茶叶及制品中农兽药残留检测方案

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Determination of PesticideResidues in Tea by an AutomatedQuEChERS SolutionPage |1 TELEDYNE TEKMAREverywhereyoulookP ag e|2 This application note validates the extraction performance ofthe AutoMate-Q40 by monitoring pesticides in tea. Thetarget residues will be determined by LiquidChromatography tandem mass spectrometry. Introduction In 2012， Americans consumed well over 79 billion servingsof tea， which is just over 3.60 billion gallons. If you factor inthe rest of the world it makes tea the most consumedtraditional beverage worldwide. Black， green， oolong andwhite teas all come from the same plant， but the differencesamong the types come from a diverse degree of processingand level of oxidization.' As with any plant based commodity， it has the potential to be vulnerable to pests and diseases. In whichthese pests and diseases are controlled by applying pesticides to the tea crop. Thus， meaning a potentialfor having pesticide residues in the final product. The QuEChERS extraction method offers superb selectivity， and sensitivity when extracting pesticideresidues in tea. This application note evaluates the performance and versatility of the AutoMate-Q40 forthe extraction of pesticides in tea. Liquid Chromatography coupled to a triple-quadrupole massspectrometry (LC-MS/MS) was employed for the detection of pesticides in tea. Quantification was basedon matrix-matched calibration curves Experimental Instrument Conditions Dry tea samples (2.0 g) were weighted into a 50.0 mL centrifuge tubes. These tubes were than placedinto the AutoMate-Q40 sample prep workstation， which then automates the QuEChERS procedure(EN15662：2008)(Figure 1) developed and defined by the European Reference Laboratory. For this analysis， the AutoMate-Q40 used 6.5 g of EN15662.2008 QuEChERS extraction salts. TheAutoMate-Q40 also used the EN version of MgSO4(900.0 mg)， PSA (150.0 mg) and GCB (15.0 mg) forthe dSPE cleanup step. Figure 1Modified EN 15662：2008 QuEChERS Extraction Sample analysis was conducted using a Shimadzu Nexera UHPLC system coupled to an AB Sciex 4500QTrap tandem mass spectrometer (MS/MS) via electrospray ionization (ESI). For separation of thecompounds of interest， a Phenomenex Kinetex 2.6um Biphenyl (50 x2.1mm) column was used. Table Iand Table II demonstrates the optimized LC-MS/MS analysis parameters for both the chromatographicseparation and optimal analyte transitions. Figure 2 shows the scheduled MRM chromatogram spiked at400.0 ng/g. Table l1 UHPLC-MS/MS Parameters Shimadzu Nexera Column Kinetix 2.6u Biphenyl 100A Column Temp 40°℃ Column Flow 0.4500mL/min Mobile Phase A： 5mM Ammonium Acetate in WaterB： 5mM Ammonium Acetate in Methanol Injection Volume 10uL AB Sciex 4500 QTrap Curtain Gas 30 lon Spray (V) 4500 Temperature (C) 500 GS1 50 GS2 60 www.teledynetekmar.com Compounds Q1Mass (Da) Q3 Mass (Da) RT DP CEI CXP Metalaxyl 279.995 220.0 8.49 21 19 165 279.995 192.0 8.49 21 25 14 Methiocarb 225.920 168.9 8.51 41 13 14 225.920 121.0 8.51 41 25 10 Methomyl 162.981 87.9 3.29 6 13 12 162.981 106.0 3.29 6 13 16 Metolachlor 283.976 252.0 9.77 16 21 20 283.976 176.0 9.77 16 33 14 Metribuzin 214.943 187.0 6.34 46 25 14 214.943 168.8 6.34 46 13 14 Monocrotophos 223.934 192.8 3.25 41 11 14 223.934 126.9 3.25 41 21 12 Myclobutanil 288.931 70.0 9.02 16 47 10 288.931 124.9 9.02 16 39 10 Profenofos 372.850 302.7 10.72 56 25 22 372.850 344.8 10.72 56 17 26 Pyrimethanil 200.017 107.0 8.31 70 31 10 200.017 82.0 8.31 70 21 11 Simazine 201.965 131.9 5.89 46 25 10 201.965 124.0 5.89 46 25 10 Thiodicarb 355.808 107.9 9.07 30 21 10 355.808 163.0 9.07 30 11 12 Figure 2 Tea MRM chromatogram spiked at 400.0 ng/g. Experimental Results By automating the QuEChERS extraction， it enables an easy， reliable and more reproducible extraction.The AutoMate-Q40 offer significant labor savings， while it improves the repeatability and consistencybetween the samples. A precision and accuracy study was performed using the AutoMate-Q40. A 0.2 and 2.0 pg/mL stockpesticide solution was used to fortify the tea samples. Using the AutoMate-Q40， the system is able tospike the following samples with 50.0 and100.0 pL of the pesticide standard that yielded a 10.0， 50.0 and100.0 ng/g check samples. These QC samples were quantitated against their