APPLICATION NOTE 10726 Simultaneous screening and quantification of pesticide residues in potato using GC-Orbitrap MS Authors: Sarvendra Pratap Singh, Subodh Kumar Budakoti, Deepti Maheshwari and Dasharath Oulkar Customer Solution Center, Thermo Fisher Scientific, Ghaziabad, India Keywords: TraceFinder, pesticide residues, potato, QuEChERS, Exactive GC Orbitrap, high-resolution accurate mass (HRAM), mass spectrometry, screening, quantification Goal To develop a combined targeted screening and quantitation method for pesticide residues in potato using gas chromatography coupled to Orbitrap ™ mass spectrometry. The optimized method performance was evaluated following the SANTE/12682/2019 guidelines and assessed for compliance with maximum residue levels (MRLs) for potato from the Food Safety and Standards Authority of India (FSSAI) and the European Commission (EC). Introduction Potato (Solanum tuberosum) is a major root crop that contributes to food security in developing countries. 1 Often, potato cultivation involves unregulated applications of pesticides, thereby leading to non-compliance issues related to trade and potential health hazards to consumers. According to a report provided by the United States Food and Drug Administration, every year around 10% of the imported potato samples fail to comply with the MRLs. Despite these concerns, there are very few reported validated analytical methods for the analysis of pesticide residues in potato. 2 With available technologies like GC-MS/MS, it is possible to detect and quantify the presence of pesticides in potato with unit mass resolution as per the SANTE/12682/2019 quantitation and identification criteria. 3 When using triple quadrupole MS, the selectivity required to separate target pesticides from the chemical background is achieved by the use of selected reaction monitoring (SRM). SRM is used in targeted experiments in which the mass spectrometer is pre-programmed utilizing a list of predefined pesticides. During acquisition, the target-specific list of compounds
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APPLICATION NOTE 10726
Simultaneous screening and quantification of pesticide residues in potato using GC-Orbitrap MS
Customer Solution Center, Thermo Fisher Scientific, Ghaziabad, India
Keywords: TraceFinder, pesticide residues, potato, QuEChERS, Exactive GC Orbitrap, high-resolution accurate mass (HRAM), mass spectrometry, screening, quantification
GoalTo develop a combined targeted screening and quantitation method for pesticide residues in potato using gas chromatography coupled to Orbitrap™ mass spectrometry. The optimized method performance was evaluated following the SANTE/12682/2019 guidelines and assessed for compliance with maximum residue levels (MRLs) for potato from the Food Safety and Standards Authority of India (FSSAI) and the European Commission (EC).
Introduction Potato (Solanum tuberosum) is a major root crop that contributes to food security in developing countries.1 Often, potato cultivation involves unregulated applications of pesticides, thereby leading to non-compliance issues related to trade and potential health hazards to consumers. According to a report provided by the United States Food and Drug Administration, every year around 10% of the
imported potato samples fail to comply with the MRLs. Despite these concerns, there are very few reported validated analytical methods for the analysis of pesticide residues in potato.2
With available technologies like GC-MS/MS, it is possible to detect and quantify the presence of pesticides in potato with unit mass resolution as per the SANTE/12682/2019 quantitation and identification criteria.3 When using triple quadrupole MS, the selectivity required to separate target pesticides from the chemical background is achieved by the use of selected reaction monitoring (SRM). SRM is used in targeted experiments in which the mass spectrometer is pre-programmed utilizing a list of predefined pesticides. During acquisition, the target-specific list of compounds
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limits the scope of analysis so pesticides present in the sample but not included in the acquisition list will not be detected and will result in non-detection (false negative) for additional compounds. This limitation has increased the interest for developing methods using high-resolution full scan mass spectrometry, which offers better selectivity due to accurate mass measurement and equal sensitivity. Sample preparation is also equally important for the analysis of residues in food matrices. For sample preparation, there are few generic multi-residue extraction methods reported in the literature. The QuEChERS acetonitrile approach is the most popular and was selected for this study of multi-residue pesticides analysis with respect to their scope.4
This work aimed to develop and validate an analytical method for simultaneous screening and quantification of pesticide residues in potato by using the QuEChERS extraction method in combination with the Thermo Scientific™ Exactive™ GC Orbitrap™ GC-MS system operated in full scan mode. The data acquisition and processing were carried out by using Thermo Scientific™ TraceFinder™ software. The optimized method was validated as per the SANTE/12682/2019 guidelines3.
