AutoMate-Q40 Automated QuEChERSExtraction for Pesticide Residues inBotanicalsApplication NoteTyler Trent, Applications Sales Specialist, Teledyne TekmarP a g e 1AbstractQuEChERS is a Quick-Easy-CheapEffective-Rugged-Safe extraction methodthat has been developed for thedetermination of pesticide residues inagriculturalcommodities.Sinceitsdevelopment, QuEChERS has beenapplied to many different classes ofcommodities ranging from fruits andvegetables to dry grains and teas. In 2007,the United States Food and DrugAdministration (USFDA) announced thatthe makers of dietary supplements(botanicals) must adhere to stricterregulations.1 Since a majority of the dietarysupplements are sourced from botanicals, itcalled for the determination of pesticideresidues in samples. It was determined thatthe QuEChERS extraction was suitable toextract pesticide residues from thebotanical samples.This study will evaluate the performance and versatility of the AutoMate-Q40, an automated QuEChERSextraction platform. Liquid Chromatography coupled to a triple-quadrupole Mass Spectrometer (LCMS/MS) was used to determine pesticide residues from the botanical samples. Quantification was basedon matrix-matched calibration curves with the use of internal standard to ensure method accuracy. QCsamples were evaluated at various levels to ensure precision and accuracy of the AutoMate-Q40extraction.IntroductionBotanical products are used to improve health and energy, and in 2015 reached sales of 93.15 billionworldwide. 2 Often the popular perception is that botanicals are harvested “in the wild”, but in reality manybotanicals are farmed using conventional agricultural practices, including the application of pesticides andfungicides.3 Consequently, in 2007 the United States Food and Drug Administration announced thatmakers of dietary supplements (botanicals) must adhere to more stringent regulations.4The QuEChERS extraction method offers superb selectivity and sensitivity when extracting pesticideresidues in botanical samples. This study evaluates the performance and versatility of the AutoMate-Q40for the extraction of pesticides in botanicals using an automated QuEChERS extraction method. LiquidChromatography coupled to a triple-quadrupole Mass Spectrometer (LC-MS/MS) was employed for thedetection of the pesticides. Quantification was based on matrix-replacement calibration curves.Experimental Conditions“Real World” Pepin and Ginkgo samples were submitted to Teledyne Tekmar for this study. For methoddevelopment, a pesticide-free green coffee matrix was used to establish the method validation. Thesamples were stored at ambient temperature until the time of extraction. Figure 1 lists the samplepreparation and extraction steps used for this analysis. The AutoMate-Q40 performed a modifiedunbuffered QuEChERS method using 5.0 g of unbuffered QuEChERS extraction salts (4.0 g MgSO 4 and1.0 g NaCl). 5,6AutoMate-Q40 QuEChERS Pesticides in Botanicals.docx; 3-Nov-16Sales/Support: 800-874-2004 Main: 513-229-70004736 Socialville Foster Rd., Mason, OH

P a g e 2Figure 1 AutoMate-Q40 Sample Preparation and ExtractionSample analysis was conducted using a Shimadzu Nexera LC interfaced to an AB Sciex 4500 QTrap Triple-Quad Mass Spectrometer (LC-MS/MS). For separation of the compounds of interest, aPhenomenex Kinetex 2.6u Biphenyl column was used. Table I and Table II show the optimizedLC-MS/MS analysis parameters for both the chromatographic separation and optimal analyte transitions.Figure 2 shows the scheduled Multiple Reaction Monitoring (MRM) chromatogram spiked at 0.4 mg/kg.Table ICritical LC-MS/MS SRM Transitions and Parameters for AB Sciex 4500 QTrap Curtain Gas (CUR)20Ion Spray Voltage (IS) /-4500Temperature (TEM)500 CCollision Gas (CAD)HighAnalyte TransitionsCompoundsRT (min)Precursor Ion (m/z)Quantization Product Ion n9.54388.1193.9AutoMate-Q40 QuEChERS Pesticides in Botanicals.docx; 3-Nov-16Sales/Support: 800-874-2004 · Main: 513-229-70004736 Socialville Foster Rd., Mason, OH

P a g e 3Table IIShimadzu Nexera LC ParametersColumnPhenomenex Kinetex 2.6u BiphenylDimensions50 X 2.1 mmA: 5 mM Ammonium Acetate in WaterB: 5 mM Ammonium Acetate in MethanolMobile PhaseGradientFlow Rate0.500 mL/minColumn Temperature35 1StopFigure 2 Spiked Matrix Match Calibration Sample 0.400 mg/kgResultsBy automating the QuEChERS extraction, an easy, reliable and more reproducible extraction is possible.The AutoMate-Q40 offers significant labor savings, while improving the repeatability and consistencybetween samples.A precision and accuracy study was performed using the AutoMate-Q40. A 2.0 µg/mL stock pesticidesolution was used to fortify the botanical samples. Using the AutoMate-Q40’s ability for spike addition,25.0, 50.0 and 100.0 µL of the pesticide standard was added to each sample, yielding 5.0, 10.0 and 20.0ng/g Quality Control (QC) samples. These QC samples were quantitated against their correspondingmatrix replacement calibration curve.AutoMate-Q40 QuEChERS Pesticides in Botanicals.docx; 3-Nov-16Sales/Support: 800-874-2004 · Main: 513-229-70004736 Socialville Foster Rd., Mason, OH

