Utilization of Rapid LC-MS for Screening and Quantitative Analysis of Pesticides in Food Matrix using an Exactive Plus Benchtop Orbitrap Platform

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1 Utilization of Rapid LC-MS for Screening and Quantitative Analysis of Pesticides in Food Matrix using an Exactive Plus Benchtop Orbitrap Platform Charles Yang, Leo Wang, Dipankar Ghosh, Jonathan Beck, Jamie Humphries; Thermo Fisher Scientific, San Jose, CA, USA

2 Introduction The demand for quick and simple analysis of large numbers of samples in agriculture analysis is growing year by year. Throughout the world pesticides are used to control pests that are harmful to crops, humans and animals. These substances can pose a significant health threat and therefore, need to be accurately detected at the lowest levels requested by the governmental authorities typically at low part per billion (ppb) or low part per trillion (ppt) levels. Traditionally, triple stage quadrupole mass spectrometers have been used by the food industry to identify and quantitate these residues. The Thermo Scientific Exactive Plus Orbitrap mass spectrometer provides high-resolution, accurate mass (HR/AM) to unequivocally identify compounds without time-consuming analysis. The results of this unique solution are improved sensitivity and precision. Mass spectrometric detection for screening experiments with HR/AM technology using full scan experiments can detect as many analytes as necessary using only one chromatographic run. An Exactive Plus mass spectrometer with the proven power of the Thermo Scientific Orbitrap mass analyzer and Thermo Scientific TraceFinder software for unified quantitative and targeted screening data processing fulfills these demands with higher confidence and precision. Methods Sample Preparation Onion matrix were prepared for analysis by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, which is a sample preparation procedure used to extract pesticides from food. The QuEChERS extracts were obtained from the California Department of Food and Agriculture (CDFA). For the QuEChERS extraction, 15 g of homogenized sample and 15 ml of acetonitrile were used. Then, 200 L of final QuEChERS extract, 300 L of acetonitrile, and 500 L of water were transferred into an autosampler vial, spiked with 20 L of the pesticides standard, and mixed well. A mixture of 60 pesticides with different starting concentrations was also provided by CDFA to make the standard calibration curve in neat matrix and onion matrix. Calibration of the Exactive Plus mass spectrometer was done with external calibration. There is no need to do lock masses because of the proven stability of the Orbitrap mass analyzer. Hardware The new Exactive Plus mass spectrometer has the proven S-Lens configuration for more ion transmission for more sensitivity. The neutral ions are removed with the bent flatapoles prior to entering the C-Trap. Then the ions are transported into the Orbitrap mass analyzer for HR/AM detection. The system also has an HCD collision cell past the C-trap where all ion fragmentation (AIF) happens. All the fragmented ions are then transported back into the C-Trap and sent into the Orbitrap mass analyzer for detection in accurate mass. (Figure 1) FIGURE 1. Exactive Plus schematics Liquid Chromatography Conditions System: Thermo Scientific UltiMa Column: Thermo Scientific Hypers (50 x 2.1 mm, 1.9 μm pa Mobile Phase A: Water with 0.1% formic a Mobile Phase B: Methanol with 0.1% form Flow Rate: 300 μl/min Column Temp: 40 C Sample Injection: 5 μl Gradient Time A B min 0.00 min 0.25 min 70% 30% min 0% 100% min min Mass Spectrometry Conditions Method: full MS scan Mass Range: -120 to 1000 Ion Mode: positive/negative Resolution: 70,000 Ion Source: heated electrospray ioniz Spray Voltage: 2200v Capillary Temp: 295 ºC Vaporizer Temp: 280 ºC Sheath Gas: 32 Auxiliary Gas: 7 The ease-of-use of HR/AM instrumentation requ transitions and collision energies unlike that of tr HR/AM method development is focused only on Results Data processing was performed with TraceFinde was achieved by applying a mass window of 5 p analytes. All analytes gave very good linear res 0.10 ng/ml depending on starting concentration TraceFinder software comes with many features show how the yellow flags next to the name of th was not found in the sample. The red flag indica with the calibration curve and it did not meet the sign indicates there is an issue with one or more to be reviewed. FIGURE 1. TraceFinder software displaying B matrix R^2, list of compounds, and chromat 2 Utilization of Rapid LC-MS for Screening and Quantitative Analysis of Pesticides in Food Matrix using an Exactive Plus Benchtop Orbitrap Platform

