Quantitative Analysis of Carbendazim & other Pesticides in Fruit Juices by Direct

Analysis in Real Time (DART®) Mass Spectrometry

IonSense, Inc. 999 Broadway Suite 404

Saugus, MA 01906 USA

Elizabeth Crawford and Brian Musselman

60th ASMS Conference Vancouver, BC Canada

20-24th May 2012

DART-SVP Ionization Source

2

3

1. Objectives:

• Generate standard curve data for carbendazim in orange juice ranging from 1 ppb – 5000 ppb.

• Screen for carbendazim in a variety of orange juices from the EU (2 countries) and the USA. – European maximum residue limit* (MRL) is set at 200 ppb for oranges and

carbendazim is not approved for use on citrus fruits in the US . The US FDA has set an action limit of 10 ppb.

2. Analysis Overview:

• No sample preparation – liquid from consumer juice beverages directly analyzed by transmission mode direct analysis in real time (DART) source coupled with triple quad.

• Positive ion mode; carbendazim transition m/z 192.2 → 160.2.

* http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2011:152:0001:0021:EN:PDF

Carbendazim: Unapproved Fungicide for Citrus Fruits in US

API 4000 QTRAP with DART-SVP

4

MRM Settings • Scan Parameters:

• Positive Ion Mode

• Resolution: Unit

• Fragmentation:

carbendazim major transition monitored m/z 192.2 → 160.2

• DP 56 V

• CE 27 V

• Dwell Time: 100 ms

DART-SVP Settings • DART Source:

• Positive Ion Mode

• Heater Temperature: 250° C

• Motorized Linear Rail:

• Sample Introduction Speed: 0.5 mm/s

4

5

3 µL of liquid sample pipetted onto

stainless steel mesh screen and allowed to completely dry

before DART analysis

Stainless steel mesh screens: Used as the sampling

surface for juice samples

Transmission DART Workflow

Juice is directly

applied to the mesh

6

y = 65.454x R² = 0.9988

0.00E+00

5.00E+04

1.00E+05

1.50E+05

2.00E+05

2.50E+05

3.00E+05

3.50E+05

4.00E+05

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000

Ave

rage

Pe

ak A

rea

(co

un

ts)

Concentration (ppb)

Carbendazim Standard Curve in Orange Juice

n = 3 per conc.

0.00E+00

1.00E+04

2.00E+04

3.00E+04

4.00E+04

5.00E+04

0 100 200 300 400 500

Concentration Range: 1 ppb – 5,000 ppb n = 3

7

0

5

10

15

20

25

USA Apple USA FruitPunch

USA OJ 1 USA OJ 2 USA OJ 3 France OJ 1 Germany OJ1

Germany OJ2

Me

asu

red

Le

vel

(p

pb

)

Fruit Juice Consumer Beverage

Carbendazim Levels: Screening Fruit Juices

n = 8; except n = 16 for German OJs

ND ND ND ND ND

ND = Not Detected

8

• DART-SVP ion source coupled to the API 4000 QTRAP yielded LOD of 2 ppb and LOQ of 15 ppb. • DART-SVP contact closure capability allows the user to acquire data directly through

an Analyst sample queue.

• The standard curve generated for cardbendazim using the transmission DART method was very good with R2 value 0.9988 across 10 – 5000 ppb.

• The reproducibility for an n = 3 across the concentration ranges was generally less than 18 % based on peak areas without internal standard correction.

• The two orange juices from Germany from the same manufacturer, but different lots yielded estimated carbendazim levels of 20 ppb, 10x below the Europe MRL, where the French orange juice contained only trace amounts.

Summary

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Settings •Scan Parameters:

•Positive Ion Mode: m/z 100 -1000

•Resolution: “High” - 50,000 @ 2 Hz

•Fragmentation: None, HCD Gas Off

•Scan Settings: 1 µ-scan by 250 ms max inject time

•AGC Target: Ultimate Mass Accuracy (5e5)

•Exactive Inlet Parameters: •Capillary Temp: 200° C

•Capillary Voltage: 25 V

•Tube Lens Voltage: 120 V

•Skimmer Voltage: 26 V

•All of the following parameters were set to zero: •Sheath Gas Flow, Aux Gas Flow, Sweep Gas Flow, Spray Voltage

DART-SVP Settings • DART Source:

• Positive Ion Mode

• Heater Temperature:

