Multiresidue Analysis of 204 Pesticides in Fresh Produce Using the QuEChERS Method Followed by LC-MS/MS 2015 ACIL P2 Conference April 21, 2015 Crystal City, VA Kelli Sikorski Simon, PhD Food and Drug Administration Center for Food Safety and Nutrition [email protected] Acknowledgements Jon Wong, PhD Kai Zhang, PhD James Wittenberg, PhD Alex Krynitsky, PhD Paul Yang, PhD – MOECC Kaushik Banerjee, PhD – NRCG, India Manjusha Jadhav – NRCG, India Kai-Chih Yang, PhD – Taiwan FDA Linda Lissemore, PhD – Univ. of Guelph Research Focus • Multi-lab, multiresidue pesticide method for fresh produce, labs explore 2 additional commodities of interest • Evaluate of the statistical difference (if any) in results obtained from five laboratories executing the same protocol and using the same materials and instrumentation • Serves as template for other matrices, processes Introduction • Multi-laboratory validation project – U.S. Food and Drug Administration, CFSAN – Taiwan Food and Drug Administration – Ministry of the Environment and Climate Change - Ontario, Canada – University of Guelph - Ontario, Canada – National Research Centre for Grapes, Pune India • Method Details – Five commodities (3 required, 2 optional) – Spike with mixture of 204 pesticides – Use of QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) – Use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for separation and detection – Four Excel templates used for reporting results • Single Laboratory Results and Limitations of the Method • Conclusions and Future Work Experimental Materials *provided to all participating laboratories • Previously comminuted, homogenized commodities* – spinach, carrot, orange - 10.0 ± 0.1 g (or similar) – raisin - 5.0 ± 0.1 g; wheat flour - 1.0 ± 0.1 g • ABSciex iDQuant Standards Kit (204 pesticides in 10 ampoules)* • QuEChERS Extraction Solvent: 50 ng/mL d10-diazinon in acetonitrile (1:50 sample dilution, calibration range: 0.05 to 20 ppb) • Restek Q-Sep QuEChERS Kits* – Extraction Salts: 50 mL centrifuge tube; 4 mg MgSO4, 1 mg NaCl – dSPE Cleanup: 15 mL centrifuge tube; (1,200 mg MgSO4, 400 mg PSA) • Waters Acquity UHPLC with ABSciex Qtrap 5500 mass spectrometer – Restek Ultra-Aqueous C18 Column (100 mm x 2.1 mm, 3 µm) – 10 mM NH4HCO2 buffer (A: water, B: methanol) with 0.1% formic acid – 0.5 mL/min flow rate, 5 µL sample injection Overall Workflow Prepare pesticide standards, optimize LC/MS/MS Cryogrind, homogenize commodities; transfer to centrifuge tubes Spike with pesticides at 3 levels, let sit 15 minutes Add 10 mL ACN containing 10 ng/mL ISTD, vortex Add QuEChERS salts; shake, centrifuge Transfer 5 mL to a 15 mL dSPE tube; vortex, shake, centrifuge Transfer 100 µL of ACN top layer, dilute with ACN, buffer Prepare Solvent Only (SO) and Matrix-Matched (MM) standards Analyze by LC/MS/MS Preparation of Pesticide Standards and Fortification Solutions Ten ~1 mL ampoules Transfer 1.0 mL of 10 µg/mL 100 µg/mL each standard to Intermediate Individual Pesticide one 10 mL Pesticide Standards volumetric flask