3 ple analysis of large numbers of samples in agriculture r. Throughout the world pesticides are used to control, humans and animals. These substances can pose a erefore, need to be accurately detected at the lowest mental authorities typically at low part per billion (ppb) or. Traditionally, triple stage quadrupole mass by the food industry to identify and quantitate these ic Exactive Plus Orbitrap mass spectrometer provides (HR/AM) to unequivocally identify compounds without results of this unique solution are improved sensitivity etric detection for screening experiments with HR/AM eriments can detect as many analytes as necessary ic run. An Exactive Plus mass spectrometer with the cientific Orbitrap mass analyzer and Thermo Scientific d quantitative and targeted screening data processing her confidence and precision. r analysis by using a modified quick, easy, cheap, EChERS) method, which is a sample preparation icides from food. The QuEChERS extracts were epartment of Food and Agriculture (CDFA). For the homogenized sample and 15 ml of acetonitrile were EChERS extract, 300 L of acetonitrile, and 500 L of autosampler vial, spiked with 20 L of the pesticides ixture of 60 pesticides with different starting ed by CDFA to make the standard calibration curve in Calibration of the Exactive Plus mass spectrometer was There is no need to do lock masses because of the mass analyzer. pectrometer has the proven S-Lens configuration for sensitivity. The neutral ions are removed with the bent C-Trap. Then the ions are transported into the Orbitrap ction. The system also has an HCD collision cell past ntation (AIF) happens. All the fragmented ions are then p and sent into the Orbitrap mass analyzer for detection ematics Liquid Chromatography Conditions System: Thermo Scientific UltiMate 3000 UHPLC system Column: Thermo Scientific Hypersil GOLD aq C18 column (50 x 2.1 mm, 1.9 μm particle size) Mobile Phase A: Water with 0.1% formic acid and 10 mm ammonium formate Mobile Phase B: Methanol with 0.1% formic acid and 10 mm ammonium formate Flow Rate: 300 μl/min Column Temp: 40 C Sample Injection: 5 μl Gradient Time A B min 0.00 min 0.25 min 70% 30% min 0% 100% min min Mass Spectrometry Conditions Method: full MS scan Mass Range: -120 to 1000 Ion Mode: positive/negative Resolution: 70,000 Ion Source: heated electrospray ionization (H-ESI) Spray Voltage: 2200v Capillary Temp: 295 ºC Vaporizer Temp: 280 ºC Sheath Gas: 32 Auxiliary Gas: 7 The ease-of-use of HR/AM instrumentation requires no optimization of mass transitions and collision energies unlike that of triple stage quadrupole methods. HR/AM method development is focused only on the HPLC. Results Data processing was performed with TraceFinder software. Specificity of analysis was achieved by applying a mass window of 5 ppm to the theoretical mass of the analytes. All analytes gave very good linear response in the calibration range (0.01 to 0.10 ng/ml depending on starting concentration in mixture). TraceFinder software comes with many features including flagging. Figures 2 and 3 show how the yellow flags next to the name of the compound indicate the compound was not found in the sample. The red flag indicates that the compound has an issue with the calibration curve and it did not meet the flagging requirement. The caution sign indicates there is an issue with one or more compounds in the sample that needs to be reviewed. FIGURE 1. TraceFinder software displaying Boscalid calibration curve plot of matrix R^2, list of compounds, and chromatogram FIGURE 2. TrceFinder displaying Imidaclopr R^2, list of compounds, and chromatogram In addition to the quantitation, TraceFinder soft page where you can see all the reports prior to of reports are available with the ability to perfor tool you can search for a particular compound i every page (Figure 3). Figure 4 shows a popular feature of TraceFinde step, last minute changes can be accomplished to return to the method development step. Table 1 shows the list of >90 targeted pesticide shows the retention time, R 2 and LOQ for the c FIGURE 3. A simple high-density calibratio up to 9 calibration curves on one sheet. In a particular compound by clicking on the bi Thermo Scientific Poster Note PN63685_e 08/12S 3