corresponding matrixmatched calibration curve. Table Ill documents the AutoMate-Q40 ability to extract pesticide residues from tea samples with 90% of all the recoveries falling within 70-120%I.nTheese spike recoveries fall well within the recommended valuesin Document N°Sanco/12495/2011 which states the mean recoveries must fall within 70% to 120% anda RSD <20%. The AutoMate-Q40， also， demonstrated great precision on average of 6.6%RSD for thespiked QC samples. Table IllRecovery and Reproducibility of the AutoMate-Q40 in Fortified Tea Low Spike 10ng/g Mid Spike 50ng/g High spike 100ng/g Compounds %Recovery %RSD %Recovery %RSD %Recovery %RSD Atrazine 99.7 4.5 87.3 3.3 89.8 2.1 Boscalid 65.7 14.3 95.6 5.0 103.2 4.2 Buprofezin 97.2 4.7 88.9 3.2 92.0 1.8 Carbaryl 81.2 7.6 89.8 3.3 92.2 4.6 Carbendazim Nd Nd 74.6 19.0 93.1 14.3 Chlorpyrifos 94.0 9.9 91.5 4.3 98.2 2.4 Cyprodinil 100.6 5.9 90.4 5.9 95.9 0.7 Imazalil 61.0 11.4 84.4 6.3 89.5 5.4 Imidacloprid 96.4 6.2 90.6 5.9 93.9 3.9 Malathion 97.4 4.6 97.8 3.3 101.3 5.6 Metalaxyl 93.0 5.1 93.3 4.4 97.4 4.1 Methiocarb 92.4 5.2 89.7 4.8 92.9 2.5 Methomyl 88.4 8.7 87.9 6.6 96.2 1.9 Metolachlor 97.3 3.9 92.5 2.5 96.9 3.1 Metribuzin 63.0 19.9 82.5 4.9 91.9 2.9 Monocrotophos 100.9 6.3 91.3 96.6 0.9 Myclobutanil 94.9 16.2 89.6 91.7 5.8 Profenofos 59.9 14.2 82.8 5.9 92.2 2.2 Pyrimethanil 77.9 11.4 85.8 88.3 4.1 Thiodicarb 115.6 17.9 96.7 87.7 4.9 Nd = not detected This developed method was then applied to the quantitation of pesticides in real tea. Figure 3 shows anexample of the tea chromatogram that was collected. Table IV shows the pesticides concentration in saidtea samples (n=3) Figure 3Tea sample (extract 10x dilution) screen for pesticides 2.8e52.5e5-2.4e5.2.3e5·2.2e5-2.1e52.0e5.1.9e5·1.8e5.1.7e5-1.8e5·1.5e5.1.4e51.3e5·1.2e51.1e5.1.0e59.0e4.8.0e4-7.0e4·8.0e4.5.0e4..4.0e43.0e42.0e4. ace 1.0e40.0 A 15411.9712.23 12.6813.25 14.17、14.4114.52.14.93 16.06 Table IVSummary of Pesticide Findings in Real Tea Samples Pesticide Concentration (ng/g) Tea Methomyl 37.5 Metalaxyl 7.90 Imidacloprid 861 Metolachlor 1.50 Buprofezin 9.00 Chlorpyrifos 176 Despite having the high selectivity of the MRM detection there is always a risk for false positives due tomatrix interference. For improved accuracy and identification of the pesticides， the MS/MS was run inEnhanced Product lon (EPl) mode of the 4500 QTRAP. This allowed for spectral library matching toimprove the confidence of detection. Figures 4-9 show the found pesticide MRM and spectra compared toa known standard MRM and spectra. P age |6 Figure 5Metalaxyl Example Spectra Figure 66Imidacloprid Example Spectra Figure7Metolachlor Example Spectra Page |8 t imn mid tDa Figure 99Chlorpyrifos Example Spectra Conclusion This study demonstrates the Automate-Q40's ability to successfully process tea samples for pesticideresidue by the QuEChERS extraction method. By automating the liquid handling， addition of salt/buffers，sample mixing， pipetting， and liquid level sensing using the patent pending VialVision TM， the AutoMate-Q40 frees the scientist from a labor-intensive extraction method and exposure to unhealthy chemicals. The automated extraction process enables an easy， reliable and more reproducible extraction. Thisenables time and labor savings， while improving consistency and reproducibility of the extraction. Asshown above in Table Ill the combined pesticide spikes recoveries of 90.71%， with an average RSD of6.61% exceed the requirement outlined in The Document N°Sanco/12495/2011. These numbers indicatesuperb precision and accuracy thus validating the performance of the AutoMate-Q40 to adequatelyperform the QuEChERS pesticide extraction method for tea. 1. The State of The U.S. Tea industry， Parpared by Joseph P. Simrany Peter F. Gggihttp：//www.teausa.com/14654/state-of-the-industry 2. European Committee for Standardization/Technical Committee CEN/TC275 (2008)，Foods of plant origin： Determination of pesticide residues using GC-MS and/or LC-MS/MS following Acetonitrile extraction/ partitioning and cleanup by dispersive SPEQuEChERS-method. 3. Method Validation and Quality Control Procedure for Pesticide Residues Analysis in Foodand Feed (Document N°SANCO/12495/2011) www.teledynetekmar.com Sales/Support： Main： Socialville Foster Rd.， Mason， OH esticide Residues in Tea； Nov-ww.teledynetekmar.com