled MRM™ transitions and acquisition of EPI spectra to search against a library for identification in a single analysis
The analytical workflow used and an example chromatogram and corresponding EPI mass spectrum are illustrated in Figure 1.
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XIC of +MRM (1064 pairs) from Sample 8 (1) of Data ...
Max. 7.8e5 cps. 3.9
7.8e5 Intensity, cps
6.0e5 4.0e5 2.0e5 0.0
1.0
XIC ...
Max. 1.3e4 cps. 0.6
1.3e4
3.0
XIC ...
4.0
5000.0
Azoxystrobin
4.0e4 2.0e4
0.0
0.0 Time, min Max. 4.8e5 cps.
XIC ...
5 3.0
XIC ...
4.0 5.0 Time, min Max. 3.2e4 cps.
2.2
4.8e5 4.0e5
3.0 3.0e4
Carbendazim
3.0e5
Carbofuran
2.0e4
9258 8000 6000
5.0
Max. 9257.8 cps.
6.0 XIC...
4.3 4000
Bendiocarb
2000
1000
6.0e4 4.0e4
2.0
3.0 4.0 Time, min Max. 6.0e4 cps.
Bromuconazole
3000 2000
XIC ...
Max. 4250.3 cps.
3.0
4000
0
7.0
0
3.0
XIC ...
4.0 5.0 Time, min Max. 4.3e4 cps.
0.7
4.7 4.0e4
Cyromazine
Diazinon
3.0e4 2.0e4
2.0e5
2.0e4
1.0e4
1.0e5 0.0
1.0
2.0 3.0 Time, min Max. 1.6e4 cps.
XIC ...
4.1 1.5e4
0.0
2.0
XIC ...
4.0
3.0 4.0 Time, min Max. 1.1e4 cps. 4.4
1.15e4 1.00e4
Dimethomorph
1.0e4
Flufenacet
1.0e4
0.0 XIC ... 6.0e4 4.0e4
0.0 Time, min Max. 6.2e4 cps. 4.6
5
XIC ...
Imazalil
4.0 5.0 6.0 Time, min Max. 2.4e4 cps. 1.8
2.4e4 2.0e4
Imazaquin
1.5e4
5000.00
1.0e4
2.0e4
5000.0
5000.0
0.0
3.0
XIC ...
4.0 5.0 Time, min Max. 1.9e4 cps. 1.6
1.9e4 1.5e4
Imidacloprid
0.00 XIC ...
4.0 5.0 6.0 Time, min Max. 5.3e4 cps. 1.8
5.3e4
0.0 XIC... 4891
4.0 5.0 6.0 Time, min Max. 4891.0 cps. 1.1
0.0 XIC ...
1.0 2.0 3.0 Time, min Max. 5.9e4 cps. 0.7
5.9e4
4000
4.0e4
Metamitron
1.0e4
3000
Methomyl
Omethoate
4.0e4
2000
2.0e4 5000.0
2.0e4
1000
0.0 XIC ...
1.0 2.0 3.0 Time, min Max. 3.0e5 cps. 4.3
3.0e5 2.0e5
Prometryn
0.0
1.0 2.0 3.0 Time, min Max. 2857.5 cps.
XIC...
5.2
2858
3.0
4.0 5.0 Time, min
0
0 XIC ... 1.29e4
0.0 Time, min Max. 1.3e4 cps. 5.5
Quinoxyfen
Spinosyn A + D 5000.00
4.0
5.0 6.0 Time, min
0.00
5
XIC ... 2.8e5
1.00e4
2000 1000
1.0e5 0.0
4.0
Time, min Max. 5.8e4 cps. XIC...
5.8e4
Acephate
1.0e4
2.0
2.0e5
Time, min Max. 2.8e5 cps. 2.7
Thiabendazole
1.0e5
5.0 6.0 Time, min
7.0
0.0
2.0 3.0 4.0 Time, min
Figure 2. Pesticide Screening using 1064 Scheduled MRM™ Algorithm transitions in less than 10 min (top) and extracted chromatograms of individual pesticides at a concentration of 1 ng/mL (bottom).
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Results
The new Linear Accelerator™ Trap of the QTRAP ® 5500 System allows acquisition of EPI spectra with unparalleled sensitivity and scan speed of 20000 Da/s. The hardware and software algorithm of Dynamic Fill Time (DFT) was improved to enable a wider range of fill times starting as low as 0.05 ms. This ability to use such short fill times greatly improves quality of mass spectra by reducing space charge effects and allows acquisition of quality EPI spectra over a large dynamic range. In addition, EPI spectra generated with Collision Energy Spread (CES) are well known to contain maximum structural information for best identification.