P a g e 4Table III exhibits the AutoMate-Q40’s ability to extract pesticide residues from botanical samples. Allrecoveries fall easily within the recommended 70-120% mean recovery and 20% RSD. TheAutoMate-Q40 also demonstrated great precision, averaging 5.0%RSD for the spiked QC samples.Table IIISpike Recovery Table for Botanical SamplesCompound5.0 ng/g10.0 ng/g20.0 ole115.07.5110.06.596.05.4The developed method was then applied to the quantitation of pesticides in the submitted, “real-world”botanical samples. Figure 3 shows an example of the Pepin chromatogram that was collected. Table IVshows the pesticides concentration in the “real world” botanical samples (n 3).Figure 3 Pepin (A) Sample (extract 10X Dilution)AutoMate-Q40 QuEChERS Pesticides in Botanicals.docx; 3-Nov-16Sales/Support: 800-874-2004 · Main: 513-229-70004736 Socialville Foster Rd., Mason, OH

P a g e 5Table IV Summary of Pesticide Findings in “Real World” Botanical Samples (ng/g)Sample NameGinkgo AGinkgo BGinkgo CPepin APepin BPepin usionThis study demonstrates the Automate-Q40's ability to successfully process botanical samples forpesticide residue by the QuEChERS extraction method. By automating the liquid handling, addition ofsalt/buffers, sample mixing, pipetting, and liquid level sensing using the VialVision feature, theAutoMate-Q40 frees the analyst from the labor-intensive extraction method as well as exposure tounhealthy chemicals.The automated extraction process enables an easy, reliable and more reproducible extraction,significantly saving time and labor and improving consistency and reproducibility of the extraction. Asshown in Table III the combined pesticide spike recoveries of 98.3% with an average RSD of 5.0%exceed the requirement for this study. These numbers indicate superb precision and accuracy, thusvalidating the performance of the AutoMate-Q40 to perform the QuEChERS pesticide extraction methodfor botanical samples.References1. FDA Issues Dietary Supplements Final Rule. [Online] 2007. cements/2007/ucm108938.htm (accessed September 09, 2016).2. Daniells, S. Herbal Supplement Sales to Hit 93.15 Billion by 2015: Report. Nutra [Online] 2011. rt (accessed September 09, 2016).3. Multiresidue Pesticide Analysis of Dried Botanical Dietary Supplements Using an AutomatedDispersive SPE Cleanup for QuEChERS and High-Performance Liquid Chromatography–TandemMass Spectrometry. Yang Chen, Fadwa Al-Taher, Rima Juskelis, Jon W. Wong, Kai Zhang, DouglasG. Hayward, Jerry Zweigenbaum, Joan Stevens, and Jack Cappozzo. Journal of Agricultural andFood Chemistry 2012 60 (40), 9991-9999 DOI: 10.1021/jf301723g.4. Current Good Manufacturing Practice in Manufacturing, Packaging, Labeling, or Holding Operationsfor Dietary Supplements. U.S. Food and Drug Administration (FDA). Docket No. 1996N-0417(formerly No. 96N-0417), CFSAN 200441 (2007) 34752.5. European Committee for Standardization. Foods of Plant Origin - Determination of PesticideResidues Using GC-MS and/or LC-MS/MS Following Acetonitrile Extraction/Partitioning and Clean-upby Dispersive SPE (QuEChERS-method), EN 15662:2008; Brussels, Belgium, 2008.6. Anastassiades, M; Lehotay, SJ; Stajnbaher, D; Schenck, FJ. Fast and Easy Multiresidue MethodEmploying Acetonitrile Extraction/Partitioning and "Dispersive Solid-Phase Extraction" for theDetermination of Pesticide Residues in Produce. JAOAC International 2003 Mar-Apr; 86 (s): 412-31.AutoMate-Q40 QuEChERS Pesticides in Botanicals.docx; 3-Nov-16Sales/Support: 800-874-2004 · Main: 513-229-70004736 Socialville Foster Rd., Mason, OH

AutoMate-Q40 also demonstrated great precision, averaging 5.0%RSD for the spiked QC samples. Table III Spike Recovery Table for Botanical Samples Compound 5.0 ng/g 10.0 ng/g 20.0 ng/g %Recovery %RSD %Recovery %RSD %Recovery %RSD Acetamiprid 105.5 4.6 119.9 14.0 102.1 2.6 A