Standard Solution (IPSS) Transfer remainder (~100 µL) to individual Transfer 400 µL to one 10 mL Transfer 0.5 mL, 2.0 mL and 5.0 mL vials for LC volumetric flask to prepare to individual 10 mL volumetric and MS Level 1 (400 ng/mL) flasks to prepare 0.5, 2.0 and 5.0 optimization Calibration Stock Solution µg/mL Fortification Solution See Table 1 for See Table 2 for preparation of Levels 2-9 preparation of all Calibration Stock Solutions Fortification Solutions Preparation of Fortification Solutions Transfer 0.5 mL of 0.5 200 µL 10 IPSS to one 10 mL µg/mL per 10g ng/mL volumetric flask, fill F.S. sample Spike 10 µg/mL to mark with ACN Intermediate Pesticide Transfer 2.0 mL of 2.0 250 µL 50 Standard IPSS to one 10 mL µg/mL per 10g ng/mL Solution (IPSS) volumetric flask, fill F.S. sample Spike to mark with ACN Transfer 5.0 mL of 5.0 500 µL 250 IPSS to one 10 mL µg/mL per 10g ng/mL volumetric flask, fill F.S. sample Spike to mark with ACN These fortification solutions are used to spike commodities at three levels. Once diluted 1:10, they provide concentrations of 1.0, 5.0 and 25 ng/mL. Preparation of Calibration Stock Solutions 10 µg/mL Intermediate 400 200 100 1 Pesticide ng/mL ng/mL ng/mL ng/mL Standard Solution (IPSS) Solution Used to Volume of Add ACN to Final Volume Calibration Concentration Make New Level Solution mark - Total Remaining Level (ng/mL) (ng/mL) Required (mL) Volume (mL) (mL) 1 400 IPSS (10 µg/mL) 0.4 10 5.0 2 200 400 5.0 10 4.0 3 100 200 5.0 10 4.0 4 50 100 5.0 10 4.0 5 20 200 1.0 10 9.0 6 10 100 1.0 10 9.0 7 5 50 1.0 10 10 8 2 20 1.0 10 10 9 1 10 1.0 10 10 These calibration solutions are used to prepare the SOCS and MMCS Calibration Standards. Once diluted 1:10, they will provide a range of calibration solutions from 0.1 to 40 ng/mL LC Optimization Time Time Number of % Total (min.) A (%) B (%) (min.) Analytes Analytes 0 90 10 4 40 60 0 to 4 15 7 8 30 70 4 to 6 62 30 11 0 100 6 to 8 76 37 12 0 100 12.01 90 10 10 to 14 51 25 15 90 10 204 pesticides • MS/MS Parameters 5 internal standards – Source: 500 °C – Ion Spray Voltage: 5.0 kV – Curtain Gas: 30 psi; Ion Source Gas 1 and 2: 60 psi; Collision Gas: high – ESI positive mode – 30 second detection window and 0.5 second target scan time MS/MS Optimization Pesticide standard mixes are individually optimized Q1 m/z Q3 m/z tR Declustering Collision Cell Exit Std # (amu) (amu) (min) Component Name Potential (V) Energy (V) Potential (V) 9 329.1 125 8.72 Pencycuron.1 71 31 8 9 329.1 218.1 8.72 Pencycuron.2 86 23 14 9 209.1 152 3.33 Aminocarb.1 71 21 8 9 209.1 137.1 3.33 Aminocarb.2 66 33 12 9 318.1 136 5.6 Desmedipham 1 41 33 10 9 318.1 182 5.6 Desmedipham.2 51 19 14 9 222.1 165 3.3 Formetanate HCl.1 71 23 14 9 222.1 120 3.3 Formetanate HCl.2 56 37 10 9 223.2 151 4.84 Mexacarbate.1 76 33 10 9 223.2 166.1 4.84 Mexacarbate.2 51 21 10 9 301.1 168.1 5.6 Phenmedipham.1 50 14 4 9 301.2 168 5.6 Phenmedipham.2 91 15 10 9 189.2 144 3.24 Propamocarb.1 61 19 14 9 189.2 102 3.24 Propamocarb.2 66 29 10 Overall Workflow Prepare pesticide