Multiresidue Analysis of 204 Pesticides in Fresh Produce ...c.ymcdn.com/sites/ · PDF fileMultiresidue Analysis of 204 Pesticides in Fresh Produce Using the QuEChERS Method Followed

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 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.


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





Preparation of Calibration Stock Solutions


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









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



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









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





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









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%)


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%)


r2 Average # Analytes

X > 0.99 152 ± 25 (74%)

X < 0.99 46 ± 25 (23%)


• 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


Matrix Spike 50


Matrix Spike 250


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

Documents

Multiresidue Analysis of 204 Pesticides in Fresh Produce ...c.ymcdn.com/sites/ · PDF fileMultiresidue Analysis of 204 Pesticides in Fresh Produce Using the QuEChERS Method Followed