Experimental GC-Orbitrap analysisThe instrument used was the Thermo Scientific™ TRACE™ 1310 GC coupled to the Exactive GC Orbitrap high-resolution accurate mass mass spectrometry (HRAM MS) system, with electron impact (EI) ionization and VPI technology. The optimized GC-MS conditions are given in Table 1.
Sample preparationReagents and chemicals• Acetonitrile, Optima™ LC/MS Grade, Fisher Scientific™
Instrumentation Exactive GC Orbitrap system with Thermo Scientific™ TriPlus™ RSH Autosampler
ColumnThermo Scientific™ TraceGOLD™ TG-5SIL MS (30 m × 0.25 mm i.d. × 0.25 µm) (P/N 26096-1420)
Injector Split/Splitless (SSL)
Liner Thermo Scientific™ LinerGOLD™ single taper (P/N 453A1345)
Injector temperature 250 °C
Injector mode Splitless
Splitless time 2.0 min
Split flow 50.0 mL/min
Purge flow 5.0 mL/min
Injection volume 1 µL
Column flow 1.20 mL/min
Carrier gas and purity Helium (99.999%)
Vacuum compensation On
Total run time 35.6 min
GC oven program
40 °C, 1.5 min hold, 25 °C/min to 90 °C, 1.5 min hold, 25 °C/min to 180 °C, 5 °C/min to 280 °C, 10 °C/min to 300 °C, 5 min hold
Orbitrap mass spectrometry method
Acquisition mode Full Scan
Filament on delay 5.0 min
MS transfer line temp 280 °C
Ion source temp 250 °C
Electron energy 70 eV
Resolving power (FWHM at m/z 200) 60,000
Scan range 50–550 Da
Ionization Electron Ionization (EI)
Sample extraction and cleanupProcedure 1: The EN 15662 citrate buffered QuEChERS method5 • Weigh 10 g homogenized sub-sample into a 50 mL
extraction tube.
• Prepare recovery spike samples (n=6 for each level) by spiking blank samples before the addition of any extraction solvent and salts with the pesticides mix at 0.005, 0.010, and 0.025 mg/kg.
• Add 10 mL acetonitrile.
• Shake vigorously for 1 min on a vortex mixer.
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• Add EN 15662 QuEChERS Extraction salts to the tube, and immediately shake vigorously for 1 min on a vortex mixer.
• Centrifuge at 3000 g for 5 min at room temperature.
• Transfer supernatant (1 mL) into a tube containing 150 mg MgSO4 and 25 mg PSA.
• Vortex for 1 min and centrifuge samples with 5000 rpm for 5 min.
• Transfer the supernatant into a GC vial for instrumental analysis.
Procedure 2: The AOAC 2007.01 QuEChERS method6
• Weigh 15 g homogenized sample into a 50 mL extraction tube.
• Prepare recovery spike samples (n=6 for each level) by spiking blank samples with the pesticide mix at 0.025 mg/kg. Recovery samples were spiked before the addition of the extraction solvent.
• Add 15 mL 1% acetic acid in acetonitrile.
• Shake vigorously for 1 min on a vortex mixer.
• Add 6 g MgSO4 and 1.5 g of sodium acetate, again mix vigorously for 1 min on a vortex mixer.
• Centrifuge at 5000 rpm for 5 min.
• Transfer supernatant (1 mL) into a tube containing 150 mg MgSO4 and 50 mg PSA.
• Vortex for 1 min and centrifuge samples with 5000 rpm for 5 min.
