Multiresidue Analysis of 204 Pesticides in Fresh Produce Using the QuEChERS Method Followed by LC-MS/MS

Transcription

1 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

2 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

3 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

4 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

5 Experimental Materials *provided to all participating laboratories Previously comminuted, homogenized commodities* spinach, carrot, orange ± 0.1 g (or similar) raisin ± 0.1 g; wheat flour ± 0.1 g ABSciex idquant Standards Kit (204 pesticides in 10 ampoules)* QuEChERS Extraction Solvent: 50 ng/ml d 10 -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 MgSO 4, 400 mg PSA) Waters Acquity UHPLC with ABSciex Qtrap 5500 mass spectrometer Restek Ultra-Aqueous C 18 Column (100 mm x 2.1 mm, 3 µm) 10 mm NH 4 HCO 2 buffer (A: water, B: methanol) with 0.1% formic acid 0.5 ml/min flow rate, 5 µl sample injection

6 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

7 Preparation of Pesticide Standards and Fortification Solutions Ten ~1 ml ampoules 100 µg/ml Individual Pesticide Standards Transfer 1.0 ml of each standard to one 10 ml volumetric flask 10 µg/ml Intermediate Pesticide Standard Solution (IPSS) Transfer remainder (~100 µl) to individual vials for LC and MS optimization Transfer 400 µl to one 10 ml volumetric flask to prepare Level 1 (400 ng/ml) Calibration Stock Solution See Table 1 for preparation of Levels 2-9 Calibration Stock Solutions Transfer 0.5 ml, 2.0 ml and 5.0 ml to individual 10 ml volumetric flasks to prepare 0.5, 2.0 and 5.0 µg/ml Fortification Solution See Table 2 for preparation of all Fortification Solutions

8 Preparation of Fortification Solutions 10 µg/ml Intermediate Pesticide Standard Solution (IPSS) Transfer 0.5 ml of IPSS to one 10 ml volumetric flask, fill to mark with ACN Transfer 2.0 ml of IPSS to one 10 ml volumetric flask, fill to mark with ACN 0.5 µg/ml F.S. 2.0 µg/ml F.S. 200 µl per 10g sample 250 µl per 10g sample 10 ng/ml Spike 50 ng/ml Spike Transfer 5.0 ml of IPSS to one 10 ml volumetric flask, fill to mark with ACN 5.0 µg/ml F.S. 500 µl per 10g sample 250 ng/ml Spike 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.

9 Preparation of Calibration Stock Solutions 10 µg/ml Intermediate Pesticide Standard Solution (IPSS) 400 ng/ml 200 ng/ml 100 ng/ml 1 ng/ml Calibration Level Solution Used to Make New Level (ng/ml) Volume of Solution Required (ml) Add ACN to mark - Total Volume (ml) Final Volume Remaining (ml) Concentration (ng/ml) IPSS (10 µg/ml) 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

10 LC Optimization Time (min.) A (%) B (%) Time (min.) Number of Analytes % Total Analytes 0 to to to to MS/MS Parameters 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 204 pesticides 5 internal standards

11 MS/MS Optimization Pesticide standard mixes are individually optimized Std # Q1 m/z (amu) Q3 m/z (amu) tr (min) Component Name Declustering Potential (V) Collision Energy (V) Cell Exit Potential (V) Pencycuron Pencycuron Aminocarb Aminocarb Desmedipham Desmedipham Formetanate HCl Formetanate HCl Mexacarbate Mexacarbate Phenmedipham Phenmedipham Propamocarb Propamocarb

12 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

13 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 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 ng/ml 250 µl 2.0 µg/ml Matrix Spike XS ng/ml 500 µl 5.0 µg/ml ppb WB 10 WB 10 WS 10 WS ppb 10 ppb MB MB MB MB 0 ppb 0 ppb WB WB WS WS 50 ppb

14 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

15 SOCS or MMCS Cal. Standards SOCS and MMCS Preparation Changes to Spike Volumes at 1:50 Dilution Volume of Blank or Matrix Solvent to Add* Volume of Calibration Stock to Add Calibration Stock Standard Volume of ACN to Add Volume of Buffer to Add Total Volume 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

16 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

17 LC-MS/MS Analysis 1. Samples (SOCS, MMCS, Samples, MMCS, SOCS) EXCEL Template A LOD/LOQ/R 2 Template B Method Detection Limit Template C Recovery Template D Uncertainty 2. MDL (MMCS Level 1 and 2, Matrix Spike 10-1; 8 replicates) 3. LOD/LOQ/R 2 (SOCS Levels 1-9; 8 replicates)

18 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

19 Results 1: Limit of Detection Determined from eight replicate injections of lowest level solvent-only calibration solution LOD = X (standard deviation) exp LOD (ng/g) Average # Analytes 0 < X < ± 4 (72%) 0 < X < ± 2 (88%) X > ± 3 ( 9%) Not Detected 6 ± 2 ( 3%)

