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- 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 .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 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 .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. Samples (SOCS, MMCS, Samples, MMCS, SOCS) 2. MDL (MMCSEXCE LevelL 1 and 2, Matrix Spike 10-1; 8 replicates) Template A – LOD/LOQ/R2 3. LOD/LOQ/R2Template (SOCS Levels B 1–- 9;Method 8 replicates) Detection Limit Template C – Recovery Template D – Uncertainty Results

1. Instrument Detection Limit – LOD, LOQ, Linearity – 8 Replicate injections of solvent-only calibration stds

2. Method Detection Limit – Method Detection Limit – 8 Replicate injections of Levels 1 and 2 MMCS and Level 1 Matrix Spike

3. Samples – Percent Recoveries and Matrix Effects – Injection of SOCS, MMCS, Samples

Results 1: Limit of Detection

• Determined from eight replicate injections of lowest level solvent-only calibration solution

• LOD = 2.998 X (standard deviation)exp

LOD (ng/g) Average # Analytes 0 < X < 0.05 147 ± 4 (72%) 0 < X < 0.20 179 ± 2 (88%) X > 0.20 19 ± 3 ( 9%) Not Detected 6 ± 2 ( 3%) Results 1: Limit of Quantitation

• LOQ = 2 X LOD Results 1: Linearity

• Determined from eight replicate injections of nine levels of solvent-only calibration solutions ranging from 0.05 to 20 ng/g

All Commodities r2 Average # Analytes X > 0.99 152 ± 25 (74%) X < 0.99 46 ± 25 (23%) Not Detected 6 ± 3 ( 3%) Results 2: Method Detection Limit

• Determined from eight replicate injections of MMCS Levels 1 and 2 and Matrix Spike at 10 ng/g

MDL (ng/g) Average # Analytes 0 < X < 0.20 179 ± 3 (88%) X > 0.20 20 ± 3 (10%) Not Determined 5 ± 2 (2%) Limitations of the Method

LOD (ng/g) Average # Analytes 0 < X < 0.05 147 ± 4 (72%) • Abamectin 0 < X < 0.20 179 ± 2 (88%) • Hydramethylnon X > 0.20 19 ± 3 ( 9%) • Alanycarb • Isocarbophos Not Detected 6 ± 2 ( 3%) • Benfuracarb • Ivermectin • Chlorfluazuron • r2 Average # Analytes • Clofentazine • X > 0.99 152 ± 25 (74%) Moxidectin • Difenoconazole • Novaluron X < 0.99 46 ± 25 (23%) • Doramectin • Oxadixyl Not Detected 6 ± 3 ( 3%) • Eprinomectin • Prothioconazole MDL (ng/g) Average # Analytes • • Etoxazole Spiromesifen 0 < X < 0.20 179 ± 3 (88%) • Thiofanox • Fenazaquin X > 0.20 20 ± 3 (10%) • Fluazinam Not Determined 5 ± 2 (2%) Results 3: Percent Recovery

• Determined from analysis of 3 matrix spikes prepared in quadruplicate

Recovery Percent Relative Error

Summary of Recoveries

Matrix X < 70 % 70% to 110% X > 110% Not Determined Broccoli 8 (4%) 170 (83%) 10 (5%) 16 (8%)

Carrot 10 (5%) 167 (82%) 11 (5%) 16 (8%)

Orange 6 (3%) 167 (82%) 15 (7%) 16 (8%)

Raisin 9 (4%) 165 (81%) 12 (6%) 18 (9%)

Wheat Flour 8 (4%) 164 (80%) 17 (8%) 15 (7%)

Overall ~8 (4%) ~166 (81%) ~13 (6%) ~16 (8%) Results 3: Matrix Effects

ME (%) = slope of cal. curve in matrix - 1 * 100% slope of cal. curve in solvent

• 93% of all analytes exhibit less than 20% ME • Matrix-matched calibration curve used for quantitation of all analytes

ME Trends: Number of Analytes Exhibiting a Negative or Positive ME Value Summary

• LOD/LOQ/MDL ranges 0 ppb to 0.05 ppb, most < 0.1 ppb

• R2 > 0.995 except orange and wheat flour R2 > 0.990

• Percent Recovery ranges 70% < x < 110%; RSD < 10% – Except wheat flour – larger RSD at all spike levels

• Signal suppression - broccoli, carrot, wheat flour • Signal enhancement – orange, raisin

• Limitations of the Method – 21 analytes not detected or out of analytical range Conclusions and Future Work

• Present multiresidue pesticide method for fresh produce using QuEChERS and LC/MS-MS techniques

• Future publication of results from all participating laboratories

• Method employed to evaluate AutoMate Q40 (automated QuEChERS system) – LC-MS/MS optimization complete – Templates available for data processing

• Serves as template for larger multi-lab validation studies 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 Guleph

Thank you for your attention! QUESTIONS? 8. Appendix – Analyte Number (1 of 2)

