使用AutoMate-Q40分析菠菜中的农药残留——一种全自动QuEChERS解决方案

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Analysis of Pesticide Residue inSpinach using the AutoMate-Q40an Automated QuEChERSSolution TELEDYNE TEKMAREverywhereyoulookP a ge |2 Application Note Page|1 Abstract QuEChERS is a Quick-Easy-Cheap-Effective-Rugged-Safe extraction method that has been developedfor the determination of pesticide residues in agricultural commodities. The rise in popularity of theQuEChERS technique and the increase in sample testing have driven the need for automation for thisextraction technique. By using the AutoMate-Q40， it streamlines the two part QuEChERS extraction fromthe liquid/solid extraction to the cleanup step. The aim of this project is to evaluate the performance and versatility of the AutoMate-Q40. A liquid massspectrocopy/ mass spectroscopy (LC/MS/MS) was used to determine 14 pesticides from spinach using，the AutoMate-Q40， an automated QuEChERS solution. Quantification was based on matrix-matchedcalibration curves with the use of internal standard to ensure method accuracy. QC samples wereevaluated at levels of 20， 40， 100 ng/g to ensure precision and accuracy of the AutoMate-Q40.Recoveries of all but one pesticide fell outside of the 70% to 120% range with sample reproducibility<8%. Introduction With the increasing globalization of the food industry there are consequently more concerns about oursafety. As a result， the number of pesticides which must be regulated and monitored has increased.QuEChERS is a Quick-Easy-Cheap-Effective-Rugged-Safe extraction method developed in 2003 for theextraction of pesticide residues in agricultural commodities. Modifications to the method have expandedthe scope to include many additional matrices and target analytes. Even though the QuEChERS is a simplified extraction technique， it still requires many manual stepsranging from addition of solvent， extraction salts， centrifugation， shaking， decanting and performing thedSPE cleanup.To modernize the traditional QuEChERS extraction through the use of automation，Teledyne Tekmar has developed the AutoMate-Q40. This automated platform will streamline the two partQuEChERS method from the liquid extraction through the cleanup. The goal of this work is to utilize the AutoMate-Q40 an automated QuEChERS extraction for thedetermination of pesticides in spinach. Pesticide residues were extracted from the spinach by using theAutoMate-Q40 following a modified AOAC 2007.01 extraction. Quantification was based on matrix-matched calibration curves with the use ofinternal standard to ensure method accuracy. QC sampleswere evaluated at levels of 20， 40， 100 ng/g to ensure the precision and accuracy of the AutoMate-Q40. Experiment -Instrument Conditions Sample Preparation/Extraction Spinach samples were prepared according to the procedure described in the“AOAC Official Method2007.01 Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with MagnesiumSulfate." The samples were stored in the freezer until sample analysis. Figure 1 shows the sample preparation and extraction steps that are needed to extract the pesticideresidues from apple juice. For this analysis， the AutoMate-Q40 used AOAC QuEChERS extraction salts(MgSO4 and NaOAc). The AutoMate-Q40， also， used a version of MgSO4， PSA， and GCB (GraphitizeCarbon Black) for the dSPE cleanup step. Figure 11AutoMate-Q40 Extraction Parameters. Place Samples into AutoMate-Q40 Add 100pL of IS (TPP) solution and QC spike solution if necessary Analyze by LC-MS/MS Transfer 8.0 mL to AOAC dSPE 15mLtube Add 15.0ml of ACN Shake for 1 minute， centrifuge for 1 minutes ADD 7.5g of AOAC QuEChERS extraction salt Transfer 2.0 ml to final extract vial Cap and shake vigorously for 1 minute www.teledynetekmar.com Instrumentation and Analytical Conditions The analysis was conducted on the Shimadzu Nexera LC interfaced to an AB Sciex 4500 QTrap triple-quad mass spectrometer (LC-MS/MS). For separation of the compounds of interest， a PhenomenexSynergi 4u Fusion-RP (50x 2.0 mm， and 80A pore size) column was used. Table l and Table Ildemonstrates the optimized LC-MS/MS analysis parameters for both the chromatographic separation andoptimal analyte transitions. Figure 2 shows the scheduled MRM chromatogram spiked at 400.0 pL/L. LCsamples were prepared by adding 100.0 pL of final extract into 900.0 pL of HPLC grade water. Tablel LC-MS/MS SRM Transitions and Parameters for AB Sciex 4500 QTrap Curtain Gas (CUR) 30 lon Spray Voltage (IS) 5500 Temperature (TEM) 400 Collision Gas (CAD) Medium Analyte Transitions Compounds Precursorlon (m/z) Quantizationproduct lon(m/z) DP(V) CE(V) CXP(V) Confirmationproduct ion(m/z) DP(V) CE(V) CXP(V) Atrazine 215.9 173.8 36.0 23.0 