®
The new QTRAP 5500 System allows the use of short MRM dwell times and pause times while maintaining highest selectivity, sensitivity and reproducibility. These short times, in combination with the Scheduled MRM™ Algorithm offered in Analyst® Software 1.5, make it possible to detect several hundreds — or even thousands — of MRM transitions in a single analytical run. Figure 2 shows an example of an IDA chromatogram monitoring over 1000 transitions with a run time of less than 10 min. The representative extracted chromatograms highlight the superior sensitivity allowing detection limits at sub ng/mL concentrations.
Figures 3 and 4 show examples of using the described method to screen for pesticides in fruit samples. A number of compounds were identified and confirmed using automated library searching.
XIC of +MRM (1064 pairs): Exp 1, 236.0/87.0 amu Expected RT: 3.2 ID: Carboxine...
Max. 4.6e4 cps.
3.0
4.6e4
Matrix
4.0e4 Intensity, cps
211 µg/kg Trifloxystrobin
3.0e4 2.0e4
8 µg/kg Methomyl 1.0e4 0.0
1.0
2.0
+EPI (163.10) CE (35) CES (15) FT...
4.0 5.0 6.0 7.0 Time, min Max. 1.9e4 cps. +EPI (409.00) CE (35) CES (15) FT... Max. 7.7e5 cps.
Methomyl FIT = 91.5%
98.9
1.9e4
3.0
1.5e4
Trifloxystrobin FIT = 93.5%
6.0e5 Intensity, cps
Intensity, cps
1.0e4 88.0
5000.0
73.0 57.1
0.0
186.1
7.7e5 7.0e5
60
106.1
77.0 102.8 80
100
117.1
4.0e5 3.0e5 116.0 145.1
2.0e5
163.1
206.2 91.0101.0 162.0
1.0e5
145.1
120 140 m/z, Da
5.0e5
160
180
200
0.0
50
409.3
100 150 200 250 300 350 400 450 500 m/z, Da
Figure 3. Detection of 8 μg/kg Methomyl and 211 μg/kg Trifloxystrobin in a grape sample and identification by library searching of Enhanced Product Ion spectra. The library match was excellent, with a FIT of >90%.
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XIC of +MRM (1064 pairs): Exp 1, 202.1/175.1 amu Expected RT: 2.6 ID: Thiabendazole...
Max. 1.4e6 cps.
2.7
1.4e6
310 µg/kg Thiabendazole
1.2e6 Intensity, cps
1.0e6 8.0e5 6.0e5
100 µg/kg Imazalil 6 µg/kg Spiroxamine
4.0e5 2.0e5
Matrix
0.0
1.0
+EPI (202.10)...
2.0
Max. 2.3e5 cps.
202.1 Thiabendazole FIT = 97.5%
2.3e5 2.0e5
3.0
4.0 Time, min +EPI (297.10)... Max. 2.5e5 cps.
Imazalil FIT = 92.6%
64.9
1.0e5 5.0e4
104.2 0.0
100
173.0 81.0
92.3
5.0e4
Intensity, cps
Intensity, cps
Intensity, cps
1.0e5
1.5e5 69.1
255.0 109.1
142.9
187.2
200 m/z, Da
300
0.0
100
Max. 4.1e4 cps.
FIT = 96.9%
3.5e4
297.1
7.0
144.1 Spiroxamine
4.0e4
3.0e4
131.2 175.0
6.0
+EPI (298.40)...
159.1
2.5e5 2.0e5
1.5e5
5.0
100.1
2.5e4
298.4
2.0e4 1.5e4 1.0e4 72.1
280.9
200 300 m/z, Da
5000.0 0.0
400
81.0 100
200 300 m/z, Da
400
Figure 4. Detection of Detection of 6 μg/kg Spiroxamine, 100 μg/kg Imazalil, and 310 μg/kg Thiabendazole in a banana sample and identification by library searching of Enhanced Product Ion spectra. The library search results were again excellent, with FIT of >90% for all compounds
Summary
References
The new QTRAP® 5500 LC/MS/MS System incorporates the proven technology of the Turbo V™ source and the Curtain Gas™ interface for ultimate sensitivity and robustness. The unmatched speed of monitoring MRM transitions and acquiring EPI spectra is made possible by advanced technologies including the new Qurved LINAC® collision cell and the Linear Accelerator™ Trap. These performance criteria, together with sophisticated software algorithms such as Scheduled MRM™ ® Algorithm and Collision Energy Spread, make the QTRAP 5500 System the ideal instrument for high throughput multi-pesticide screening at required sensitivity levels for analysis of food and drinking water. In addition, library search of EPI spectra gives highest degree of confidence in analytical results.
1 L. Alder, K. Greulich, G. Kempe, B. Vieth: Mass Spectrometry Reviews 25 (2006) 838-865
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