1 3-Hydroxycarbofuran 26 Butoxycarboxim 51 76 2 Abamectin 27 52 77 Fenpropimorph 3 28 53 Dimethomorph Isomer 78 Fenpyroximate 4 29 Carbetamide 54 Dimoxystrobin 79 Fenuron 5 Acibenzolar-S-methyl 30 55 Diniconazole 80 6 Alanycarb 31 Carboxin 56 81 Flonicamid 7 32 Carfentrazone-ethyl 57 Dioxacarb 82 Fluazinam 8 Aldicarb sulfone 33 58 Diuron 83 9 Aldicarb sulfoxide 34 Chlorfluazuron 59 Doramectin 84 Fludioxinil 10 Ametryn 35 Chlorotoluron 60 Emamectin-benzoate 85 Flufenacet 11 Aminocarb 36 Chloroxuron 61 Epoxiconazole 86 12 37 Clethodim Isomer 62 Eprinomectin 87 Fluometuron 13 Azoxystrobin 38 Clofentezine 63 Etaconazole Isomer 88 Fluoxastrobin 14 Benalaxyl 39 64 89 Fluquinconazole 15 40 Cyazofamid 65 Ethiprole 90 Flusilazole 16 Benfuracarb 41 Cycluron 66 Ethirimol 91 Flutolanil 17 Benzoximate 42 Cymoxanil 67 Ethofumesate 92 Flutriafol 18 Bifenazate 43 Cyproconazole Isomer 68 Etoxazole 93 Forchlorfenuron 19 Bitertanol 44 Cyprodinil 69 Famoxadone 94 Formetanate HCl 20 Boscalid 45 70 Fenamidone 95 Fuberidazole 21 Bromucanozole Isomer 46 Desmedipham 71 Fenarimol 96 Furalaxyl 22 Bupirimate 47 Diclobutrazol 72 Fenazaquin 97 Furathiocarb 23 48 73 Fenbuconazole 98 Halofenozide 24 Butafenacil 49 Diethofencarb 74 Fenhexamid 99 Hexaconazole 25 50 Difenoconazole Isomer 75 100 Hexaflumuron 8. Appendix – Analyte Number (2 of 2)

101 Hexythiazox 126 151 Prochloraz 176 Spirotetramat 102 127 Methoprotryne 152 177 Spiroxamine Isomer 178 Sulfentrazone 103 Imazalil 128 Methoxyfenozide 153 Prometon 179 Tebuconazole 104 129 Metobromuron 154 Prometryne 180 105 130 Metribuzin 155 Propamocarb 181 106 Ipconazole Isomer 131 Isomer 156 Propargite 182 Tebuthiuron 107 Iprovalicarb Isomer 132 Mexacarbate 157 Propham 183 Teflubenzuron 108 Isocarbophos 133 158 Propiconazole Isomer 184 Temephos 109 Isoprocarb 134 Monolinuron 159 185 Terbumeton 186 Terbutryn 110 Isoproturon 135 Moxidectin 160 Prothioconazole 187 Tetraconazole 111 Ivermectin 136 Myclobutanil 161 Pymetrozine 188 Thiabendazole 112 Kresoxim-methyl 137 Neburon 162 Pyracarbolid 189 113 Linuron 138 163 Pyraclostrobin 190 114 139 Novaluron 164 Pyridaben 191 Thidiazuron 115 Mandipropamid 140 Nuarimol 165 Pyrimethanil 192 Thiobencarb 193 116 Mefenacet 141 166 194 Thiophanate-methyl 117 Mepanipyrim 142 Oxadixyl 167 Quinoxyfen 195 Triadimefon 118 Mepronil 143 168 Rotenone 196 Triadimenol 119 Mesotrione 144 Paclobutrazol 169 Secbumeton 197 Trichlorfon 120 Metaflumizone 145 Penconazole 170 Siduron 198 Tricyclazole 121 Metalaxyl 146 Pencycuron 171 Simetryn 199 Trifloxystrobin 200 Triflumizole 122 Metconazole 147 Phenmedipham 172 Spinetoram 201 Triflumuron 123 Methabenzthiazuron 148 Picoxystrobin 173 (Spinosyn A) 202 Triticonazole 124 149 Piperonyl butoxide 174 Spirodiclofen 203 Vamidothion 125 150 175 Spiromesifen 204 Zoxamide Sample Preparation at 1:50 Dilution

Original Volume of Volume Volume of Total Desired Final Sample Spike Conc. post-dSPE of ACN Buffer To Volume Conc. Sample to Add to Add Add Water Blank 0 ng/mL 100 µL 900 µL 4000 µL 5000 µL 0 ng/mL (WB) Water Spike 50 ng/mL 100 µL 900 µL 4000 µL 5000 µL 1.0 ng/mL (WS) Matrix Blank 0 ng/mL 100 µL 900 µL 4000 µL 5000 µL 0 ng/mL (XB) Matrix Spike 10 ng/mL 100 µL 900 µL 4000 µL 5000 µL 0.2 ng/mL 10 Matrix Spike 50 ng/mL 100 µL 900 µL 4000 µL 5000 µL 1.0 ng/mL 50 Matrix Spike 250 ng/mL 100 µL 900 µL 4000 µL 5000 µL 5.0 ng/mL 250

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