• 250° C

• Motorized Linear Rail: • Sample Introduction Speed: 0.5 mm/s

Thermo Exactive with DART-SVP

10

192.06 192.08 192.10 192.12 192.14 192.16 192.18

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Rela

tive

Ab

un

da

nce

0

20

40

60

80

100192.07675

192.08685

192.07664192.12313

192.09582 192.13880 192.15934

192.07666

192.12279192.08670

192.15833192.13769

NL:2.09E4

C 9 H 9 O 2 N 3 +H: C 9 H 10 O 2 N 3

p (gss, s /p:40) Chrg 1R: 70000 Res .Pwr . @FWHM

NL:5.80E4

blk,10,20ngml_carbendazimspike_graninioj_70k_e+full_pos_03_20120307#439-475 RT: 2.04-2.20 AV: 37 SB: 64 4.16-4.44 T: FTMS + p NSI Full ms [150.00-1000.00]

NL:2.65E5

BLK,10,20ngmL_CarbendazimSpike_MeOH_70K_E+Full_POS_01_20120307#445-477 RT: 2.07-2.21 AV: 33 SB: 56 4.17-4.41 T: FTMS + p NSI Full ms [150.00-1000.00]

192.06 192.08 192.10 192.12 192.14 192.16 192.18

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Rela

tive

Ab

un

da

nce

0

20

40

60

80

100192.07675

192.08685

192.07664192.12313

192.09582 192.13880 192.15934

192.07666

192.12279192.08670

192.15833192.13769

NL:2.09E4

C 9 H 9 O 2 N 3 +H: C 9 H 10 O 2 N 3

p (gss, s /p:40) Chrg 1R: 70000 Res .Pwr . @FWHM

NL:5.80E4

blk,10,20ngml_carbendazimspike_graninioj_70k_e+full_pos_03_20120307#439-475 RT: 2.04-2.20 AV: 37 SB: 64 4.16-4.44 T: FTMS + p NSI Full ms [150.00-1000.00]

NL:2.65E5

BLK,10,20ngmL_CarbendazimSpike_MeOH_70K_E+Full_POS_01_20120307#445-477 RT: 2.07-2.21 AV: 33 SB: 56 4.17-4.41 T: FTMS + p NSI Full ms [150.00-1000.00]

192.06 192.08 192.10 192.12 192.14 192.16 192.18

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

0

20

40

60

80

100192.07675

192.08685

192.07664192.12313

192.09582 192.13880 192.15934

192.07666

192.12279192.08670

192.15833192.13769

NL:2.09E4

C 9 H 9 O 2 N 3 +H: C 9 H 10 O 2 N 3

p (gss, s /p:40) Chrg 1R: 70000 Res .Pwr . @FWHM

NL:5.80E4

blk,10,20ngml_carbendazimspike_graninioj_70k_e+full_pos_03_20120307#439-475 RT: 2.04-2.20 AV: 37 SB: 64 4.16-4.44 T: FTMS + p NSI Full ms [150.00-1000.00]

NL:2.65E5

BLK,10,20ngmL_CarbendazimSpike_MeOH_70K_E+Full_POS_01_20120307#445-477 RT: 2.07-2.21 AV: 33 SB: 56 4.17-4.41 T: FTMS + p NSI Full ms [150.00-1000.00]

192.06 192.08 192.10 192.12 192.14 192.16 192.18

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Rela

tive

Ab

un

da

nce

0

20

40

60

80

100192.07675

192.08685

192.07664192.12313

192.09582 192.13880 192.15934

192.07666

192.12279192.08670

192.15833192.13769

NL:2.09E4

C 9 H 9 O 2 N 3 +H: C 9 H 10 O 2 N 3

p (gss, s /p:40) Chrg 1R: 70000 Res .Pwr . @FWHM

NL:5.80E4

blk,10,20ngml_carbendazimspike_graninioj_70k_e+full_pos_03_20120307#439-475 RT: 2.04-2.20 AV: 37 SB: 64 4.16-4.44 T: FTMS + p NSI Full ms [150.00-1000.00]

NL:2.65E5

BLK,10,20ngmL_CarbendazimSpike_MeOH_70K_E+Full_POS_01_20120307#445-477 RT: 2.07-2.21 AV: 33 SB: 56 4.17-4.41 T: FTMS + p NSI Full ms [150.00-1000.00]

