Determination of Neonicotinoid Insecticides and Strobilurin Fungicides

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Determination of neonicotinoid insecticides and strobilurin fungicides in particle phase atmospheric samples by liquid chromatography-tandem mass spectrometry Renata Raina-Fulton J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01347 • Publication Date (Web): 11 May 2015 Downloaded from http://pubs.acs.org on May 17, 2015

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Journal of Agricultural and Food Chemistry

Determination of neonicotinoid insecticides and strobilurin fungicides in particle phase atmospheric samples by liquid chromatography-tandem mass spectrometry

Renata Raina-Fulton1* 1*

University of Regina, Department of Chemistry & Biochemistry, 3737 Wascana Parkway,

Regina, SK, S4S 0A2, corresponding author e-mail: [email protected]

1 ACS Paragon Plus Environment


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Abstract A liquid chromatography/tandem mass spectrometry method has been developed for the

3

determination of neonicotinoids and strobilurin fungicides in the particle phase fraction of

4

atmosphere samples. Filter samples were extracted with pressurized solvent extraction, followed

5

by a clean-up step with solid phase extraction. Method detection limits for the seven

6

neonicotinoid insecticides and six strobilurin fungicides were in the range of 1.0 to 4.0 pg/m3.

7

Samples were collected from June-September 2013 at two locations (Osoyoos and Oliver) in the

8

southern Okanagan Valley Agricultural Region of British Columbia where these insecticides and

9

fungicides are recommended for use on tree fruit crops (apples, pears, cherries, peaches,

10

apricots) and vineyards. This work represents the first detection of acetamiprid, imidacloprid,

11

clothianidin, kresoxim-methyl, pyraclostrobin, and trifloxystrobin in particle phase atmospheric

12

samples collected in the Okanagan Valley in Canada. The highest particle phase atmospheric

13

concentrations were observed for imidacloprid, pyraclostrobin, and trifloxystrobin at 360.0

14

pg/m3, 655.6 pg/m3, and 1908.2 pg/m3, respectively.

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Key Words: neonicotinoid insecticides, strobilurin fungicides, atmospheric particle

16

concentrations of pesticides


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INTRODUCTION In this paper a new method for the simultaneous analysis of neonicotinoid insecticides

19

and strobilurin fungicides in particles in the atmosphere was developed. Table 1 shows that

20

these pesticides have low volatility such that particle transport in the atmosphere may play a role

21

in their movement in the environment. Both of these classes of pesticides have not been

22

previously detected in atmospheric samples (gas or particle phase) in North America and a

23

suitable method was required to be developed for their analysis in atmospheric samples capable

24

of detection in the low pg/m3 range. The range of volatility of these pesticides and similar

25

solubility in organic solvents that are required to extract pesticides from solid matrices such as

26

particles makes development of a simultaneous method for both of these classes of pesticides

27

feasible. Pressurized solvent extraction was used for their extraction as this approach is

28

commonly used in our lab and others for analysis of pesticides and other related contaminants in

29

atmospheric samples (1-4).

30

Neonicotinoids are a class of neuro-active insecticides that have high binding affinity to

31

insect acetylcholine receptor (5). Neonicotinoids registered for use in Canada include

32

acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam (see Table 1) (6). They

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are commonly used on crops within Canada including corn, soybeans, canola, sunflowers, and a

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wide variety of fruit tree crops as well some are used in commercial products such as flea collars

35

for dogs and cats (6, 7). Strobilurin fungicides are a newer class of fungicides of the quinine

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outside inhibitors (Qol) group or act in a similar way to the natural strobilurin A, produced by the

37

strobilurus tenacellus fungus (8). This class includes azoxystrobin, fluoxastrobin, kresoxim-

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methyl, and pyraclostrobin, and trifloxystrobin which are also registered for use in Canada (see

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Table 1) (6). 3 ACS Paragon Plus Environment


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In recent years, liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods

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have been developed for the analysis of selected neonicotinoid insecticides with the number of

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applications of these methods increasing due to concerns of their harmful effects on honey bees

43

and potential transport in the environment in surface waters due to their higher water solubility’s

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(7-12). Little attention has been given to studying the presence of neonicotinoids or strobilurin

45

fungicides in the atmosphere. Due to the low volatility and high polarity of neonicotinoids,

46

liquid chromatography is preferred over more complex GC methods. LC with the use of

47

photodiode array or electrochemical detectors has also been used as well as capillary

48

electrophoresis with mass spectrometry (13-15). However, most recent methods of analysis of

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selected neonicotinoid insecticides have LC-MS/MS with electrospray ionization in positive ion

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mode (ESI+) and included their analysis in honey liqueur, bee pollen, fruits, and vegetation (9,

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14, 16-23). These sample matrices differ significantly from atmospheric samples.

