Advanced Quantification of Pesticides in Black Tea

Application Note Food Authors Anastasia A. Andrianova and Limian Zhao Agilent Technologies, Inc. Abstract This application note presents results for the sensitive and robust quantitation of 246 pesticides in black tea extract with the Agilent 7010D Triple Quadrupole Mass Spectrometer (GC/TQ) featuring a second-generation High Efficiency Source 2.0 (HES 2.0), that addresses the challenges posed by residual pesticide analysis in complex matrices. By optimizing sample preparation and using state-of-the-art GC/MS hardware, including ion source technology and midcolumn backflushing, excellent calibration performance and sensitivity at low-ppb levels were achieved. The method demonstrated exceptional ruggedness and robustness over 800 consecutive injections of a black tea extract spiked with pesticides at 2 ppb, with high precision and low RSDs, ensuring prolonged instrument uptime and maximum throughput. The demonstrated limits of quantitation (LOQs) were as low as 0.01 ppb for over a third of evaluated compounds, and the calibration range spanned up to five orders of magnitude while meeting SANTE 11312/2021 guidelines. This application note highlights intelligent GC/TQ features, such as early maintenance feedback and an instrument health status dashboard, maintaining confidence in the results for a high-throughput analysis. The updated data acquisition platform provides enhanced user experience, including a new implementation of the retention time locking functionality. Brewing Excellence: Quantitating Over 200 Pesticides in Black Tea with Steady Performance and Maximized Uptime by GC/MS/MS 2 Introduction Tea is among the most common nonalcoholic beverages consumed worldwide. Like many foods, tea cultivation relies heavily on pesticide application to combat pests, leading to concerns over pesticide residues intensifying.1 Assessing pesticide levels in tea is essential for evaluating safety, and is required by many regulatory bodies, including the European Commission and the US Environmental Protection Agency.2,3 A complete workflow for pesticide testing in tea includes sample extraction via QuEChERS, followed by extract cleanup, and subsequent testing with liquid and gas chromatography coupled with triple quadrupole mass spectrometry (LC/TQ and GC/TQ).4 Workflow performance should enable sufficient method sensitivity, calibration range, pesticide recovery from the extraction, and precision. Sensitivity requirements are set based on the maximum residue levels (MRLs), which are the highest levels of pesticide residue that are legally allowed in or on food or feed when pesticides are applied correctly. The ability to calibrate over a wide dynamic range allows the varying MRLs for individual compounds monitored in the commodity, which can vary from 10 ppb to 100 ppm. When a specific pesticide lacks an established MRL, a default limit of 10 ppb is commonly applied. Efficiency of extraction and cleanup are characterized in terms of recovery of matrix spikes, and precision is expressed in terms of relative standard deviation (RSD) of repeat analyses. This application note presents a complete GC/TQ workflow solution for the accurate and reliable analysis of 246 volatile and semivolatile pesticides in black tea. Excellent analytical performance of the workflow was achieved through a combination of cutting-edge technology and optimized methodology that included: – Sample preparation using QuEChERS extraction, followed by EMR mixed-mode pass-through cleanup using Agilent Captiva EMR–GPD cartridges – Agilent 8890 GC hardware and GC supplies – Novel electron ionization (EI) source technology with HES 2.0 – Built-in GC/TQ MS intelligence and new software functionality for method setup, maintenance, and system health evaluation The presented workflow allowed for quantitating 246 pesticide residues in black tea with LOQs as low as 0.01 ppb for 34% of the targets, at or below 0.1 ppb for 74% of compounds, and below 2 ppb for 96%. Matrix-matched calibrations demonstrated excellent accuracy over a wide dynamic range, spanning up to five orders of magnitude over 0.01 to 1,000 ppb in the complex black tea extract. Method ruggedness was demonstrated through maintaining measurement accuracy with good precision (RSDs < 20% for 176 compounds) for black tea extract spiked at 2 ppb sequentially analyzed over 800 runs spanning 17 days of continuous analysis. The new HES 2.0 ion source is equipped with a novel dipolar radiofrequency (RF) lens that redirects the carrier gas ions and, as a result, enables improved system robustness and maximizes uptime while maintaining unparalleled analytical sensitivity. Experimental GC/TQ analysis The 8890 GC and 7010D GC/TQ systems (Figure 1A) were used and configured to achieve the best sensitivity, maintain a wide calibration range, and provide the most rugged method performance. The GC was configured with the Agilent 7693A automatic liquid sampler (ALS) and 150-position tray. The system used a multimode inlet (MMI) operated in temperature-programmed splitless injection mode (also known as cold splitless). The injection parameters were optimized for maximizing sensitivity while limiting carryover. Midcolumn backflush capability was provided by the Agilent Purged Ultimate Union (PUU) installed between two identical 15 m columns, and the Agilent 8890 pneumatic switching device (PSD) module (Figure 1B). The instrument operating parameters are listed in Table 1. Data were acquired in dynamic MRM (dMRM) mode, which provides the capability for large multi-analyte assays and the accurate quantitation of narrow peaks by an automatically determined most-efficient dwell time distribution. The dMRM capability enabled successful analysis for a large panel of 246 pesticides, with 749 total MRM transitions with up to 64 concurrent MRMs. Furthermore, dMRM allows the analyst to add and remove additional analytes with ease. The acquisition method was retention time locked to match the retention times in the Agilent MassHunter Pesticides and Environmental Pollutants MRM Database 4.0 (P&EP 4.0)5 , 3 Figure 1. The Agilent 8890 GC system with the Agilent 7010D GC/TQ system (A) and system configuration (B). PSD (helium) Agilent 8890 GC Liquid Injector Multimode inlet (helium) Agilent 7010D TQ MS HES 2.0 Agilent HP-5ms UI, 15 m × 0.25 mm, 0.25 µm Agilent HP-5ms UI, 15 m × 0.25 mm, 0.25 µm A B which was used to seamlessly create the MS method. The use of P&EP 4.0 increased the ease and speed of setting up a targeted dMRM method. High method selectivity in the presence of coeluting matrix components was achieved by selecting the best MRM transitions from up to nine transitions available for each compound in the P&EP 4.0 database. Three targets were not available in the P&EP 4.0 database (flonicamid, bioallethrin, and cycloxydim). For these compounds, MRM transitions were developed using Agilent MassHunter Optimizer for GC/TQ, operating in Start from Full Scan mode. The Optimizer is fully integrated into MassHunter Acquisition 13.0 for GC/MS (Figure 2). The acquisition method was retention time locked to the P&EP database with chlorpyrifos-methyl eluting at 9.143 minutes. The retention time locking functionality, integrated in MassHunter Acquisition 13.0 for GC/MS, has an updated user-friendly and intuitive interface (Figure 3). It allows for semi-automated or manual compound selection, provides a choice to use three or five points for retention time locking calibration, and features both a visual and quantitative assessment of the calibration curve fit, while providing the tools to maintain excellent