20 Results 1: Limit of Quantitation LOQ = 2 X LOD

21 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 r 2 Average # Analytes X > ± 25 (74%) X < ± 25 (23%) Not Detected 6 ± 3 ( 3%)

22 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 < ± 3 (88%) X > ± 3 (10%) Not Determined 5 ± 2 (2%)

23 Limitations of the Method LOD (ng/g) Average # Analytes 0 < X < ± 4 (72%) 0 < X < ± 2 (88%) X > ± 3 ( 9%) Not Detected 6 ± 2 ( 3%) r 2 Average # Analytes X > ± 25 (74%) X < ± 25 (23%) Not Detected 6 ± 3 ( 3%) MDL (ng/g) Average # Analytes 0 < X < ± 3 (88%) X > ± 3 (10%) Not Determined 5 ± 2 (2%) Abamectin Alanycarb Benfuracarb Chlorfluazuron Clofentazine Difenoconazole Doramectin Eprinomectin Etoxazole Fenazaquin Fluazinam Hydramethylnon Isocarbophos Ivermectin Metaflumizone Moxidectin Novaluron Oxadixyl Prothioconazole Spiromesifen Thiofanox

24 Results 3: Percent Recovery Determined from analysis of 3 matrix spikes prepared in quadruplicate Recovery Percent Relative Error

25

26 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%)

27 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

28 Summary LOD/LOQ/MDL ranges 0 ppb to 0.05 ppb, most < 0.1 ppb R 2 > except orange and wheat flour R 2 > 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

29 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

30 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?

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

32 8. Appendix Analyte Number (2 of 2) 101 Hexythiazox 102 Hydramethylnon 103 Imazalil 104 Imidacloprid 105 Indoxacarb 106 Ipconazole Isomer 107 Iprovalicarb Isomer 108 Isocarbophos 109 Isoprocarb 110 Isoproturon 111 Ivermectin 112 Kresoxim-methyl 113 Linuron 114 Lufenuron 115 Mandipropamid 116 Mefenacet 117 Mepanipyrim 118 Mepronil 119 Mesotrione 120 Metaflumizone 121 Metalaxyl 122 Metconazole 123 Methabenzthiazuron 124 Methamidophos 125 Methiocarb 126 Methomyl 127 Methoprotryne 128 Methoxyfenozide 129 Metobromuron 130 Metribuzin 131 Mevinphos Isomer 132 Mexacarbate 133 Monocrotophos 134 Monolinuron 135 Moxidectin 136 Myclobutanil 137 Neburon 138 Nitenpyram 139 Novaluron 140 Nuarimol 141 Omethoate 142 Oxadixyl 143 Oxamyl 144 Paclobutrazol 145 Penconazole 146 Pencycuron 147 Phenmedipham 148 Picoxystrobin 149 Piperonyl butoxide 150 Pirimicarb 151 Prochloraz 152 Promecarb 153 Prometon 154 Prometryne 155 Propamocarb 156 Propargite 157 Propham 158 Propiconazole Isomer 159 Propoxur 160 Prothioconazole 161 Pymetrozine 162 Pyracarbolid 163 Pyraclostrobin 164 Pyridaben 165 Pyrimethanil 166 Pyriproxyfen 167 Quinoxyfen 168 Rotenone 169 Secbumeton 170 Siduron 171 Simetryn 172 Spinetoram 173 Spinosad (Spinosyn A) 174 Spirodiclofen 175 Spiromesifen 176 Spirotetramat 177 Spiroxamine Isomer 178 Sulfentrazone 179 Tebuconazole 180 Tebufenozide 181 Tebufenpyrad 182 Tebuthiuron 183 Teflubenzuron 184 Temephos 185 Terbumeton 186 Terbutryn 187 Tetraconazole 188 Thiabendazole 189 Thiacloprid 190 Thiamethoxam 191 Thidiazuron 192 Thiobencarb 193 Thiofanox 194 Thiophanate-methyl 195 Triadimefon 196 Triadimenol 197 Trichlorfon 198 Tricyclazole 199 Trifloxystrobin 200 Triflumizole 201 Triflumuron 202 Triticonazole 203 Vamidothion 204 Zoxamide

33 Sample Preparation at 1:50 Dilution Sample Original Spike Conc. Volume of post-dspe Sample to Add Volume of ACN to Add Volume of Buffer To Add Total Volume Desired Final Conc. Water Blank (WB) Water Spike (WS) Matrix Blank (XB) Matrix Spike 10 Matrix Spike 50 Matrix Spike ng/ml 100 µl 900 µl 4000 µl 5000 µl 0 ng/ml 50 ng/ml 100 µl 900 µl 4000 µl 5000 µl 1.0 ng/ml 0 ng/ml 100 µl 900 µl 4000 µl 5000 µl 0 ng/ml 10 ng/ml 100 µl 900 µl 4000 µl 5000 µl 0.2 ng/ml 50 ng/ml 100 µl 900 µl 4000 µl 5000 µl 1.0 ng/ml 250 ng/ml 100 µl 900 µl 4000 µl 5000 µl 5.0 ng/ml 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