Theoretical [M+H]+

[M+H]+

C9H10N3O2

-0.6 ppm

[M+H]+

C9H10N3O2

-0.5 ppm

Spiked in Orange Juice

Spiked in Methanol

Nearing LOD for detection directly out of orange juice on Exactive

Carbendazim: 10 ppb in Orange Juice & MeOHCarbendazim Positive Ion

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Gerstel Twister Spin Bar: Sample Concentration

Twister was spun overnight in orange juice with

50 ppb level of 10 pesticides

Higher Log Kow = greater uptake onto the Twister sorptive surface. 12

10 Pesticide Mix [Exact Mass + H] + Log Kow

Carbaryl 202.08626 2.4

Metalaxyl 280.15433 1.7

Spiroxamine 298.27406 Unknown

Fenhexamid 302.07091 3.5

Diazinon 305.10833 3.8

Pyriproxyfen 322.14377 5.6

Pyridaben 365.14489 5.5

Dimethomorph 388.13101 2.7

Azoxystrobin 404.12410 2.5

Difenoconazole 406.07197 4.2

RT: 0.00 - 8.20

0 1 2 3 4 5 6 7 8

Time (min)

0

100

0

100

0

100

0

100

0

100

Rela

tive

Ab

un

da

nce

0

100

0

100

0

1005.03

5.60 5.756.75 7.47 7.502.75 4.333.33 3.990.79

5.035.60 5.75

6.75 7.47 7.502.75 4.333.33 3.990.79

5.00 5.035.60

5.85 6.65 7.27 7.47

4.99 5.055.595.58 6.75 7.135.85 7.43

4.99 5.055.59

5.85 7.04 7.27 7.50

5.055.035.59 5.85 7.216.74 7.31

5.055.00 5.087.275.60 7.367.13

5.087.315.03 5.59 7.477.045.77

NL: 4.23E4

m/z= 202.08512-202.08714 MS 120424-01_Twister19&20_OSCranLt_Blank&50ppbSpin

NL: 4.23E4

m/z= 202.08512-202.08714 MS 120424-01_Twister19&20_OSCranLt_Blank&50ppbSpin

NL: 2.20E5

m/z= 280.15286-280.15567 MS 120424-01_Twister19&20_OSCranLt_Blank&50ppbSpin

NL: 6.78E4

m/z= 302.06818-302.07120 MS 120424-01_Twister19&20_OSCranLt_Blank&50ppbSpin

NL: 1.88E6

m/z= 305.10634-305.10939 MS 120424-01_Twister19&20_OSCranLt_Blank&50ppbSpin

NL: 2.62E6

m/z= 322.14134-322.14456 MS 120424-01_Twister19&20_OSCranLt_Blank&50ppbSpin

NL: 9.00E5

m/z= 365.14233-365.14598 MS 120424-01_Twister19&20_OSCranLt_Blank&50ppbSpin

NL: 1.71E5

m/z= 388.12772-388.13161 MS 120424-01_Twister19&20_OSCranLt_Blank&50ppbSpin

10 Pesticides Concentrated onto Twister: 50 ppb in orange juice spun overnight → DART/MS

Exactive XICs generated with +/- 5 ppm m/z window.

Twister Blank Spun in Blank OJ

Twister Spun in Spiked OJ

50 ppb level mixture of pesticides were well concentrated onto the Twister and were then directly desorbed from the Twister sorptive material for direct exact mass analysis with DART.

120424-01_Twister19&20_OSCranLt_Blank&50ppbSpin #598-654 RT: 4.99-5.41 AV: 57 SB: 116 0.46-1.46 NL: 1.34E6T: FTMS {1,1} + p NSI Full ms [100.00-500.00]

200 220 240 260 280 300 320 340 360 380 400 420

m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive

Ab

un

da

nce

322.14321

305.10788

365.14431

406.07125

280.15399 388.13052217.17969

372.09673

298.27375

234.20614 355.33151

338.34136

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Direct Twister DART-MS Analysis

10 Pesticide Mix (50 ppb) in orange juice DART Heater 325

C; linear rail movement 0.2 mm/s

• There is a very good concentration effect on the Gerstel Twister for direct screening for low levels (50 ppb) of pesticides with ambient ionization DART source.

• Long lasting signal from the Twister experiments indicates that a large amount of material is collected on the Twister surface • A temperature ramp of the DART heater would yield thermal

separation and long lasting signal is beneficial for MS/MS experiments.

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Summary