• Transfer the supernatant into a GC vial for instrumental analysis.
Solvent standard calibration• The solvent standard calibration was prepared in a range
of 0.001 to 0.1 mg/L.
• Prepare matrix blank (un-spiked) extract by following the above protocol for matrix-matched calibration standards.
• Matrix-matched calibration standards: Prepare the matrix-matched calibration standards as per the procedure given in Thermo Scientific Application Note 730396.
• Inject the final extract as well as matrix-matched standards into the Exactive GC Orbitrap system.
Data acquisition and processingThe data acquisition and processing were carried out using Thermo Scientific™ TraceFinder™ 4.1 software. The data were acquired in full scan mode. For data processing, the identification criteria of the analyte, the mass error (±5.0 ppm) for the base peak and confirmatory ion, retention time (±0.10 min), and linearity (>0.99 with back-calculated concentration difference ±20%), recovery (70–120%), and precision (±20%) were set for quantitation with user-defined filters per the SANTE guidelines3.
Results and discussionSample preparationPotatoes contain 80% water and have low fat content (0.1%) and protein levels (2%). Most of the remaining matter is the edible starch portion of the plant. Because of this high starch content, it is a challenge to extract the pesticides from the potato. The recovery of spiked analyses in potato was evaluated with both methods by using a pre-spiked sample at 0.025 mg/kg. The results showed that there is no significant difference between methods.
In both methods, the mass accuracy observed was within the acceptance criteria of mass error (±5 ppm). The EN 15662 method has been utilized for extraction and analysis. Signal enhancement was observed due to matrix interferences when the TIC was compared between solvent standards and the matrix-matched standard equivalent (Figure 1A and 1B) at the concentration of 0.01 mg/kg. The high-resolution extracted ion chromatogram (EIC) filtered out the matrix interferences and provided a symmetrical peak. The high selectivity provided by HRAM is illustrated for chlorpropham (exact mass m/z 213.05510 in Figure 1C. At 15,000 and 30,000 chlorpropham could not be fully mass resolved from a co-eluting matrix co-extractive compound correctly due to high mass error (>5 ppm). At a resolving power of 60,000, the m/z 213.06366 impurity mass was isolated from m/z 213.05462, and m/z 213.05511 for the chlorpropham was observed with a mass accuracy of 0.4 ppm (Figure 1C).
The matrix effect was checked by comparing the peak area of the target analytes at a solvent calibration concentration equivalent to 0.01 mg/kg against the matrix-matched standard at 0.01 mg/kg. Ninety-two analytes showed <20% matrix effect (defined as acceptable matrix influence on the analyte as per the SANTE guidelines), with 105 other analytes showing >20% ion enhancement that was observed in the range of 20% to 264%. To obtain accurate results, it is necessary to use matrix-matched standards for the accurate quantitation.
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Figure 1. Comparison of total ion chromatogram for solvent standard (A) and matrix-matched standard (B), and the impact of resolving power on chlorpropham (C10H12ClNO2 theoretical mass m/z 213.055116) selectivity at various resolving power settings of 15,000, 30,000, and 60,000 (C)
A
B
C
Matrix-matched standard 0.01 mg/kg
Solvent standard 0.01 mg/kg
Mass error = 0.15 ppm
Mass error = -5.9 ppm
Mass error = -7.9 ppm
R=30,000
R=60,000
R=15,000
Chlorpropham Matrix
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GC-Orbitrap analysisGenerally, a non-polar solvent is preferred for GC analysis. In this experiment, acetonitrile was used for extraction and as the final solvent prior to GC-MS analysis. The advantage of using acetonitrile as the final solvent is that samples prepared this way can be analyzed on both GC-MS and LC-MS systems without further time-consuming solvent exchange steps. Acetonitrile has a low molecular weight and high polarity. It has a relatively high expansion volume and carries high matrix co-extractives that may disturb the chromatography. By considering these challenges, the splitless injection volume was reduced to 1.0 µL.