14.0 103.8 36.0 37.0 10.0 Azoxystrobin 403.9 372.0 41.0 21.0 10.0 343.9 41.0 35.0 26.0 Boscalid 342.8 306.7 81.0 27.0 24.0 139.7 81.0 25.0 14.0 Carbaryl 202.0 144.9 11.0 13.0 22.0 126.9 11.0 39.0 10.0 Carbendazim 191.9 159.9 91.0 27.0 16.0 131.9 91.0 41.0 12.0 Cyprodinil 225.9 92.9 111.0 47.0 8.0 76.9 111.0 65.0 8.0 Imazalil 296.9 158.8 36.0 35.0 12.0 254.6 36.0 25.0 20.0 Kresoxim-methly 313.9 205.7 10.0 9.0 16.0 115.9 10.0 19.0 10.0 Propiconazole 341.9 158.8 81.0 35.0 12.0 122.8 81.0 81.0 20.0 Pyrimetanil 200.0 107.0 86.0 31.0 10.0 77.0 86.0 59.0 10.0 Tebuconazole 308.0 69.9 71.0 61.0 10.0 124.8 71.0 55.0 12.0 Thiabendazole 296.8 158.7 56.0 31.0 16.0 200.8 56.0 25.0 16.0 TPP 326.9 214.7 91.0 35.0 16.0 167.7 91.0 47.0 14.0 Triflumazole 345.9 277.6 1.0 15.0 24.0 72.8 1.0 21.0 6.0 Table ll Shimadzu Nexera LC Parameters Column Synergi 4u Fusion RP 80A Dimensions 50.0 X2.00 mm Mobile Phase A： 5 mM Ammonium Formate in H2O B：5mM Ammonium Formate in MeOH Gradient Time (min) %B 0.1 5.0 9.0 90.0 12.0 100.0 15.0 100.0 15.1 20.0 17.0 Stop Flow Rate (mL/min) 0.3 Column Temperature (C) 30.0 Figure 22400.0 ng/g Spike of Compounds in Spinach Experimental Results Automation of the QuEChERS extraction allows for a fast， easy reliable and more reproducible extraction.By using the AutoMate-Q40， it offers significant labor savings， while improving the repeatability andconsistency between the samples. A precision and accuracy study was performed on all three commodities using the AutoMate-Q40. A 6.0pg/mL stock pesticide solution was used to fortify the spinach samples. Using the AutoMate-Q40， thesystem is able to spike the following samples with 50.0，100.0 and 250.0 pL of the pesticide standard thatyielded a 20.0， 40.0， and 100.0 ng/g check samples. These QC samples were quantitated against acorresponding matrix matched calibration. Table IIlData Table For Spinach 20.0 ng/g Spike 40.0 ng/g Spike 100.0 ng/g Spike Compounds R2 % Recovery %RSD % Recovery %RSD % Recovery %RSD Atrazine 0.9992 102.30 1.52 95.67 2.62 88.45 1.94 Azoxystrobin 0.9997 98.34 1.61 97.64 2.76 93.32 2.18 Boscalid 0.9983 109.95 8.37 105.28 4.84 92.31 2.74 Carbaryl 0.9988 100.94 2.06 95.23 2.94 90.14 1.91 Carbendazim 0.9655 n/d n/d n/d n/d n/d n/d Cyprodinil 0.9987 94.93 2.73 87.98 1.97 83.46 3.93 Imazalil 0.9995 87.93 1.89 85.35 2.48 80.85 1.43 Kresoxim-methly 0.9995 103.63 3.17 96.32 3.25 93.32 2.58 Propiconazole 0.9991 101.72 1.17 96.67 2.98 91.14 2.50 Pyrimethanil 0.9987 97.81 1.72 91.91 3.64 85.73 1.19 Tebuconazole 0.9993 100.65 1.64 95.68 2.46 88.81 2.04 Thiabendazole 0.99946 90.85 2.94 86.46 1.89 81.09 2.69 Triflumazole 0.9991 92.58 2.11 82.34 1.82 77.58 2.78 Table Ill shows that when using the AutoMate-Q40 to extract pesticide residues from spinach samples， itexhibits recoveries ranging from 77.58% to 109.95%. Since GCB was used in the cleanup plainercompounds such as carbendazim were absorbed into the loose media. These spiking recoveries fellwithin the recommend values for the Document N° Sanco/12495/2011.4 This document also states thatthe %RSD must be below 20%. By using the AutoMate-Q40 for the QuEChERS extraction it showedgreat precision ranging from 1.17% to 8.37%RSD for the spiked QC samples. Conclusion This study demonstrates the feasibility of automating the QuEChERS extraction method using theAutoMate-Q40. By automating the liquid handing， addition of salt/buffers， sample mixing， pipetting， andliquid level sensing using the patent pending VialVisionTM. The extraction process is faster， more reliable，and easier. This enables time and labor savings， while improving consistency and repeatability of theextraction. As shown above in Table Ill combined pesticide spikes recovered at 90.99% with an averageRSD of 2.57%RSD. These numbers indicate superb precision and accuracy thus validating theperformance of the AutoMate-Q40 and its use as an excellent analytical tool. 1..European Committee for Standardization/Technical Committee CEN/TC275 (2008)， Foods of plantorigin： Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrileextraction/ partitioning and cleanup by dispersive SPE QuEChERS-method. 2.AOAC Official Method 2007.07 Pesticide Residues in Food by Acetonitrile Extraction and Partitioningwith Magnesium Sulfate. Gas Chromatography/Mass Spectrometry and LiquidChromatography/Tandem Mass Spectrometry， First Action 2007 3.M. Anastassiades： QuEChERS a mini-multiresidue method for the analysis of pesticide residues inlow-fat products 4. Method Validation and Quality Control Procedure for Pesticide Residues Analysis in Food and Feed(Document N°SANCO/12495/2011) Sales/Support：Main： Socialville Foster Rd.， Mason， OH ww.teledynetekmar.comAnalysis of Pesticide Residue in Spinach； -Apr-