precision of retention times, even after column trimming. Full scan data acquisition mode was used for the initial screening of the matrix extract. This screening was used to evaluate in-source loading, and for monitoring the efficiency of the sample cleanup procedure that followed QuEChERS extraction. Agilent MassHunter Workstation software, including Agilent MassHunter Acquisition 13.0 for GC/MS, MassHunter Quantitative Analysis 12.1, and MassHunter Qualitative Analysis 12.0 packages were used in this work. 4 Table 1. Agilent 8890 GC system with Agilent 7010D gas chromatograph and mass spectrometer conditions for pesticide analysis. Parameter Value GC Agilent 8890 with fast oven, auto injector and tray Inlet MMI Mode Cold splitless Purge Flow to Split Vent 60 mL/min at 3 min Septum Purge Flow 3 mL/min Septum Purge Flow Mode Switched Injection Volume 1.0 µL Injection Type Reversed 2-layer (L2, L1) L1 Airgap 0.2 µL L2 Volume (ISTD) 0.2 μL L2 Airgap 0.2 μL Gas Saver On at 30 mL/min after 5 min Inlet Temperature 60 °C for 0.1 min, then to 280 °C at 600 °C/min, hold for 5 min, then to 325 °C at 600 °C/min Postrun Inlet Temperature 310 °C Postrun Total Flow 25 mL/min Carrier Gas Helium Inlet Liner Agilent Ultra Inert 2 mm dimpled liner (p/n 5190-2297) Oven Oven Program 60 °C for 1 min; 40 °C/min to 170 °C; Hold 0 min; 10 °C /min to 310 °C; Hold 2.25 min Total Run Time 20 min Postrun Time 1.5 min Equilibration time 0.5 min Column 1 Type Agilent HP-5ms UI, 15 m × 0.25 mm, 0.25 µm (p/n 19091S-431UI-KEY) Control Mode Constant flow Flow 1.0 mL/min (then retention time locked) Inlet Connection MMI Outlet Connection PSD (PUU) PSD Purge Flow 5 mL/min Postrun Flow (Backflushing) –7.873 Parameter Value Column 2 Type Agilent HP-5ms UI, 15 m × 0.25 mm, 0.25 µm (p/n 19091S-431UI-KEY) Control Mode Constant flow Flow 1.2 mL/min (then retention time locked) Inlet Connection PSD (PUU) Outlet Connection MSD Postrun Flow (Backflushing) 8.202 MSD Model Agilent 7010D Source HES 2.0 Vacuum Pump Performance turbo Tune File atunes.eihs2.jtune.xml Solvent Delay 3.75 min Quad Temperature (MS1 and MS2) 150 °C Source Temperature 280 °C Mode dMRM or Scan He Quench Gas 2.25 mL/min N2 Collision Gas 1.5 mL/min MRM Statistics Total MRMs (dMRM mode) 749 Minimum Dwell Time 5.42 ms Minimum Cycle Time 85.01 ms Maximum Concurrent MRMs 64 EM Voltage Gain Mode 10 Scan Parameters Scan Type MS1 Scan Scan Range 45 to 450 m/z Scan Time (ms) 220 Step Size 0.1 amu Threshold 0 EM Voltage Gain Mode 1 5 Figure 2. Agilent MassHunter Optimizer software for GC/TQ, used for the automated development of MRM transitions. 6 Figure 3. New Agilent retention time locking software in Agilent MassHunter Acquisition 13.0 for GC/MS. 7 Sample preparation Black tea powder was obtained from a local grocery store. Black tea powder (2 g) was extracted with a modified QuEChERS extraction using acetonitrile (ACN) with 2% formic acid and EN extraction salt. The crude tea extract then was mixed with 2% of acidic buffer. The sample mixture was cleaned by EMR mixed-mode pass-through cleanup using Agilent Captiva EMR–GPD 6 mL. The sample eluent was dried with anhydrous MgSO4 to remove water residue completely before GC/MS/MS analysis. The sample preparation procedure flowchart is shown in Figure 4, and details will be discussed in a separate application note. The entire sample preparation procedure resulted in a 5x dilution factor. Pesticide standards Agilent GC pesticide standards 1 through 12 (part numbers PSM-100-A through -L) and Agilent GC/LC pesticide standards 1, 2, and 3 (part numbers PSM-100‑AA, PSM-100-AB, PSM-100-AC) were used for preparing matrix matched calibration standards. A combination of the 15 used standards yielded a mix of 246 pesticides commonly regulated by the FDA, USDA, and other global governmental agencies. Figure 4. QuEChERS sample preparation and cleanup method for black tea. 2 g of black tea Agilent QuEChERs EN extraction kit Mechanical shaker Centrifuge – Dry sample hydration and equilibration – Addition of extraction solvent Sample cleanup Sample extraction Black tea extract on Agilent Captiva EMR–GPD Sample eluent drying with MgSO4 Sample analysis on the Agilent 7010D GC/TQ Crude extract 2 g of – Premixing with 2% acidic buffer – Loading 2.5 to 3 mL on cartridge 8 Matrix-matched calibration Calibration performance was evaluated using a series of matrix matched calibration standards, ranging from 0.01 to 1,000 ppb, including 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 50, 100, 200, 500, and 1,000 ppb. The standard parathion-d10 (Agilent QuEChERS IS standard number 6, part number PPS-610-1) was used as the internal standard for quantitation of the target pesticides. It was added at 0.2 μL through reversed sandwich injection with the ALS to a final concentration of 10 ppb in the injected sample. An appropriate calibration function, either linear or quadratic, guided by the lower value of the relative standard error (RSE) was used. A weighting factor of 1/x allowed for maintaining accuracy across the entire calibration range. The deviation of the back-calculated concentrations of the calibration standards from the true concentrations, using the calibration curve in the relevant region, did not exceed ± 20%. The concentrations expressed in ppb (w:v) correspond to the pesticide concentration in the injected sample. The QuEChERS sample preparation procedure described in the Sample preparation section resulted in a dilution factor of 5. Hence, the concentrations measured in the injected samples were five times lower than the corresponding concentration in the black tea sample, expressed in µg/kg. Analyte protectants were not used in this work. The preliminary investigation showed that analyte protectants did not have a response enhancement effect on most of the compounds when analyzed in the rich and complex black tea matrix. It is of note that analyte protectants often significantly enhance target analyte response and stability as is described in-depth in the peer-reviewed literature.6 Recovery evaluation Sample preparation efficiency was evaluated by performing recovery studies. The surrogate black tea matrix was spiked at two different levels, 10 and 50 ppb, with six replicates at each level. The samples were extracted and cleaned up. Assuming 100% recovery, pesticide concentrations in the prespiked samples were expected to be 2 and 10 ppb due to a 5x dilution rate. A blank black tea extract was postspiked with the pesticide standard to achieve final concentrations of 2 and 10 ppb. The prespiked and postspiked samples were analyzed, and the response areas were compared. The recovery was measured as the ratio of the pesticide peak area in the prespiked sample to the area in the postspiked samples. Results and discussion As contemporary GC/MS technology continues to advance, so do the expectations for high sample throughput, intuitive user-friendly system setup and configuration, and streamlined maintenance. The demands for enhanced analytical performance are driven by the evolving regulations in pesticide residue analysis and food safety. Several best practices to achieve the best GC/TQ performance in pesticide residue analyses were described in Agilent application note 5994-4965EN.7 This work presents a complete workflow for analyzing 246 pesticides in black tea, while implementing the previously described best practices and offering further method and technology enhancements. The innovative HES 2.0 yielded enhanced GC/TQ performance stability, as evidenced by precise results at the low concentration of 2 ppb over 800 consecutive injections of complex black tea extract. The