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LC/MS/MS methods for neonicotinoids have not included the simultaneous analysis of

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strobilurin fungicides nor do the majority of the methods contain as complete list of individual

54

neonicotinoid insecticides as this study. Strobilurin fungicides can be analyzed by GC or LC

55

methods. GC/MS for analysis of strobilurin fungicides have utilized selected ion monitoring

56

(SIM) mode with electron impact ionization (EI), while only selected strobilurin fungicides have

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been included in multiresidue LC/MS/MS methods or specific LC/MS/MS methods for

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individual fungicides (24-28). Although many of the strobilurin fungicides can be analyzed by

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GC/MS with EI, confirmation of fungicide identity is limited (29) and atmospheric sample

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extracts are more prone to MS interference issues with EI as compared to negative chemical

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ionization GC/MS at low atmospheric concentrations (30, 31). LC/MS/MS methods also provide

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added confirmation ability with a four point identification approach (two selected reaction 4 ACS Paragon Plus Environment

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monitoring (SRM) transitions) rather than monitoring of a single ion for each strobilurin

64

fungicide in GC/MS. In addition the use of LC/MS/MS allows for an opportunity to

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simultaneous analyze both strobilurin fungicides and neonicotinoids for atmospheric particles

66

where sample extract volume available for chemical analysis is limited.

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The sample preparation methods developed for use for LC/MS/MS methods for analysis

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of neonicotinoids are largely designed for water analysis rather than solid matrices that are

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required for collection of atmospheric samples. Extraction of pesticides from these solid

70

matrices used in air sampling has generally utilized pressurized solvent extraction with organic

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solvents rather than water prior to any clean-up steps such as solid-phase extraction (1-4).

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Selected neonicotinoid insecticides or strobilurin fungicides in solid food product samples have

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also been extracted with pressurized solvent extraction and particle phase atmospheric samples

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have been extraction with microwave-assisted extraction (17, 18, 32, 33). A variety of other

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chemical classes of pesticides (organochlorines, organophosphorus pesticides, and azole

76

fungicides) have been extracted from solid matrices used in atmospheric sampling using

77

pressurized solvent extraction (1-4). Due to the high solubility of these insecticides and

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fungicides in organic solvents generally ethyl acetate, acetonitrile, or acetone are selected as the

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extraction solvent or for sample preparation (14, 17-20, 24-28, 32-33). It should be noted that it

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is extremely difficult to selectivity extract specific chemical classes of pesticides in a specific

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organic solvent without loss of other target compounds and thus multiple pressurized solvent

82

extraction steps are generally not preferred. Solid phase extraction (SPE) methods are generally

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used to provide the clean-up of sample extracts (in organic solvents from pressurized solvent

84

extraction) and often designed for specific chemical classes of pesticides due to large range in

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polarities of pesticides and solubility’s in different organic solvents. Atmospheric sample 5 ACS Paragon Plus Environment


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extract volume is limited with often only 1 mL of final extract available for SPE stages for all

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chemical classes of pesticides of interest.

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Table 1 shows that the vapour pressures of neonicotinoid insecticides and strobilurin

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fungicides are generally 97.0% purity) were obtained from

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VWR Scientific (West Chester, PA, USA). Solids or stock solutions at 100 µg/mL in

113

acetonitrile or methanol of strobilurin fungicides (azoxystrobin, dimoxystrobin, fluoxastrobin,

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kresoxim-methyl, pyraclostrobin, and trifloxystrobin) and neonicotinoid insecticides

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(acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiamethoxam, and

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thiacloprid) were supplied by Chem Service Inc. (West Chester, PA, USA). Solid of

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imidacloprid-d4, diazinon-d10 and malathion-d10 were purchased from C/D/N Isotopes Inc.

118

(Pointe-Claire, Quebec, QC, Canada).

119

Pesticide Standards

120

Deuterated internal standard (diazinon-d10) had previously been used as a surrogate

121

standard for other chemical classes in our laboratory (1) and was found to behave with similar

122

retention characteristics as the pesticides under study on C18 SPE sorbent tubes and was used to

123

evaluate recoveries. Diazinon-d10 also provides excellent sensitivity with both LC/MS/MS and

124

GC/MS methods and thus can be used as a multi-class surrogate compound. No deuterated

125

strobilurin fungicides were commercially available. The only commercial available deuterated

126

neonicitinoid insecticide at the time of this study was imidacloprid-d4 which was used as the

127

internal standard for LC/MS/MS analysis. Malathion-d10 can also be used as an internal standard



128

and was used to determine the final volume of the dried fraction F1 from the SPE clean-up after

129

addition of internal standard from the SPE stage which was ∼1.0 mL.