The GC oven program was taken from Thermo Scientific Application Note 10586, which offered excellent chromatographic separation for all target analytes7.
Instrument sensitivityThe limit of identification (LOI) was estimated in potato matrix by following retention time criteria and one diagnostic ion with mass accuracy within ±5 ppm. The IDL was 0.0005 mg/kg for 169 molecules, 0.001 mg/kg for 189 molecules, and 0.0025 for 199 molecules. The total 197 compounds in the range of 0.001–0.005 mg/kg in potato matrix-matched standards were successfully complying the identification and confirmation criteria as per the SANTE/12682/2019 guidelines. All the molecules pass the acceptance criteria for mass accuracy of <5 ppm. All the parent ions overlap with confirmatory ions at defined retention time (±0.1 min). An isotopic pattern of chlorpropham showed the chlorinated pattern (m/z 127.01833 and 129.01542) having the chlorinated structure with confirmatory ions m/z 171.00815 and m/z 213.05510 with the mass accuracy 0.26 and 0.07 ppm, respectively, which were within acceptance criteria3 (Figure 2).
Figure 2. Extracted ion chromatogram along with spectra, isotopic pattern, and fragments for the chlorpropham of the sample post-spiked at 0.01 mg/kg concentration
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Targeted quantificationFor reliable and confident quantitation, good and symmetrical peak shape is a requirement. An accurate quantitation is reliant upon several factors, one of which is that an acquisition speed should be fast enough to provide at least 12 points across the chromatographic peak. At a resolution of 60K, the Exactive GC Orbitrap system has a scan speed of approximately 7.4 Hz. Because of the high number of scans per peak, better repeatability was achieved.
As a productivity benefit, based on the user-defined criteria, the data was processed automatically with flagging. These flags indicate through color codes whether results pass or fail based on the acceptance criteria given in the processing method. The results that passed under user-defined criteria (SANTE/12682/2019 guidelines) are shown in green (Figure 3).
The parent/base ion (m/z 127.01832) at 0.005 mg/kg was considered the quantitation ion for the chlorpropham. Further, linearity was assessed using matrix-matched standards across a concentration of 0.001–0.1 mg/kg.
The coefficient of determination (R2) was >0.99 with RF RSD residual values <20% for all the target analytes in the matrix by plotting the calibration curve.
The matrix effect was checked by injecting the solvent standards linearity and matrix-matched standard linearity (Table 2, page 10). Response enhancement has been observed in matrix-matched standards as compared to solvent standards linearity.
The optimized method was tested for repeatability. A long-term single sequence was assessed for the average mass accuracy by injecting a spiked potato at 0.025 mg/kg level (n=35). The mass accuracy observed for molecules along with the isomers between -2.6 and 0.8 ppm without lock mass correction was found to be within an acceptable range (±5 ppm). The mass error observed for all molecules in spiked potato samples is presented in Figure 4. The ion ratios stability values were also monitored throughout the batch, and all values were within the acceptance criteria (±30%) (Figure 5) and are presented in Table 3, page 15. The ion ratio variation was 3.5% for chlorpropham in one sequence (n=35 injections) (Figure 6).
Figure 3. Screening and quantification of chlorpropham based on confirmation criteria
%Residuals
R2= 0.9991 RF RSD = 8.0%
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Figure 4. Observed average mass accuracy for all 197 molecules in n=35 replicates of a 0.025 mg/kg pre-spiked potato sample.
Figure 5. The difference in ion ratio % against the standard reference value in pre-spiked potato matrix at 0.005 mg/kg and 0.01 mg/kg
Figure 6. Chlorpropham ion ratio stability across n=35 injections of a potato spiked matrix at 0.025 mg/kg. .