technology and method enhancements that enable unparalleled GC/TQ performance while ensuring stable and reliable results in a high-throughput setting are outlined in this application note and grouped into four categories: sample preparation, GC instrumentation and supplies, MS electron ionization technology advancements, and instrument intelligence and software functionality. Effective matrix cleanup Sample preparation is a key component of performing successful pesticide analysis. Performing analysis of samples prepared by QuEChERS extraction, particularly when analyzing complex pigmented samples such as black tea without adequate cleanup, can lead to increased system maintenance. The parts of the system that are affected without adequate sample cleanup include liner replacement, GC column trimming, and inlet and MS source cleaning. As a result, throughput is decreased. Further, the presence of large amounts of matrix can affect the accuracy of results, often most pronounced with difficult to analyze pesticides. The EMR mixed-mode pass‑through cleanup using Captiva EMR with Carbon S cartridges is a simplified procedure that demonstrates an improvement on both sample matrix removal, and overall recovery and reproducibility of targets. As shown in Figure 5, the abundance of the TIC signal in full scan data acquisition mode was noticeably reduced for black tea extract after cleanup when comparing the crude extracts before cleanup. 9 Performing matrix screening in full scan data acquisition mode, as shown in Figure 5, facilitates the evaluation of in-source matrix loading, as discussed in 5994-4965EN.7 Every MS source has a limitation on the amount of material present in the source, at any point in time, to maintain optimal performance. Quantitation accuracy of the analysis can be significantly compromised if the source is overloaded with matrix. Hence, it is essential to analyze the matrix in full scan mode to evaluate the TIC and maintain optimal GC/TQ performance. For best performance with the HES 2.0 source, it is recommended to have a TIC full scan abundance below 7 × 107 counts when analyzing with an EM gain set to 1. As shown in Figure 5, black tea extract is complex, featuring abundant matrix components. Cleaning up the extract is key to lowering the matrix background that leads to adequate in‑source loading, enhancing selectivity and sensitivity, widening the dynamic range, and allowing for less frequent system maintenance, increasing productive uptime. GC instrumentation and supplies Midcolumn backflushing: The Agilent 8890 GC provides an easy-to-use midcolumn backflushing functionality that results in increased sample throughput through shorter analysis times and less frequent column maintenance. Midcolumn backflush allows for the elution of the high boiling point matrix components from the column in a shorter time and without eluting high-boiling matrix into the MS. Midcolumn backflushing is a technique in which the carrier gas flow is reversed after the last analyte has exited the column and all MS data are collected. The oven is then held at the final temperature in postrun mode, with the reversed carrier gas flow through the first column. The high boilers are eluted back out the head of the column and into the split vent trap. The ability to reverse the flow is provided by the PUU. The PUU is a tee that is inserted, in this case, between two identical 15 m columns. During the analysis, a small makeup flow of carrier gas from the 8890 PSD is used to sweep the connection. During backflushing, the Figure 5. Scan TIC of black tea extract. The green trace corresponds to matrix sample with Agilent Captiva-EMR cleanup, and the red trace corresponds to matrix sample without cleanup. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 15 min ×108 Acquisition time (min) Counts Extract before cleanup After cleanup 10 makeup flow from the PSD is raised to a much higher value, sweeping high boilers backward out of the first column while simultaneously providing forward flow in the second column. For the configuration in this application, the backflushing time was 1.5 minutes. More details about using PSD for backflushing in the Agilent 8890 GC system can be found in Agilent application note 5994‑0550EN.8 Figure 6 illustrates the effectiveness of the backflush technique in reducing cycle time without carryover of the black tea matrix. The cycle time was reduced by 50% and the columns did not have to be exposed to the higher bake‑out temperatures for an extended time. Using backflushing, excess column bleed and heavy residues are not introduced into the MSD, thereby reducing ion source contamination. 1 1 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 It took an additional 20 minutes with the columns heated to 310 °C to remove these high boilers Run stopped at 20 minutes and backflushed for 1.5 minutes The following blank has no carryover peaks Run stopped at 20 minutes, no backflushing, no bakeout The following blank has carryover peaks Siloxanes from the septum Siloxanes from the septum Acquisition time (min) Figure 6. TIC Scan chromatograms of black tea extract, followed by analysis of an instrument blank with: column bake-out, with backflush, and without backflush or bake-out. 11 The backflush setup process has been simplified with the introduction of new tools that allow for making a capillary flow technology (CFT) connection with ease. These tools include the gold-plated flexible metal ferrules (part number G2855-28501) and the GC column installation preswaging tool for Flexible Metal ferrules into Capillary Flow Technology devices (part number G3440-80227)—shown in Figure 7. Figure 7. Flexible metal ferrules (part number G2855-28501) (A) and the GC column installation preswaging tool for Flexible Metal ferrules into Capillary Flow Technology devices (part number G3440-80227) (B). A B Additionally, MassHunter Acquisition 13.0 for GC/MS provides intuitive guides for backflushing setup and review. Figure 8 shows the backflush overview tab in the GC Method Editor in MassHunter Acquisition 13.0 for GC/MS. Figure 8. Backflush summary in Agilent MassHunter Acquisition 13.0 for GC/MS. 12 GC injection optimization: Efficiently volatilizing the sample in the GC inlet is an essential component of successful GC/MS analysis. Various sample introduction techniques are aimed at preserving thermally labile and active compounds. In this work, cold splitless and solvent vent injection modes were evaluated. As shown on the left in Figure 9, the use of solvent vent mode for analyzing black tea extract resulted in very large caffeine carryover into the subsequent analyses. To reduce caffeine carryover, cold splitless injection mode was used (Figure 9, at right). Increase of the splitless purge time to 3 minutes resulted in enhanced method sensitivity without deteriorating the chromatographic peak shape for the targets. 0 1 2 3 4 5 0 1 2 3 4 5 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 0 1 2 3 4 5 0 1 2 3 4 5 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Black tea in solvent vent mode Following ACN blank in solvent vent mode Black tea in cold splitless mode Following ACN blank in cold splitless mode 1 2 3 4 8.6 8.7 8.8 8.9 9.0 9.1 Five sequential blanks Caffeine Caffeine Clean blank Same scale ×108 ×108 ×108 Same scale ×108 ×108 Acquisition time (min) Counts Counts Counts Counts Figure 9. Injection optimization for analyzing black tea: cold splitless (on the right) reduces carryover of caffeine compared to solvent vent mode (on the left). 