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Solids of individual pesticides (∼1 mg) were dissolved in 1 mL methanol with further

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diluted to prepare individual stock solutions at 1.00 X105 ng/mL in methanol and stored at -4ºC.

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Suitable calibration standards were prepared by dilution of a standard mixture at 1000 ng/mL and

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internal standard (IS) with pesticide grade methanol with final concentration of the internal

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standard, imidacloprid-d4, at 20 ng/mL in all standards and samples. All final diluted standards

135

and samples were prepared on day of analysis. The calibration range typically examined was

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MDL-100 ng/mL with lowest standard prepared at 0.5 ng/mL.

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Sample collection and preparation of air extracts

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Air samples were collected in the southern Okanagan Valley agricultural region at two

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field sites (BC Ministry of Environment/Canada Customs Border station in Osoyoos, BC; and

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Environment Canada Water Quality Site near Oliver, BC). The distance between the Osoyoos

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and Oliver field site is ∼20 km. Air samples are collected using a PUF (polyurethane foam) high-

142

volume air sampler (Tisch Environmental, Cleves, OH) operated continuously at a flow rate of

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225 L/min. The start dates for the samples collected for this study at Osoyoos were June 28,

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2013; July 29, 2013; August 6, 2013; August 9, 2013, and August 20, 2013. The last sampling

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period end date was September 3, 2013. At the Oliver site sampling start dates were the same

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except no sample was available for the August 6 or 9, 2013 sampling period due to instrument

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issues. Average duration of samples collected at the Osoyoos sampling location was 13.2 days.

148

Due to restrictions on use of the sampler at night from noise concerns from the motor of the air

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sampler to nearby public homes adjacent to the customs station the air sampler at Osoyoos was

150

operated continuously from the hours of 7 AM to 9 PM local time (no operation overnight) with 8 ACS Paragon Plus Environment

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counter time reported for duration the sample motor was operated. At the Oliver site the air

152

sampler was operated continuously (24 hours daily throughout the sampling period).

153

The sampling module contained the PUF/sorbent cartridge (for gas-phase) and a glass

154

fiber filter (for particle fraction) as previously described (1). The sampling head is equipped

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with a PM10 cyclone with levels of PM10 (particles < 10 µm in diameter) determined from

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gravimetric analysis of the filters ranging from 13.1X106-22.8X106 pg/m3 and 15.3X106-

157

22.4X106 pg/m3 at Osoyoos and Oliver, respectively. There was a weak relationship of PM10

158

levels and temperature over the agricultural season, however for this period under investigation

159

the average temperature during the sampling period ranged from 21.3-26.0 °C. Precipitation

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amounts were low at both sites with rainfall amounts 129.2

15, 13

1.19±17.1

203.1>113.0

15, 12

0.665±9.96

271.2>56.0

25, 30

271.2>99.0

25, 17

1.02±10.9

271.2>189.1

25, 10

0.425±20.5

271.2>225.7

25, 11

0.0154±19.8

292.2>211.4

19, 12

292.2>132.0

19, 20

0.556±22.8

292.2>181.3

19, 27

0.408±14.3

250.1>169.3

17, 13

250.1>132.0

17, 15

256.1>175

20, 15

256.1>209.5

20, 15

0.887±6.72

0.261±11.0



Acetamiprid

Thiacloprid

222.68

252.72

5.06

7.23

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223.1>126.1

20, 20

223.1>56.0

20, 15

0.619±14.7

223.1>90.0

20, 30

0.160±22.9

253.1>126.0

25, 22

253.1>186.2

25, 20

404.1>372.4

20, 13

404.1>329.4

20, 30

327.6>205.3

15, 10

327.6>238.5

15, 10

459.2>427.4

25, 15

459.2>188.2

25, 30

314.1>206.3

15, 7

314.1>116.0

15, 15

0.799±12.3

314.1>267.4

15, 5

0.672±13.4

388.1>163.5

20, 22

388.1>194.5

20, 11

409.1>186.4

20, 20

409.1>206.4

20, 20

0.0350±32.6

Strobilurin Fungicides Azoxystrobin

Dimoxystrobin

Fluoxastrobin

Kresoxim-Methyl

Pyraclostrobin

Trifloxystrobin

403.39

326.39

458.83

313.35

387.82

408.37

10.95

11.27

12.89

12.96

14.32

15.75

0.152±19.1

0.0554±21.4

0.457±9.91

0.573±24.8

0.433±15.7

Deuterated Internal Standards Imidacloprid-d4 (IS)

