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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
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 100 µg/mL Individual Pesticide Standards
Transfer 1.0 mL of each standard to one 10 mL volumetric flask
Transfer remainder
(~100 µL) to individual vials for LC
and MS optimization
10 µg/mL Intermediate
Pesticide Standard
Solution (IPSS)
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
Transfer 0.5 mL of IPSS to one 10 mL volumetric flask, fill to mark with ACN
Preparation of Fortification Solutions
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.
10 µg/mL Intermediate
Pesticide Standard
Solution (IPSS)
0.5 µg/mL
F.S.
2.0 µg/mL
F.S.
5.0 µg/mL
F.S.
200 µL per 10g sample
10 ng/mL Spike
50 ng/mL Spike
250 ng/mL Spike
250 µL per 10g sample
500 µL per 10g sample
Transfer 2.0 mL of IPSS to one 10 mL volumetric flask, fill to mark with ACN
Transfer 5.0 mL of IPSS to one 10 mL volumetric flask, fill to mark with ACN
Preparation of Calibration Stock Solutions
10 µg/mL Intermediate
Pesticide Standard
Solution (IPSS)
Calibration Level
Concentration (ng/mL)
Solution Used to Make New Level
(ng/mL)
Volume of Solution
Required (mL)
Add ACN to mark - Total Volume (mL)
Final Volume Remaining
(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
400 ng/mL
200 ng/mL
100 ng/mL
1 ng/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
LC Optimization
• 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
Time (min.)
Number of Analytes
% Total Analytes
0 to 4 15 7
4 to 6 62 30
6 to 8 76 37
10 to 14 51 25
Time (min.) A (%) B (%)
0 90 10
4 40 60 8 30 70
11 0 100
12 0 100 12.01 90 10
15 90 10
204 pesticides 5 internal standards
MS/MS Optimization
Std # Q1 m/z (amu)
Q3 m/z (amu)
tR (min) Component Name
Declustering Potential (V)
Collision Energy (V)
Cell Exit 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
Pesticide standard mixes are individually optimized
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
WB WB WS WS
MB MB MB MB
WB WB WS WS 10 10 10 10
50 50 50 50
250 250 250 250 250 ppb
50 ppb
10 ppb
0 ppb
0 ppb 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 or MMCS Cal. Standards
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
SOCS and MMCS Preparation – Changes to Spike Volumes at 1:50 Dilution
• 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
EXCEL Template A – LOD/LOQ/R2
Template B – Method Detection Limit Template C – Recovery Template D – Uncertainty
1. Samples (SOCS, MMCS, Samples, MMCS, SOCS)
2. MDL (MMCS Level 1 and 2, Matrix Spike 10-1; 8 replicates)
3. LOD/LOQ/R2 (SOCS Levels 1-9; 8 replicates)
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
r2 Average # Analytes
X > 0.99 152 ± 25 (74%)
X < 0.99 46 ± 25 (23%)
Not Detected 6 ± 3 ( 3%)
All Commodities
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
• Abamectin
• Alanycarb
• Benfuracarb
• Chlorfluazuron
• Clofentazine
• Difenoconazole
• Doramectin
• Eprinomectin
• Etoxazole
• Fenazaquin
• Fluazinam
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%)
r2 Average # Analytes
X > 0.99 152 ± 25 (74%)
X < 0.99 46 ± 25 (23%)
Not Detected 6 ± 3 ( 3%)
• Hydramethylnon
• Isocarbophos
• Ivermectin
• Metaflumizone
• Moxidectin
• Novaluron
• Oxadixyl
• Prothioconazole
• Spiromesifen
• Thiofanox
MDL (ng/g) Average # Analytes
0 < X < 0.20 179 ± 3 (88%)
X > 0.20 20 ± 3 (10%)
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
QUESTIONS?
Thank you for your attention!
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
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
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
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)
0 ng/mL 100 µL 900 µL 4000 µL 5000 µL 0 ng/mL
Water Spike (WS)
50 ng/mL 100 µL 900 µL 4000 µL 5000 µL 1.0 ng/mL
Matrix Blank (XB)
0 ng/mL 100 µL 900 µL 4000 µL 5000 µL 0 ng/mL
Matrix Spike 10
10 ng/mL 100 µL 900 µL 4000 µL 5000 µL 0.2 ng/mL
Matrix Spike 50
50 ng/mL 100 µL 900 µL 4000 µL 5000 µL 1.0 ng/mL
Matrix Spike 250
250 ng/mL 100 µL 900 µL 4000 µL 5000 µL 5.0 ng/mL
Sample Preparation at 1:50 Dilution
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