13 HES 2.0: Novel electron ionization (EI) source technology Equipped with the novel HES 2.0 EI source, the 7010D demonstrated sensitivity that allows for ultra-trace level detection when analyzing pesticides. The new HES 2.0 ion source is equipped with a novel dipolar RF lens that redirects the carrier gas ions and, as a result, enables improved system robustness and unparalleled analytical sensitivity. Figure 10 shows MRM chromatograms for selected pesticides at 0.01 ppb in black tea extract. The overlaid chromatograms show repeatability over seven replicate injections, and the response RSD% as a measure of precision. Appendix Table 1 shows the LOQs for all analyzed pesticides. LOQs as low as 0.01 ppb were observed for 34% of the targets, at or below 0.1 ppb for 74% of compounds, and below 2 ppb for 96%. The number of compounds expressed in percent, with their respective LOQs, are plotted in Figure 11. Figure 10. MRM chromatograms with seven replicate injections for the selected pesticides at the LOQ of 0.01 ppb in black tea extract and their calibration curves. Bromophos-ethyl Concentration (ng/mL) 0 200 400 600 800 1,000 Concentration (ng/mL) 0 200 400 600 800 1,000 Concentration (ng/mL) 0 200 400 600 800 1,000 Concentration (ng/mL) 0 200 400 600 800 1,000 Concentration (ng/mL) 0 200 400 600 800 1,000 -0.2 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 y = 23,817.677338x + 874.434909 R2 = 0.99951780 R = 0.99991699 RSE = 9.1 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 11.3 11.4 11.5 11.6 Chlorfenson RSD = 9% 0.01 ppb N = 7 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 10.9 11.0 11.1 11.2 11.3 Bromophos ethyl RSD = 8% 0.01 ppb N = 7 0.01 to 1,000 ppb 0.01 to 1,000 ppb 0.01 to 1,000 ppb 0.01 to 1,000 ppb 0.01 to 1,000 ppb Chlorfenson -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 y = 103,879.642468x + 3,894.601308 R2 = 0.99936894 R = 0.99990218 RSE = 10.2 DDE-o,p' 0 1 2 3 4 5 6 7 8 y = 83,323.889419x + 6,145.523095 R2 = 0.99949459 R = 0.99992324 RSE = 6.4 DDE-p,p' 0 1 2 3 4 5 6 y = 62,964.732203x + 6,196.477029 R2 = 0.99949744 R = 0.99993069 RSE = 6.8 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 11.0 11.2 11.4 11.6 11.8 DDE -o,p' RSD = 5% 0.01 ppb DDE -p,p' RSD = 6% 0.01 ppb N = 7 Chloropropylate 0 1 2 3 4 5 6 7 y = 72,085.499690x + 3,773.703370 R2 = 0.99957751 R = 0.99991508 RSE = 7.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 12.05 12.15 12.25 12.35 Chloropropylate RSD = 12% 0.01 ppb N = 7 ×102 ×103 ×103 ×107 ×107 ×107 ×107 ×103 ×108 Acquisition time (min) Acquisition time (min) Acquisition time (min) Acquisition time (min) Counts Counts Counts Counts Responses Responses Responses Responses Responses 14 Figure 11. Percentage of compounds with their respective LOQ levels (in ppb) in black tea extract. 0% 20% 40% 60% 80% 100% 120% 0.01 0.05 0.1 0.5 1 2 10 Percent of compounds (out of 246) with LOQ at or belo w LOQ (in ppb) Figure 10 also shows the matrix-matched calibration performance in black tea extract with excellent linearity maintained over five orders of magnitude, ranging from 0.01 to 1,000 ppb. All calibration curves were inspected, and if needed, were trimmed to comply with SANTE 11312/2021 guidelines.2 Appendix Table 1 provides information on calibration ranges and quality of calibration fit for all compounds. The R2 correlation coefficient for all targets was > 0.99. The RSE was used as an additional criterion for demonstrating the calibration curve quality. The RSE provides an improved criterion for evaluation of calibration curves, as it is consistent for evaluation of all curve fitting types.9 In this work, the calibration curves for all compounds had RSE values below 20. For the compounds for which quadratic calibration fit was used, a linear calibration curve fit can be used instead by narrowing the calibration range. For example, oxyfluorfen could be calibrated over five orders of magnitude, from 0.01 to 1,000 ppb using quadratic calibration fit with R2 = 0.9995 and RSE = 14. Alternatively, a linear calibration fit could be applied over a calibration range of 0.01 to 500 ppb with R2 = 0.9960 and RSE = 26. The selection of the calibration curve fit was guided by the lower RSE value. Some pesticides are known to present a particular challenge for analysis. As stated in the EURL Analytical Observation Report10, captan and folpet are analytically among the most challenging pesticides due to their nonamenability to LC/TQ, and their tendency to degrade both in solution as well as in the GC inlet. Figure 12 demonstrates that captan and folpet could be quantitated with great precision at LOQs as low as 2 and 0.5 ppb, respectively. Freshly diluted standard, acidified sample extracts, and optimized injection conditions with cold splitless injection were key to achieving high recoveries and precision in analyzing captan and folpet. Deltamethrin, a synthetic pyrethroid, elutes at the end of the chromatographic run and is also known to be difficult for GC/MS analysis.11 As shown in Figure 12, deltamethrin could be reliably quantitated down to 0.5 ppb using the developed method. Other compounds shown in Figure 12 include organochlorine pesticides, aldrin, dieldrin, and endrin, and the two most widely used multipurpose pyrethroids, cypermethrin and cyfluthrin, quantitated with great precision and excellent linearity over a wide dynamic range. 15 Figure 12. MRM chromatograms with eight replicate injections for the selected challenging pesticides. Included are LOQ levels and their calibration curves. 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 9.8 9.9 10.0 Aldrin RSD = 8% 0.5 ppb N = 8 Aldrin 0 1 2 3 4 5 6 7 y = 6,985.401438x + 203.727553 R2 = 0.99952950 R = 0.99993060 RSE = 8.8 0.4 0.5 0.6 0.7 0.8 0.9 1.0 10.6 10.7 10.8 10.9 11.0 Folpet RSD = 7% 0.5 ppb Folpet 0 50 100 150 200 0 1 2 3 4 5 6 y = 2,692.336742x + 830.709066 R2 = 0.99889601 R = 0.99995851 RSE = 16.4 0.5 to 200 ppb 1 to 500 ppb 0.05 to 1,000 ppb 0.5 to 1,000 ppb 0.1 to 500 ppb 0.1 to 1,000 ppb 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 11.6 11.8 12.0 12.2 Endrin RSD = 9% 0.5 ppb Dieldrin RSD = 12% 0.5 ppb 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 10.55 10.65 10.75 10.85 10.95 Captan RSD = 10% 2 ppb N = 8 N = 8 Endrin 0 1 2 3 4 5 6 7 y = 7,758.571223x + 792.052413 R2 = 0.99958608 R = 0.99993430 RSE = 5.5 Captan -1 0 1 2 3 4 5 6 7 y = 1,198.868510x + 256.553484 R2 = 0.99734455 R = 0.99927622 RSE = 18.9 0.50 0.54 0.58 0.62 0.66 0.70 0.74 0.78 0.82 0.86 0.90 0.94 0.98 1.02 18.0 18.1 18.2 18.3 18.4 Deltamethrin RSD = 10% 0.5 ppb N = 8 Deltamethrin 0 100 200 300 400 500 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 y = 1,873.813407x + 335.177249 R2 = 0.99627763 R = 0.99887520 RSE = 17.2 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 5.0 16.1 16.3 16.5 16.7 Cyfluthrins RSD = 6% 1 ppb Cypermethrins RSD = 5% 1 ppb N = 8 Cypermethrin I 0 200 400 600 800 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 y = 12,792.103487x + 12,912.393135 R2 = 0.99849640 R = 0.99974393 RSE = 16.6 Concentration (ng/mL) 0 200 400 600 800 Concentration (ng/mL) 0 200 400 600 800 1,000 1,000 1,000 Concentration (ng/mL) Concentration (ng/mL) Concentration (ng/mL) 0 100 200 300 400 500 Concentration (ng/mL) ×103 ×103 ×103 ×105 ×105 ×106 ×103 ×103 ×103 ×106 ×106 ×107 Acquisition time (min) Acquisition time (min) Acquisition time (min) Acquisition time (min) Acquisition time (min) Acquisition time (min) Counts Counts Counts Counts Counts Counts Responses Responses Responses Responses Responses Responses 16 Recovery and precision To validate the complete workflow solution and ensure that enhanced matrix cleanup did not have a negative impact on pesticide recovery, a study aimed at recovery and precision evaluation was performed. Two concentrations were selected for the study (10 and 50 ng/g) in dry black tea, resulting in 2 and 10 ppb in the final extract due to the 5x dilution factor. Figure 13 shows target results at 10 and 50 ng/g in black tea, demonstrating the acceptable recoveries achieved for the majority of pesticides, even for the common problematic pesticides such as planar and labile. Longevity and maximized throughput with confidence The ruggedness of the analysis was demonstrated by analyzing a challenging black tea extract spiked with pesticides at 2 ppb. The area of the analyte response was monitored over 800 consecutive injections. Analyte response, normalized by the internal standards (ISTD), remained consistent over 800 injections that spanned over 400 hours of continuous running with RSDs < 20% for 176 compounds. Figure 14 shows the response for 60 compounds, normalized by the ISTD and by the average response for each analyte. 