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Figure 7. % Recovery of 197 target compounds in potato at 0.005 and 0.01 mg/kg
Figure 8. Repeatability (n= 6 injections) as %RSD of peak area for 197 compounds analyzed in potato at 0.005 mg/kg and 0.01 mg/kg, respectively
To harmonize the results the smallest concentration (0.005 mg/kg) was selected as a limit of quantitation (LOQ) which offered good identification and confirmation criteria.3 The LOQ offered excellent recoveries between 76 and 116% and <13% repeatability (precision). The recovery experiment was carried out at 0.005 (LOQ) and 0.01 (LOQ × 2) mg/kg to demonstrate the method performance
in terms of accuracy and precision (n=6). The average recovery was observed in the range of 76 to 116% with average %RSD of 4.6 and 3.5% for pre-spiked samples at concentration of 0.005 mg/kg and 0.01 mg/kg, respectively (Figures 7 and 8, and Table 2, page 10), which were within acceptance criteria (recovery 70–120% and precision <20%) of the SANTE/12682/2019 guidelines3.
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Conclusion • The experiments performed demonstrate that the
Exactive GC Orbitrap GC-MS high-resolution mass spectrometer, in combination with TraceFinder software, delivers robust and sensitive performance for routine pesticide screening and quantitation in potato in accordance with the SANTE/12682/2019 guidance document.
• The use of the QuEChERS method for extraction, followed by the instrumental analysis, increases the overall throughput and significantly increases the confidence in the results.
• Full scan acquisition allows for easy method setup and enables retrospective data analysis by HRMS.
• The limit of identification (LOI) was observed in the range of 0.0005 mg/kg to 0.0025 mg/kg.
• The observed R2 value was >0.99 for the plotted calibration curve in the range of 0.001 to 0.1 mg/kg.
• The average recovery was observed in the range of 76 to 116%, with average %RSD of 4.6% and 3.5% for pre-spiked samples at a concentration of 0.005 mg/kg and 0.01 mg/kg, respectively, which were within acceptance criteria (recovery 70–120% and precision <20%) of the SANTE/12682/2019 guidelines.
• The mass error was observed for molecules along with the isomers and metabolites between -2.6 and 0.8 ppm without lock mass correction. The average mass accuracy observed was within ±1 ppm for 94% of the compounds, whereas 6% (12) compounds were between -1 and -2.6 ppm.
• The ion ratios repeatability values were monitored throughout the batch (n=35 injections), and all values were within the acceptance criteria of SANTE guidelines (±30%).
• The method complies with the EU and the FSSAI MRLs requirements.
References1. US FDA. (2013). Pesticide Monitoring Program Fiscal Year 2013 Pesticide Report;
2. Khan, Z. Analysis of pesticide residues in tuber crops using pressurized liquid extraction and gas chromatography tandem mass spectrometry. Food Chem. 2018, 241, 250–257. doi: 10.1016/j.foodchem.2017.08.091. Epub (2017) Aug 31.
3. Guidance document on analytical quality control and method validation procedures for pesticide residues and analysis in food and feed (2017) SANTE/12682/2019, 01/01/2020 rev.0.
4. Citrate buffered QuEChERS method EN 15662:2008 (BS EN 15662:2008).
5. AOAC Official Method 2007.01 Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate.2007 AOAC international.
6. Thermo Scientific Application Note 73039 (2019): Large-scale screening and quantitation of pesticide residues in milk using GC-(EI)-MS/MS. https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-73039-gc-ei-ms-pesticides-milk-an73039-en.pdf
7. Thermo Scientific Application Note 10586 (2018): Ultra low level quantification of pesticides in baby foods using an advanced triple quadrupole GC-MS/MS system. https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-10586-gc-ms-ms-pesticides-baby-foods-an10586-en.pdf
8. FSSAI regulatory requirements (The Food Safety Standard Act, 2006). Insecticides / Pesticides Registered under section 9(3) of the Insecticides Act, 1968 for use in the Country: (As on 31/12/2018). https://fssai.gov.in/home/fss-legislation/notifications/gazette-notification.html (accessed on 7th January 2019)
9. EU MRLs for potatoes: https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/public/?event=product.resultat&language=EN&selectedID=88 (accessed on 9th August 2019).