259.66

4.59

260.1>213.1* 20, 14 260.1>179.1

20, 14


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Diazinon-d10

314.35

13.14

(SURR) Malathion-d10 (DILUTION

340.36

12.51

315.1>170.0

30, 20

315.1>154.0

30, 22

341.0>132.1

20, 14

341.0>100.0

20, 25

0.739±3.37

0.524±1.90

CHECK STD) 549

550

*SRM Transition used for determination of response ratio (area standard/area internal standard)



551

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Table 3: Method Detection Limits for Neonicotinoid Insecticides and Strobilurin Fungicides Compound

SRM1

Method

Method

Y=mx+b

SRM2

Detection

Detection

(Calibration range

(Optional

LimitA

LimitB

MDL-100 ng/mL)

SRM3 or

(ng/mL)

(pg/m3)

R2

SRM4) Neonicotinoid Insecticides Dinotefuran

203.1>157.2 1.0

1.0

0.0211x+0.0152

0.993

203.1>129.2 2.0

2.0

0.0187x-0.0005

0.996

203.1>113.0 1.0

1.0

0.0134x+0.0167

0.997

271.2>56.0

2.0

2.0

0.0573x-0.0066

0.997

271.2>99.0

2.0

2.0

0.0519x+0.0028

0.999

271.2>189.1 2.0

2.0

0.0207x-0.0086

0.995

271.2>225.7 6.0

5.9

0.00104x-0.00305

0.978

Thiamethoxam 292.2>211.4 1.0

1.0

0.00941x-0.00350

0.999

292.2>132.0 10.0

9.8

0.00585x+0.00187

0.991

292.2>181.3 1.0

1.0

0.00367x+0.00053

0.992

250.1>169.3 2.0

2.0

0.00520x+0.00510

0.996

250.1>132.0 1.0

1.0

0.00475x+0.00070

0.998

256.1>175

4.0

3.9

0.00401x+0.00230

0.996

256.1>209.5 4.0

3.9

0.00110x-0.00031

0.982

223.1>126.1 1.0

1.0

0.0289x-0.0038

0.995

Nitenpyram

Clothianidin

Imidacloprid

Acetamiprid


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223.1>56.0

2.0

2.0

0.0168x+0.0025

0.997

223.1>90.0

4.0

3.9

0.00487x-0.00228

0.982

253.1>126.0 2.0

2.0

0.0480x+0.0100

0.995

253.1>186.2 8.0

7.9

0.00111x+0.00614

0.983

404.1>372.4 0.5

0.5

0.0509x-0.0092

0.998

404.1>329.4 8.0

7.9

0.00729x+0.00814

0.995

Dimoxystrobin 327.6>205.3 2.0

2.0

0.0612x-0.045

0.994

327.6>238.5 4.0

3.9

0.00347x-0.00593

0.998

459.2>427.4 4.0

3.9

0.0855x+0.0970

0.998

459.2>188.2 2.0

2.0

0.0389x+0.0389

0.996

Kresoxim-

314.1>206.3 2.0

2.0

0.146x-0.061

0.999

Methyl

314.1>116.0 2.0

2.0

0.109x+0.0184

0.998

314.1>267.4 2.0

2.0

0.0915x-0.0064

0.999

388.1>163.5 2.0

2.0

0.0145x+0.0036

0.994

388.1>194.5 2.0

2.0

0.0102x+0.0013

0.994

Trifloxystrobin 409.1>186.4 2.0

2.0

0.0260x+0.0184

0.998

409.1>206.4 2.0

2.0

0.0123x-0.0020

0.995

Thiacloprid

Strobilurin Fungicides Azoxystrobin

Fluoxastrobin

Pyraclostrobin

552

553

A

554

fit regression line.

Based on calibration standards –lowest calibration standard within ≤ 25% deviation from best-



555

B

556

duration of sampling 13.2 days -continuous air sampling); sample extract volume 1.0 mL with

557

0.25 mL taken for SPE clean-up stage.

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Based on average air volume obtained from samples collected at Osoyoos of 4073 m3 (average

558 559 560


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561

Table 4: Percent Recoveries of Neonicotinoid Insecticides and Strobilurin Fungicides from

562

Extraction (and Drying). Accelerated solvent extraction recoveries from first extraction with

563

solvent selected as ethyl acetate. Drying step is included as it is required for concentration of

564

extracts prior to LC/MS/MS analysis. Particles on filter spiked at pesticide level of 50 ng/mL in

565

final 1 mL extract.

566

Compound

% Recoveries ± Relative Standard Deviation (N=3)

567

Dinotefuran

97.3 ± 5.8

Nitenpyram

Determination of Neonicotinoid Insecticides and Strobilurin Fungicides

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