0% 20% 40% 60% 80% 100% 120% 140% 160% 180% Dichlorvos Dichlorobenzonitrile, 2,6- Biphenyl Mevinphos, EChlormephos Propham Pebulate Etridiazole Nitrapyrin cis-1,2,3,6-Tetrahydrophthalimide Methacrifos Chloroneb Crimidine 2-Phenylphenol Isoprocarb I Pentachlorobenzene Heptenophos DEET Chlorfenprop-methyl Omethoate Thionazin Flonicamid Propachlor Ethoprophos Cycloate Chlorpropham Ethalfluralin DMSA Trifluralin Benfluralin Monocrotophos Dicrotofos Sulfotep Bromoxynil Promecarb Cadusafos Phorate BHC-alpha (benzene hexachloride) Desmedipham Hexachlorobenzene Dichloran Dimethoate Pentachloroanisole Propazine BHC-beta DMST (Tolylfluanid metabolite) Propetamphos Profluralin BHC-gamma (Lindane, gamma HCH) Cyanophos Terbufos Pentachloronitrobenzene Fonofos Diazinon Pyrimethanil Fluchloralin Phosphamidon I Dinitramine Tefluthrin Paraoxon-methyl BHC-delta Isazofos Etrimfos Triallate Chlorothalonil Iprobenfos Formothion Bromocyclen Pentachloroaniline Desmetryn Dichlofenthion Propanil 2,4,4'-Trichlorobiphenyl (BZ #28) Malaoxon Vinclozolin Transfluthrin Parathion-methyl Chlorpyrifos-methyl Cymiazole Tolclofos-methyl Alachlor Fuberidazole Heptachlor Prometryn Paraoxon Ronnel Prosulfocarb Octachlorodipropyl ether Pirimiphos-methyl 2,2',5,5'-Tetrachlorobiphenyl (BZ #52) Fenitrothion Methiocarb Dipropetryn Malathion Ioxynil Dichlofluanid Parathion-d10 Metolachlor Phorate sulfone Aldrin Anthraquinone Chlorpyrifos Parathion Flufenacet Nitrothal-isopropyl DCPA (Dacthal, Chlorthal-dimethyl) Isocarbophos Chlorthion Isobenzan Trichloronat Fenson Bromophos Pirimiphos-ethyl Fosthiazate I Isopropalin Cyprodinil Isofenphos-methyl Isodrin Pendimethalin Terbufos sulfone Chlozolinate Heptachlor exo-epoxide Esbiothrin Bioallethrin Chlordane-oxy Tolylfluanid Isofenphos Mecarbam Chlorfenvinphos Heptachlor endo-epoxide Fipronil Captan Quinalphos Phenthoate Dinobuton Procymidone Folpet Chlorbenside Methidathion Bromophos-ethyl Chlordane-trans DDE-o,p' 2,2',4,5,5'-Pentachlorobiphenyl (BZ #101) Tetrachlorvinphos Chlordane-cis Endosulfan I (alpha isomer) Ditalimfos Picoxystrobin Flutriafol Fenamiphos Nonachlor, transChlorfenson Iodofenphos Prothiofos Isoprothiolane Flubenzimine Profenofos DDE-p,p' Dieldrin Oxyfluorfen Myclobutanil DDD-o,p' Methoprotryne Azaconazole Dibromobenzophenone, 4,4'- Isoxathion Binapacryl Nitrofen Ethylan Chlorfenapyr Endrin Carbophenothion-Methyl Chloropropylate 2,3',4,4',5-Pentachlorobiphenyl (BZ #118) Endosulfan II (beta isomer) Fensulfothion Flamprop-isopropyl DDD-p,p' Aclonifen DDT-o,p' Ethion Chlorthiophos Tetrasul 2,2',4,4',5,5'-Hexachlorobiphenyl (BZ #153) Sulprofos Triazophos Famphur Carbophenothion Methoxychlor olefin Cyanofenphos Edifenphos DDT-p,p' Endosulfan sulfate 2,2',3,4,4',5'-Hexachlorobiphenyl (BZ #138) Diclofop-methyl Diflufenican Propargite Piperonyl butoxide Captafol Nitralin Mefenpyr-diethyl Benzoylprop-ethyl Iprodione Spiromesifen Tetramethrin I Pyridaphenthion Endrin ketone Dimoxystrobin Phosmet Bifenthrin Bromopropylate EPN Picolinafen Dicofol, p, p'- Fenpropathrin 2,2',3,4,4',5,5'-Heptachlorobiphenyl (BZ #180) Phenothrin I Tetradifon Furathiocarb Phosalone Azinphos-methyl Leptophos Cyhalothrin (Lambda) Cyhalofop-butyl Tralkoxydim Mirex Acrinathrin Pyrazophos Azinphos-ethyl Cycloxydim (Focus) Permethrin, (1R)-cisPermethrin, (1R)-transCoumaphos Dioxathion Butafenacil Cyfluthrin I Cypermethrin I Halfenprox Flucythrinate I Ethofenprox Silafluofen Fenvalerate I Fluvalinate-tau I Deltamethrin 10 ppb in tea → 2 ppb in the extract 50 ppb in tea → 10 ppb in the extract Figure 13. Pesticide recoveries in black tea at 10 and 50 ppb shown for all 244 pesticides. Figure 14. Stability of the peak area for pesticides spiked at 2 ppb into black tea extract, normalized by the ISTD and the average response, over 800 consecutive injections with the Agilent 8890 GC and 7010D GC/TQ systems. 100 200 300 400 500 600 700 BHC-alpha (benzene hexachloride) 11% Coumaphos 15% Fenitrothion 11% Chlorpyrifos 11% Ethalfluralin 10% Esbiothrin 9% Propazine 10% Tefluthrin 10% 2,2',4,4',5,5'-Hexachlorobiphenyl (BZ #153) 9% Aldrin 12% Myclobutanil 11% Chlordane-oxy 11% BHC-beta 10% DDD-o,p' 10% Fenpropathrin 11% Parathion 7% DMSA 10% Bioallethrin 9% Diazinon 10% Iprobenfos 9% 2,2',3,4,4',5'-Hexachlorobiphenyl (BZ #138) 9% Dieldrin 10% Nonachlor, trans- 10% Chlorfenvinphos 11% Bromophos 10% DDT-p,p' 9% Fonofos 12% Fenson 9% Trifluralin DCPA (Dacthal, Chlorthal-dimethyl) 11% Pyrimethanil 10% 2,2',5,5'-Tetrachlorobiphenyl (BZ #52) 10% 2,2',3,4,4',5,5'-Heptachlorobiphenyl (BZ #180) 9% Endrin 15% Chlorfenson 10% Heptachlor endo-epoxide 10% Prothiofos 10% DDE-o,p' 8% Piperonyl butoxide 11% EPN 11% Benfluralin 10% Metolachlor 9% Fluchloralin 8% 2,2',4,5,5'-Pentachlorobiphenyl (BZ #101) 9% Bifenthrin 11% Endrin ketone 11% Chlordane-cis 10% Heptachlor exo-epoxide 10% Chlorthiophos 11% DDE-p,p' 9% Sulfotep 12% Pentachloronitrobenzene 11% Profluralin 9% Pirimiphos-ethyl 10% Dinitramine 11% 2,3',4,4',5-Pentachlorobiphenyl (BZ #118) 9% Chloropropylate 8% Nitrofen 10% Chlordane-trans 10% Deltamethrin 25% Column trim Replaced syringe 0% 20% 40% 60% 80% 100% 120% 140% 160% 180% 200% 17 The graph shows that analyte responses were stable and within 80 to 120% throughout the entire study that lasted over 17 days of continuous analysis. The RSDs for each of the target responses are shown in the legend in Figure 14, with most of them below 12%. The absolute responses in terms of peak areas also remained consistent throughout the longevity study. For example, the RSDs on the peak areas over 800 injections for early-eluting BHC-beta, mid-range eluting fenson, and late-eluting coumaphos were 9%, 10%, and 16%, respectively. The maintenance performed during the robustness testing involved septum and liner replacement every 100 injections. With the midcolumn backflush configuration and the use of the temperature-programmed MMI inlet, inlet liner and septum replacement could be performed in under four minutes, providing a productivity boost to the workflow. Two inches of the GC column head were trimmed after 500 injections. The use of the backflushing allowed for a substantial increase in the number of injections before column head maintenance was needed when analyzing a complex black tea extract. Similar to GC inlet maintenance, column trimming could be performed efficiently in a short amount of time (5 to 10 minutes) and did not require MS cool-down and venting due to the midcolumn configuration coupled with the temperature-programmed MMI. The autoinjector syringe was replaced after 600 injections in the longevity study, as noted in Figure 14, with a total of 1,000 injections made with the syringe. The decision to replace the