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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

For Research Use Only. Not for use in diagnostic procedures. © 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license. Publication number: 1121010-01

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Fast, Selective, and Sensitive Screening for More Than 1000 Targeted Pesticides with Identification  

Fast, Selective, and Sensitive Screening for More Than 1000 Targeted Pesticides with Identification Using Automated Library Searching

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