4 nditions o Scientific UltiMate 3000 UHPLC system o Scientific Hypersil GOLD aq C18 column 2.1 mm, 1.9 μm particle size) r with 0.1% formic acid and 10 mm ammonium formate anol with 0.1% formic acid and 10 mm ammonium formate L/min FIGURE 2. TrceFinder displaying Imidacloprid calibration curve plot of matrix, R^2, list of compounds, and chromatogram FIGURE 4. The Data Review screen allows th without going back to Method Development. A B 70% 30% 0% 100% ions S scan to 1000 ve/negative 0 d electrospray ionization (H-ESI) v C C strumentation requires no optimization of mass gies unlike that of triple stage quadrupole methods. t is focused only on the HPLC. ed with TraceFinder software. Specificity of analysis mass window of 5 ppm to the theoretical mass of the ery good linear response in the calibration range (0.01 to rting concentration in mixture). with many features including flagging. Figures 2 and 3 xt to the name of the compound indicate the compound The red flag indicates that the compound has an issue it did not meet the flagging requirement. The caution ue with one or more compounds in the sample that needs In addition to the quantitation, TraceFinder software has a very easy-to-view report page where you can see all the reports prior to print out. More than 35 different types of reports are available with the ability to perform customization. With a simple find tool you can search for a particular compound in the report without having to review every page (Figure 3). Figure 4 shows a popular feature of TraceFinder software. During the Data Review step, last minute changes can be accomplished prior to report printing without having to return to the method development step. Table 1 shows the list of >90 targeted pesticides analyzed in this experiment. Table 2 shows the retention time, R 2 and LOQ for the components analyzed in onion matrix. FIGURE 3. A simple high-density calibration report is shown below allowing up to 9 calibration curves on one sheet. In this window you can also search for a particular compound by clicking on the binocular to open the search window. Table 1. List of 90 pesticides and associate theoretical m/z with calculated mass errors ftware displaying Boscalid calibration curve plot of nds, and chromatogram 4 Utilization of Rapid LC-MS for Screening and Quantitative Analysis of Pesticides in Food Matrix using an Exactive Plus Benchtop Orbitrap Platform

5 playing Imidacloprid calibration curve plot of matrix, d chromatogram FIGURE 4. The Data Review screen allows the user to perform quick changes without going back to Method Development. Table 2. Tabulated results of LOQ s per co Table 1. List of 90 pesticides and associated retention times with actual vs. theoretical m/z with calculated mass errors. n, TraceFinder software has a very easy-to-view report l the reports prior to print out. More than 35 different types the ability to perform customization. With a simple find rticular compound in the report without having to review ature of TraceFinder software. During the Data Review an be accomplished prior to report printing without having elopment step. 0 targeted pesticides analyzed in this experiment. Table 2 2 and LOQ for the components analyzed in onion matrix. -density calibration report is shown below allowing on one sheet. In this window you can also search for y clicking on the binocular to open the search window. Conclusion TraceFinder software coupled with the spectrometer powered by Orbitrap tech quantitative and screening data in one p The results show very linear R 2 with ver pesticides in difficult matrix. The power of resolution at 70,000 (FWH interferences which can cause lower-en The mass error also shows confidence Acknowledgements The authors wish to thank to Roland Carlson extracts, pesticide mixture and methodology f All trademarks are the property of Thermo Fisher Scientific a encourage use of these products in any manners that might Thermo Scientific Poster Note PN63685_e 08/12S 5

6 creen allows the user to perform quick changes Development. Table 2. Tabulated results of LOQ s per compound, retention times and curve fit R 2 s and associated retention times with actual vs. ted mass errors. Conclusion TraceFinder software coupled with the Exactive Plus benchtop mass spectrometer powered by Orbitrap technology provides easy access to full quantitative and screening data in one package. The results show very linear R 2 with very low LOQs which will assist in detecting pesticides in difficult matrix. The power of resolution at 70,000 (FWHM) at m/z 200 helps resolve matrix interferences which can cause lower-end detection issues. The mass error also shows confidence in the results of the data acquired. Acknowledgements The authors wish to thank to Roland Carlson at CDFA for providing the QuEChERS extracts, pesticide mixture and methodology for this study. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 6 Utilization of Rapid LC-MS for Screening and Quantitative Analysis of Pesticides in Food Matrix using an Exactive Plus Benchtop Orbitrap Platform

7 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Thermo Scientific Dionex products are designed, developed, and manufactured under an ISO 9001 Quality System. Australia Austria Benelux Brazil China Denmark France Germany India Ireland Italy Japan Korea Singapore Sweden Switzerland Taiwan UK USA and Canada PN63685_E 08/12S

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