syringe was driven by the decreased measurement precision resulting from a higher variability in the target and ISTD responses. The replacement procedure was performed as guided by the user manual for the 7693A ALS.12 The precision of measurements was restored after the syringe replacement. As a result, the graph in Figure 14 shows an increased response variability between 500 and 600 injections. This effect was particularly pronounced for deltamethrin, which can present a challenge for achieving good precision at low concentrations. Washing the syringe needle support foot is an additional autoinjector maintenance procedure to consider when analyzing challenging samples to minimize carryover and ensure precision. It is of note that there was no need to perform GC inlet or MS source cleaning during the entire study, which spanned over 1,000 injections, including the calibration assessment and precision and recovery studies. The exceptional method ruggedness shown in this work was achieved by: – Following the key practices to successful pesticide analysis outlined in this application note and in another application note7 – Performing effective sample preparation and cleanup – Using the state-of-the art GC and MS technology in the 8890 and 7010D GC/TQ systems GC/TQ intelligence and new software functionality The health and status of the GC/TQ system was continuously monitored though the longevity study by using the Early Maintenance Feedback functionality in MassHunter Acquisition 13.0 for GC/MS. Figure 15A shows a screenshot of the MS health status, featuring the electron multiplier (EM) voltage at last tune, filament age, pump maintenance schedule, and time since the source was cleaned. The dashboard allows for daily tracking for essential maintenance procedures and reminds the user to perform maintenance on time. In addition to the dashboard view, the built-in intelligent functionality in the 7010D GC/TQ system allows for plotting tune-related parameters over time to track the EI source health and performance. The plot for the EM voltage is shown in Figure 15B. The routine tune check procedures, which could be built into the sequence table via keywords, are helpful in guiding when the EM gain curve needs to be updated. This procedure allows for adjusting the EM voltage to maintain a stable response while not altering tune parameters and ion ratio, maintaining MS tune and method calibration validity. 18 A B Consider maintenance EMV monitored over a five-month duration of experiment preparation and execution Figure 15. The early maintenance dashboard for GC/TQ (A) and the EM voltage plot for the tune history (B) shown in Agilent MassHunter Acquisition 13.0 for GC/MS. 19 Conclusion This application note presents a workflow solution for analyzing pesticides in black tea with the new 7010D GC/TQ, allowing for the quantitation of 246 pesticide residues at trace levels with LOQs as low as 0.01 ppb for 34% of the targets, at or below 0.1 ppb for 74% of compounds, and below 2 ppb for 96%. Matrix-matched calibration allowed for excellent accuracy over a wide dynamic range, spanning up to five orders of magnitude over the 0.01 to 1,000 ppb range in a complex black tea extract. Method ruggedness was demonstrated through maintaining measurement accuracy with good precision (RSDs < 20% for 176 compounds) for black tea extract spiked at 2 ppb sequentially analyzed over 800 runs and spanning over 17 days of continuous analysis. The key components for a robust workflow included a combination of efficient sample preparation and cleanup, the Agilent 8890 GC hardware, functionality, and GC supplies, the novel EI source technology with HES 2.0, and lastly, the built‑in GC/TQ intelligence and new software functionality. References 1. Mehri, A.; Taleb, R.; Elaridi, J.; Hassan, H. F. Analytical Methods Used to Determine Pesticide Residues in Tea: A Systematic Review. Appl. Food Res. 2022, 2(1), 100131. 2. Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed. SANTE 11312/2021, 2021. 3. Tolerances and Exemptions for Pesticide Chemical Residues in Food. Title 40 U.S. Code of Federal Regulations, US EPA. 4. Lozano, A.; Rajski, L.; Belmonte-Valles N.; Uclés, A.; Uclés, S.; Mezcua, M.; Fernández-Alba, A. Pesticide Analysis in Teas and Chamomile by Liquid Chromatography and Gas Chromatography Tandem Mass Spectrometry Using a Modified QuEChERS Method: Validation and Pilot Survey in Real Samples. J. Chrom. A 2012, 1268, 109–122. 5. The Agilent MassHunter Pesticide and Environmental Pollutants MRM Database (P&EP 4.0). G9250AA. https:// www.agilent.com/en/product/gas-chromatographymass-spectrometry-gc-ms/gc-ms-application-solutions/ pesticides-environmental-pollutants-4-0-mrm-database 6. Maštovská, K.; Lehotay, S. J.; Anastassiades, M. Combination of Analyte Protectants to Overcome Matrix Effects in Routine GC Analysis of Pesticide Residues in Food Matrixes. Anal. Chem. 2005, 77, 8129–8137 7. Five Keys to Unlock Maximum Performance in the Analysis of Over 200 Pesticides in Challenging Food Matrices by GC/MS/MS. Agilent Technologies application note 5994-4965EN, 2022. 8. Using the PSD for Backflushing on the Agilent 8890 GC System. Agilent Technologies application note, publication number 5994 0550EN, 2018. 9. Burrows, R. Parr, R. Evaluating the Goodness of Instrument Calibration for Chromatography Procedures. LCGC Supplements Special Issues 2020 11-01-20, 38(11), 35–38. 10. EURL-SRM – Analytical Observation Report. Quantification of Residues of Folpet and Captan in QuEChERS Extracts Version 3.1 (last update: 06.04.17). 11. Kim, L.; Baek, S.; Son, K.; Kim, E.; Noh, H. H.; Kim, D.; Oh, M.; Moon, B.; Ro, J.-H. Optimization of a Simplified and Effective Analytical Method of Pesticide Residues in Mealworms (Tenebrio molitor Larvae) Combined with GC–MS/MS and LC–MS/MS. Mol. 2020, 25(15), 3518. 12. Agilent 7693A Automatic Liquid Sampler. Installation, Operation and Maintenance. Agilent Technologies, 2023. 20 Name RT Transition Calibration Range (ppb) CF CF R2 Relative Standard Error Methamidophos 4.520 141.0 & 64.0 0.1 – 1,000 Linear 0.9994 7.5 Dichlorvos 4.643 184.9 & 93.0 0.05 – 1,000 Linear 0.9988 11.2 Dichlorobenzonitrile, 2,6- 5.210 171.0 & 100.0 0.01 – 1,000 Linear 0.9990 10.4 Biphenyl 5.390 154.1 & 153.1 0.5 – 1,000 Quadratic 0.9991 11.8 Mevinphos, E- 5.578 127.0 & 94.9 0.5 – 1,000 Linear 0.9994 7.9 Acephate 5.679 136.0 & 94.0 5 – 1,000 Quadratic 0.9990 8.1 Chlormephos 5.687 153.9 & 121.1 0.5 – 1,000 Linear 0.9979 5.0 Propham 5.740 178.9 & 137.1 1 – 1,000 Linear 0.9986 9.6 Pebulate 5.774 128.0 & 57.1 0.5 – 1,000 Linear 0.9983 6.6 Etridiazole 5.798 213.1 & 185.0 0.1 – 500 Linear 0.9986 10.7 Nitrapyrin 5.804 194.0 & 158.0 0.05 – 1,000 Quadratic 0.9994 8.7 cis-1,2,3,6-Tetrahydrophthalimide 5.956 151.1 & 80.0 0.1 – 1,000 Linear 0.9994 6.1 Methacrifos 6.027 207.9 & 180.1 0.05 – 1,000 Quadratic 0.9996 11.4 Chloroneb 6.110 191.0 & 113.0 0.01 – 1,000 Linear 0.9995 8.2 Crimidine 6.212 170.9 & 142.1 0.1 – 1,000 Linear 0.9994 10.4 2-Phenylphenol 6.213 169.1 & 115.1 0.5 – 1,000 Linear 0.9995 5.6 Isoprocarb I 6.295 136.0 & 121.1 0.5 – 1,000 Linear 0.9994 9.3 Pentachlorobenzene 6.311 251.9 & 217.0 0.01 – 1,000 Linear 0.9984 11.4 Heptenophos 6.585 124.0 & 89.0 0.01 – 500 Linear 0.9990 12.5 DEET 6.600 191.0 & 190.0 0.5 – 1,000 Linear 0.9981 11.5 Chlorfenprop-methyl 6.696 165.0 & 102.0 0.01 – 1,000 Linear 0.9989 14.0 Omethoate 6.773 110.0 & 47.0 0.1 – 1,000 Quadratic 0.9997 8.8 Thionazin 6.781 143.0 & 79.0 0.1 – 1,000 Quadratic 0.9998 9.5 Flonicamid 6.859 173.9 & 68.9 0.01 – 1,000 Linear 0.9994 11.5 Propachlor 6.865 176.1 & 57.1 0.05 – 1,000 Quadratic 0.9997 8.5 Ethoprophos 6.996 