standards, optimize LC/MS/MS Cryogrind, homogenize commodities; transfer to centrifuge tubes Spike with pesticides at 3 levels, let sit 15 minutes Add 10 mL ACN containing 10 ng/mL ISTD, vortex Add QuEChERS salts; shake, centrifuge Transfer 5 mL to a 15 mL dSPE tube; vortex, shake, centrifuge Transfer 100 µL of ACN top layer, dilute with ACN, buffer Prepare Solvent Only (SO) and Matrix-Matched (MM) standards Analyze by LC/MS/MS Sample Spiking Using Fortification Solutions Sample Type Sample Name Desired Conc. Volume Stock to Add Fortification Solution Water Blank 1 WB-1 0 ng/mL 500 µL ACN Matrix Blank 1-4 XB-1 0 ng/mL 500 µL ACN Matrix Spike 10-1 XS-10-1 10 ng/mL 200 µL 0.5 µg/mL Water Spike 50-1 WS-1 50 ng/mL 250 µL 2.0 µg/mL Matrix Spike 50-1 XS-50-1 50 ng/mL 250 µL 2.0 µg/mL Matrix Spike 250-1 XS-250-1 250 ng/mL 500 µL 5.0 µg/mL 250 250 250 250 250 ppb 50 50 50 50 50 ppb WB10 WB10 WS10 WS10 10 ppb MB MB MB MB 0 ppb 0 ppb WB WB WS WS 50 ppb Overall Workflow Prepare pesticide standards, optimize LC/MS/MS Cryogrind, homogenize commodities; transfer to centrifuge tubes Spike with pesticides at 3 levels, let sit 15 minutes Add 10 mL ACN containing 10 ng/mL ISTD, vortex Add QuEChERS salts; shake, centrifuge Transfer 5 mL to a 15 mL dSPE tube; vortex, shake, centrifuge Transfer 100 µL of ACN top layer, dilute with ACN, buffer Prepare Solvent Only (SO) and Matrix-Matched (MM) standards Analyze by LC/MS/MS SOCS and MMCS Preparation – Changes to Spike Volumes at 1:50 Dilution SOCS or Volume of Blank Volume of Calibration Volume of Volume of Total MMCS Cal. or Matrix Calibration Stock ACN Buffer Volume Standards Solvent to Add* Stock to Add Standard to Add to Add Blank 100 µL 100 µL ACN 800 µL 4000 µL 5000 µL 0.05 ng/mL 100 µL 125 µL 2.0 ng/mL 775 µL 4000 µL 5000 µL 0.10 ng/mL 100 µL 100 µL 5.0 ng/mL 800 µL 4000 µL 5000 µL 0.20 ng/mL 100 µL 100 µL 10 ng/mL 800 µL 4000 µL 5000 µL 0.50 ng/mL 100 µL 250 µL 10 ng/mL 650 µL 4000 µL 5000 µL 1.0 ng/mL 100 µL 100 µL 50 ng/mL 800 µL 4000 µL 5000 µL 2.0 ng/mL 100 µL 100 µL 100 ng/mL 800 µL 4000 µL 5000 µL 5.0 ng/mL 100 µL 125 µL 200 ng/mL 775 µL 4000 µL 5000 µL 10 ng/mL 100 µL 125 µL 400 ng/mL 775 µL 4000 µL 5000 µL 20 ng/mL 100 µL 250 µL 400 ng/mL 650 µL 4000 µL 5000 µL • For SOCS, use combined dSPE supernatants from WB1+WB2 • FOR MMCS, use combined dSPE supernatants from MB-1 thru MB-4 Original internal standard concentration in QES = 50 ng/mL Final internal standard concentration = 1 ng/mL using a 1:50 dilution sample preparation Final Sample composition is 20% ACN and 80% Buffer Overall Workflow Prepare pesticide standards, optimize LC/MS/MS Cryogrind, homogenize commodities; transfer to centrifuge tubes Spike with pesticides at 3 levels, let sit 15 minutes Add 10 mL ACN containing 10 ng/mL ISTD, vortex Add QuEChERS salts; shake, centrifuge Transfer 5 mL to a 15 mL dSPE tube; vortex, shake, centrifuge Transfer 100 µL of ACN top layer, dilute with ACN, buffer Prepare Solvent Only (SO) and Matrix-Matched (MM) standards Analyze by LC/MS/MS LC-MS/MS Analysis 1.