157.9 & 97.0 0.1 – 1,000 Quadratic 0.9997 8.2 Cycloate 7.017 154.1 & 83.1 0.05 – 1,000 Linear 0.9991 16.2 Chlorpropham 7.080 171.0 & 127.1 0.05 – 500 Linear 0.9993 13.7 Ethalfluralin 7.109 275.9 & 202.1 0.05 – 1,000 Quadratic 0.9997 11.4 DMSA 7.169 200.0 & 108.0 2 – 1,000 Quadratic 0.9963 17.5 Trifluralin 7.217 306.1 & 264.0 0.05 – 1,000 Quadratic 0.9997 11.8 Benfluralin 7.251 292.0 & 264.0 0.1 – 1,000 Quadratic 0.9996 12.0 Monocrotophos 7.258 192.0 & 127.0 0.1 – 1,000 Quadratic 0.9998 7.4 Dicrotofos 7.264 193.0 & 127.1 0.5 – 1,000 Quadratic 0.9998 10.1 Sulfotep 7.349 321.8 & 201.9 0.05 – 1,000 Quadratic 0.9997 10.4 Bromoxynil 7.395 276.8 & 88.0 0.05 – 1,000 Quadratic 0.9997 7.1 Promecarb 7.399 135.1 & 115.1 2 – 1,000 Linear 0.9967 16.1 Cadusafos 7.405 158.8 & 97.0 0.01 – 1,000 Quadratic 0.9997 13.1 Phorate 7.475 121.0 & 47.0 0.5 – 1,000 Linear 0.9983 11.1 BHC-alpha (Benzene Hexachloride) 7.609 218.9 & 183.0 0.01 – 1,000 Quadratic 0.9998 9.9 Desmedipham 7.690 181.0 & 122.0 2 – 1,000 Linear 0.9985 11.8 Hexachlorobenzene 7.741 283.8 & 213.9 0.01 – 1,000 Quadratic 0.9996 10.7 Dichloran 7.771 160.1 & 124.1 0.01 – 1,000 Linear 0.9996 6.7 Dimethoate 7.781 87.0 & 46.0 0.01 – 1,000 Linear 0.9997 11.8 Pentachloroanisole 7.797 279.9 & 236.8 0.05 – 1,000 Linear 0.9993 8.0 Appendix Table 1. Calibration performance for 246 pesticides in black tea using the Agilent 7010D GC/TQ equipped with the Agilent High-Efficiency Source (HES) 2.0. 21 Name RT Transition Calibration Range (ppb) CF CF R2 Relative Standard Error Propazine 7.933 229.1 & 58.1 0.01 – 1,000 Linear 0.9988 13.5 BHC-beta 8.010 218.9 & 183.1 0.01 – 1,000 Linear 0.9994 12.2 DMST (Tolylfluanid Metabolite) 8.032 214.0 & 106.0 2 – 1,000 Quadratic 0.9954 17.7 Propetamphos 8.079 138.0 & 64.0 0.1 – 1,000 Linear 0.9988 6.9 Profluralin 8.087 318.1 & 199.1 0.05 – 1,000 Quadratic 0.9997 12.7 BHC-gamma (Lindane, Gamma HCH) 8.119 216.9 & 181.0 0.01 – 1,000 Linear 0.9981 15.6 Cyanophos 8.135 242.9 & 109.0 0.05 – 1,000 Linear 0.9993 12.8 Terbufos 8.137 230.9 & 129.0 0.1 – 1,000 Linear 0.9994 12.5 Pentachloronitrobenzene 8.195 141.9 & 106.9 0.01 – 1,000 Quadratic 0.9996 9.4 Fonofos 8.223 246.1 & 109.0 0.01 – 500 Linear 0.9981 11.9 Diazinon 8.264 137.1 & 84.0 0.05 – 1,000 Quadratic 0.9997 17.0 Pyrimethanil 8.269 198.0 & 118.1 0.01 – 500 Linear 0.9990 12.6 Fluchloralin 8.299 325.8 & 62.9 0.05 – 500 Quadratic 0.9995 13.5 Phosphamidon I 8.339 127.0 & 95.0 0.5 – 500 Linear 0.9981 17.4 Dinitramine 8.382 260.7 & 241.0 0.05 – 1,000 Quadratic 0.9997 11.6 Tefluthrin 8.400 177.1 & 127.1 0.01 – 1,000 Linear 0.9986 16.0 Paraoxon-methyl 8.411 229.9 & 106.1 0.05 – 500 Linear 0.9947 18.3 BHC-delta 8.489 219.0 & 183.1 0.5 – 1,000 Quadratic 0.9998 16.5 Isazofos 8.504 256.9 & 162.0 0.01 – 1,000 Quadratic 0.9997 13.8 Etrimfos 8.523 292.0 & 153.1 0.01 – 500 Linear 0.9985 16.7 Triallate 8.540 268.0 & 184.1 0.05 – 1,000 Linear 0.9995 10.8 Chlorothalonil 8.568 265.9 & 168.0 0.1 – 500 Quadratic 0.9971 10.7 Iprobenfos 8.673 203.9 & 91.0 0.01 – 1,000 Linear 0.9986 13.9 Formothion 8.763 124.9 & 47.0 0.1 – 1,000 Quadratic 0.9997 16.0 Bromocyclen 8.764 271.8 & 236.9 0.1 – 1,000 Linear 0.9996 8.6 Pentachloroaniline 8.897 158.0 & 123.0 0.5 – 1,000 Linear 0.9986 14.7 Desmetryn 8.916 213.0 & 58.1 0.05 – 1,000 Linear 0.9972 11.7 Dichlofenthion 8.961 279.0 & 223.0 0.01 – 500 Linear 0.9975 11.4 Propanil 8.980 161.0 & 99.0 0.01 – 1,000 Linear 0.9983 16.8 2,4,4'-Trichlorobiphenyl (BZ #28) 9.030 256.0 & 186.0 0.05 – 1,000 Linear 0.9981 11.7 Malaoxon 9.103 126.9 & 99.0 2 – 1,000 Quadratic 0.9989 15.0 Vinclozolin 9.128 187.0 & 124.0 0.05 – 1,000 Linear 0.9962 15.8 Transfluthrin 9.129 163.1 & 143.1 0.1 – 1,000 Linear 0.9972 14.3 Parathion-methyl 9.151 262.9 & 109.0 0.5 – 500 Quadratic 0.9991 18.2 Chlorpyrifos-methyl 9.151 288.0 & 93.0 0.05 – 1,000 Quadratic 0.9995 12.0 Cymiazole 9.213 218.0 & 144.1 2 – 1,000 Linear 0.9986 12.3 Tolclofos-methyl 9.242 267.0 & 93.0 0.5 – 1,000 Linear 0.9987 12.8 Alachlor 9.280 237.0 & 160.1 1 – 1,000 Linear 0.9969 12.2 Fuberidazole 9.306 184.0 & 156.2 5 – 500 Linear 0.9921 19.5 Heptachlor 9.330 271.7 & 236.9 0.1 – 200 Linear 0.9990 15.4 Prometryn 9.339 241.0 & 58.2 5 – 200 Quadratic 0.9967 19.4 Paraoxon 9.383 148.9 & 119.0 50 – 1,000 Quadratic 0.9982 11.3 Ronnel 9.411 286.9 & 272.0 1 – 1,000 Linear 0.9989 11.7 Prosulfocarb 9.424 251.0 & 128.2 0.1 – 1,000 Quadratic 0.9995 14.9 Octachlorodipropyl Ether 9.431 129.9 & 94.9 0.5 – 1,000 Quadratic 0.9996 10.0 Pirimiphos-methyl 9.610 290.0 & 125.0 0.01 – 1,000 Quadratic 0.9996 9.3 22 Name RT Transition Calibration Range (ppb) CF CF R2 Relative Standard Error 2,2',5,5'-Tetrachlorobiphenyl (BZ #52) 9.617 289.9 & 219.9 0.01 – 1,000 Linear 0.9997 11.9 Fenitrothion 9.622 125.1 & 47.0 0.01 – 1,000 Linear 0.9994 11.3 Methiocarb 9.628 168.0 & 109.1 2 – 1,000 Quadratic 0.9998 11.3 Dipropetryn 9.748 255.1 & 222.1 0.01 – 500 Linear 0.9982 13.5 Malathion 9.759 172.9 & 99.0 0.01 – 1,000 Quadratic 0.9997 12.1 Ioxynil 9.780 370.8 & 117.0 0.05 – 1,000 Linear 0.9982 12.2 Dichlofluanid 9.785 167.0 & 97.0 1 – 500 Quadratic 0.9991 13.8 Metolachlor 9.913 238.0 & 162.2 0.01 – 1,000 Linear 0.9993 12.5 Phorate Sulfone 9.914 199.0 & 97.0 0.1 – 500 Linear 0.9975 11.7 Aldrin 9.932 254.9 & 220.0 0.05 – 1,000 Linear 0.9995 8.8 Anthraquinone 9.941 208.0 & 152.2 0.05 – 1,000 Linear 0.9990 19.1 Chlorpyrifos 9.968 313.8 & 257.8 0.05 – 1,000 Linear 0.9994 11.7 Parathion 9.983 291.0 & 109.0 0.01 – 1,000 Linear 0.9985 15.2 Flufenacet 10.004 151.0 & 95.0 0.5 – 1,000 Linear 0.9994 11.9 Nitrothal-isopropyl 10.057 254.0 & 212.0 0.5 – 500 Linear 0.9967 16.3 DCPA (Dacthal, Chlorthal-dimethyl) 10.068 298.9 & 221.0 0.01 – 1,000 Linear 0.9993 14.1 Isocarbophos 10.114 136.0 & 69.0 0.5 – 1,000 Linear 0.9985 13.1 Chlorthion 10.156 125.1 & 47.1 0.05 – 1,000 Quadratic 0.9995 14.7 Isobenzan 10.186 274.7 & 240.0 0.1 – 1,000 Linear 0.9992 11.2 Trichloronat 10.199 296.8 & 268.9 0.01 – 1,000 Linear 0.9987 14.5 Fenson 10.210 141.0 & 77.1 0.01 – 1,000 Linear 0.9996 6.7 Bromophos 10.294 330.9 & 315.9 0.01 – 1,000 Linear 0.9986 15.8 Pirimiphos-ethyl 10.294 318.1 & 166.1 0.05 – 1,000 Linear 0.9977 14.8 Fosthiazate I 10.299 195.0 & 103.0 0.05 – 1,000 Quadratic 0.9997 12.1 Isopropalin 10.350 280.1 & 238.1 0.01 – 500 Linear 0.9975 16.4 Cyprodinil 10.413 225.2 & 224.3 0.05 – 1,000 Linear 0.9990 14.4 Isofenphos-methyl 10.420 199.0 & 121.0 0.01 – 1,000 Quadratic 0.9995 9.9 Isodrin 10.442 193.0 & 123.0 0.05 – 1,000 Linear 0.9985 10.9 Pendimethalin 10.522 251.8 & 162.2 0.05 – 1,000 Quadratic 0.9995 11.5 Terbufos Sulfone 10.573 264.0 & 199.0 0.05 – 1,000 Quadratic 0.9996 8.7 Chlozolinate 10.586 186.0 & 109.0 0.1 – 1,000 Quadratic 0.9995 8.6 Heptachlor Exo-epoxide 10.616 354.8 & 264.9 0.01 – 1,000 Linear 0.9993 15.3 Esbiothrin 10.622 123.0 & 93.0 2 – 1,000 Linear 0.9975 15.2 Bioallethrin 10.629 123.0 & 81.0 2 – 1,000 Linear 0.9924 12.2 Chlordane-oxy 10.629 114.9 & 51.1 0.5 – 1,000 Linear 0.9996 3.8 Tolylfluanid 10.639 238.0 & 137.0 0.05 – 1,000 Quadratic 0.9996 14.6 Isofenphos 10.674 212.9 & 121.1 0.01 – 1,000 Quadratic 0.9994 10.7 Mecarbam 10.675 130.9 & 86.0 0.5 – 1,000 Quadratic 0.9992 13.0 Chlorfenvinphos 10.679 266.9 & 159.0 0.01 – 200 Linear 0.9986 11.0 Heptachlor Endo-epoxide 10.683 135.0 & 99.0 0.5 – 1,000 Linear 0.9993 6.6 Fipronil 10.698 366.8 & 212.8 0.05 – 1,000 Quadratic 0.9997 8.8 Captan 10.738 149.0 & 70.0 1 – 500 Linear 0.9973 18.9 Quinalphos 10.738 298.0 & 156.0 0.01 – 500 Quadratic 0.9992 17.6 Phenthoate 10.741 274.0 & 125.0 0.01 – 1,000 Quadratic 0.9988 8.1 Dinobuton 10.743 211.0 & 163.0 0.5 – 1,000 Quadratic 0.9999 9.0 Procymidone 10.850 282.8 & 96.0 0.01 – 1,000 Linear 0.9996 8.3 23 Name RT Transition Calibration Range (ppb) CF CF R2 Relative Standard Error Folpet 10.851 259.8 & 130.1 0.5 – 200 Linear 0.9989 16.4 Chlorbenside 10.904 125.0 & 89.0 0.01 – 1,000 Linear 0.9993 8.2 Methidathion 11.007 125.0 & 47.0 0.5 – 1,000 Linear 0.9993 16.2 Bromophos-ethyl 11.022 358.7 & 302.8 0.01 – 1,000 Linear 0.9995 9.1 Chlordane-trans 11.024 271.7 & 236.9 0.05 – 1,000 Linear 0.9995 8.4 DDE-o,p' 11.073 246.0 & 176.2 0.01 – 1,000 Linear 0.9995 6.4 2,2',4,5,5'-Pentachlorobiphenyl (BZ #101) 11.111 325.9 & 255.9 0.01 – 1,000 Linear 0.9995 7.7 Tetrachlorvinphos 11.166 329.0 & 108.9 0.01 – 1,000 Linear 0.9972 16.9 Chlordane-cis 11.288 372.8 & 265.9 0.01 – 1,000 Linear 0.9994 15.4 Endosulfan I (Alpha Isomer) 11.290 194.9 & 160.0 0.1 – 1,000 Linear 0.9993 14.8 Ditalimfos 11.299 242.9 & 148.1 0.05 – 500 Linear 0.9977 14.8 Picoxystrobin 11.307 145.0 & 102.1 0.05 – 1,000 Linear 0.9994 14.1 Flutriafol 11.335 123.1 & 75.1 0.05 – 1,000 Linear 0.9995 11.9 Fenamiphos 11.360 303.0 & 154.0 0.1 – 500 Linear 0.9959 17.1 Nonachlor, trans- 11.369 406.8 & 299.8 0.05 – 1,000 Linear 0.9993 11.9 Chlorfenson 11.374 175.0 & 111.0 0.01 – 1,000 Linear 0.9994 10.2 Iodofenphos 11.466 376.8 & 361.8 0.05 – 1,000 Quadratic 0.9998 12.4 Prothiofos 11.488 308.9 & 238.9 0.05 – 500 Linear 0.9975 16.8 Isoprothiolane 11.498 162.1 & 85.0 0.01 – 1,000 Linear 0.9986 13.4 Flubenzimine 11.538 186.0 & 69.0 2 – 500 Quadratic 0.9974 10.5 Profenofos 11.544 207.9 & 63.0 0.1 – 200 Linear 0.9984 12.4 DDE-p,p' 11.613 246.1 & 176.2 0.01 – 1,000 Linear 0.9995 6.8 Dieldrin 11.713 262.9 & 193.0 0.5 – 1,000 Linear 0.9990 13.8 Oxyfluorfen 11.721 252.0 & 146.0 0.01 – 1,000 Quadratic 0.9995 14.0 Myclobutanil 11.750 179.0 & 125.1 0.01 – 1,000 Quadratic 0.9995 12.6 DDD-o,p' 11.783 235.0 & 165.1 0.01 – 1,000 Linear 0.9993 12.7 Methoprotryne 11.788 256.0 & 212.1 0.01 – 1,000 Quadratic 0.9996 12.6 Azaconazole 11.865 217.0 & 173.1 0.01 – 1,000 Linear 0.9994 11.4 Dibromobenzophenone, 4,4'- 11.913 340.0 & 183.0 0.05 – 1,000 Quadratic 0.9997 7.6 Isoxathion 11.941 313.0 & 177.0 0.05 – 1,000 Quadratic 0.9993 14.9 Binapacryl 12.003 100.0 & 82.0 5 – 1,000 Quadratic 0.9988 18.9 Nitrofen 12.011 282.9 & 253.0 0.01 – 500 Linear 0.9963 14.5 Ethylan 12.041 223.1 & 193.1 0.05 – 500 Linear 0.9976 14.7 Chlorfenapyr 12.051 328.0 & 247.0 0.5 – 1,000 Linear 0.9983 14.8 Endrin 12.108 262.8 & 193.0 0.5 – 1,000 Linear 0.9996 5.5 Carbophenothion-methyl 12.167 125.0 & 47.0 0.1 – 1,000 Linear 0.9986 19.2 Chloropropylate 12.187 139.0 & 75.0 0.01 – 1,000 Linear 0.9996 7.2 2,3',4,4',5-Pentachlorobiphenyl (BZ #118) 12.222 325.9 & 255.9 0.01 – 1,000 Linear 0.9996 7.6 Endosulfan II (Beta Isomer) 12.274 206.9 & 172.0 0.1 – 1,000 Quadratic 0.9989 13.0 Fensulfothion 12.284 293.0 & 97.0 0.01 – 1,000 Linear 0.9978 14.8 Flamprop-isopropyl 12.305 276.0 & 105.1 0.05 – 1,000 Linear 0.9991 10.4 DDD-p,p' 12.369 237.0 & 165.1 0.01 – 1,000 Linear 0.9993 13.7 Aclonifen 12.397 264.1 & 194.2 0.1 – 1,000 Quadratic 0.9995 10.3 DDT-o,p' 12.430 235.0 & 199.1 0.01 – 1,000 Linear 0.9974 19.4 Ethion 12.431 231.0 & 129.0 0.01 – 1,000 Linear 0.9962 19.9 24 Name RT Transition Calibration Range (ppb) CF CF R2 Relative Standard Error Chlorthiophos 12.484 268.9 & 205.1 0.05 – 1,000 Linear 0.9983 13.9 Tetrasul 12.572 321.7 & 252.0 0.01 – 1,000 Linear 0.9987 11.5 2,2',4,4',5,5'-Hexachlorobiphenyl (BZ #153) 12.610 359.9 & 289.9 0.01 – 1,000 Linear 0.9992 10.9 Sulprofos 12.650 322.0 & 156.0 0.01 – 500 Linear 0.9975 12.5 Triazophos 12.662 161.2 & 134.2 1 – 500 Quadratic 0.9995 15.0 Famphur 12.810 218.0 & 109.0 2 – 1,000 Linear 0.9982 14.6 Carbophenothion 12.826 342.0 & 157.0 0.05 – 500 Linear 0.9974 14.5 Methoxychlor Olefin 12.837 308.0 & 238.0 0.01 – 1,000 Linear 0.9984 13.5 Cyanofenphos 12.906 169.0 & 77.1 0.1 – 1,000 Linear 0.9988 10.7 Edifenphos 12.940 309.9 & 172.9 0.5 – 1,000 Quadratic 0.9998 13.3 DDT-p,p' 13.027 235.0 & 165.2 0.01 – 1,000 Linear 0.9976 19.9 Endosulfan Sulfate 13.032 271.9 & 237.0 0.5 – 1,000 Quadratic 0.9997 18.2 2,2',3,4,4',5'-Hexachlorobiphenyl (BZ #138) 13.118 359.9 & 289.9 0.01 – 1,000 Linear 0.9996 6.5 Diclofop-methyl 13.284 339.9 & 252.9 0.01 – 500 Linear 0.9973 13.1 Diflufenican 13.310 266.0 & 246.1 0.01 – 1,000 Linear 0.9971 18.7 Propargite 13.327 231.0 & 135.0 0.5 – 1,000 Linear 0.9993 13.7 Piperonyl Butoxide 13.380 176.1 & 103.1 0.1 – 1,000 Quadratic 0.9995 9.0 Captafol 13.440 310.8 & 78.8 10 – 1,000 Quadratic 0.9987 19.3 Nitralin 13.551 315.9 & 274.0 0.1 – 500 Quadratic 0.9993 13.9 Mefenpyr-diethyl 13.608 253.0 & 189.0 0.01 – 500 Quadratic 0.9989 15.8 Benzoylprop-ethyl 13.699 292.0 & 105.0 0.1 – 1,000 Linear 0.9991 11.8 Iprodione 13.721 313.8 & 55.9 0.1 – 500 Linear 0.9984 12.4 Spiromesifen 13.722 272.0 & 254.2 0.1 – 500 Quadratic 0.9994 15.7 Tetramethrin I 13.814 164.0 & 77.1 5 – 1,000 Quadratic 0.9990 17.1 Pyridaphenthion 13.822 340.0 & 199.0 0.05 – 1,000 Quadratic 0.9996 12.9 Endrin Ketone 13.876 316.9 & 101.0 0.01 – 500 Quadratic 0.9993 13.6 Dimoxystrobin 13.880 205.0 & 58.0 0.1 – 1,000 Quadratic 0.9996 8.4 Phosmet 13.917 160.0 & 77.1 2 – 1,000 Quadratic 0.9993 10.6 Bifenthrin 13.922 181.2 & 165.2 0.1 – 500 Quadratic 0.9990 13.8 Bromopropylate 13.928 338.8 & 182.9 0.05 – 1,000 Quadratic 0.9993 10.7 EPN 13.935 169.0 & 77.1 0.05 – 1,000 Quadratic 0.9991 13.3 Picolinafen 13.958 376.0 & 238.1 0.01 – 200 Linear 0.9978 17.1 Bifenazate 13.975 168.1 & 61.9 10 – 1,000 Quadratic 0.9990 6.6 Dicofol, p, p'- 13.976 183.9 & 141.2 1 – 1,000 Linear 0.9972 18.8 Fenpropathrin 14.056 265.0 & 89.0 0.01 – 1,000 Quadratic 0.9996 13.5 2,2',3,4,4',5,5'-Heptachlorobiphenyl (BZ #180) 14.299 393.8 & 323.8 0.01 – 1,000 Linear 0.9992 12.2 Phenothrin I 14.399 122.9 & 81.1 0.1 – 1,000 Linear 0.9987 11.5 Tetradifon 14.424 158.9 & 111.0 0.01 – 1,000 Linear 0.9992 7.4 Furathiocarb 14.437 163.1 & 135.1 2 – 1,000 Linear 0.9992 6.1 Phosalone 14.590 182.0 & 75.0 0.05 – 500 Linear 0.9958 19.2 Azinphos-methyl 14.626 160.0 & 77.0 2 – 1,000 Quadratic 0.9993 11.4 Leptophos 14.638 171.0 & 51.0 0.05 – 1,000 Quadratic 0.9995 12.4 Cyhalothrin (Lambda) 14.698 181.1 & 152.1 5 – 1,000 Quadratic 0.9979 12.9 Cyhalofop-butyl 14.703 357.1 & 229.1 0.01 – 500 Linear 0.9958 16.1 www.agilent.com DE28615044 This information is subject to change without notice. © Agilent Technologies, Inc. 2024 Printed in the USA, May 16, 2024 5994-7436EN Name RT Transition Calibration Range (ppb) CF CF R2 Relative Standard Error Tralkoxydim 14.830 137.0 & 57.0 0.05 – 1,000 Linear 0.9990 7.9 Mirex 14.865 271.8 & 236.8 0.01 – 1,000 Linear 0.9994 13.5 Acrinathrin 15.045 247.0 & 68.0 1 – 1,000 Quadratic 0.9996 12.8 Pyrazophos 15.144 221.0 & 149.0 0.01 – 1,000 Quadratic 0.9994 14.1 Azinphos-ethyl 15.228 160.0 & 77.1 0.5 – 1,000 Quadratic 0.9997 12.4 Cycloxydim (Focus) 15.500 178.0 & 80.9 0.1 – 1,000 Quadratic 0.9997 8.0 Permethrin, (1R)-cis- 15.622 163.0 & 91.0 2 – 1,000 Quadratic 0.9978 18.4 Permethrin, (1R)-trans- 15.744 163.0 & 127.0 0.01 – 1,000 Linear 0.9990 12.0 Coumaphos 15.880 361.9 & 109.0 0.05 – 500 Linear 0.9972 16.0 Dioxathion 15.963 271.0 & 96.9 0.1 – 500 Linear 0.9969 19.8 Butafenacil 15.988 331.0 & 180.0 0.01 – 500 Linear 0.9973 14.4 Cyfluthrin I 16.202 163.0 & 127.0 0.5 – 1,000 Linear 0.9980 15.8 Cypermethrin I 16.510 163.0 & 127.0 0.1 – 1,000 Linear 0.9985 16.6 Halfenprox 16.565 262.9 & 169.0 0.05 – 1,000 Quadratic 0.9994 11.9 Flucythrinate I 16.725 156.9 & 107.1 0.01 – 1,000 Linear 0.9992 13.7 Ethofenprox 16.798 163.0 & 107.1 0.1 – 1,000 Linear 0.9989 14.3 Silafluofen 16.944 286.0 & 207.0 0.1 – 1,000 Quadratic 0.9995 9.7 Fenvalerate I 17.428 167.0 & 125.1 0.05 – 1,000 Linear 0.9988 12.6 Fluvalinate-tau I 17.601 250.0 & 200.0 0.1 – 1,000 Quadratic 0.9996 15.2 Deltamethrin 18.152 252.9 & 174.0 0.1 – 500 Linear 0.9963 17.2