fast screening of over 1000 pesticides and toxins … a. sack.pdflc-ms/ms determination fast...

Fast Screening of Over 1000 Pesticides

and Toxins Using Multiple Techniques

at the US Food and Drug Administration

3rd Latin American Pesticide Residue Workshop

May 8-11, 2011

Chris A. Sack, David Eide, Michael Smoker, Jon Wong,

Kai Zhang, Greg Mercer, Lora Lin, Sara Edison

Fast Screening of Over 1000 Pesticides and Toxins Using Multiple Techniques at the US FDA

Overview

Residue methods

Future

Pesticide Residue Analysis Pilot Course

Introduction

Food and feed matrices

Imports: > 11 million per year

Domestic: ???

Pesticides and other contaminants

1000s that are known

Range: 10 ppb - ???

Analyses

Up to 50 samples per day per lab

Timeframe: 1 day for imports

Overview - Challenges



Overview6 PESTICIDE LABORATORIES

SEATTLE

LOS ANGELES

JEFFERSON

NEW YORK

ATLANTA

KANSAS CITY

Analytical Strategy


Expected Unexpected Unknown

Pesticides and Toxins

Non-targeted screening

GC-MS, LC-MS/LIT

LC-HRMS, DART-HRMS

Targeted analysis

LC-MS/MS & GC-MS/MS

Targeted Quantitative Analysis

LC-MS/MS and GC-MS/MS

>350 Selected analytes

Historical findings

Anticipated findings

Calibration Standard Mixtures

Designed by FDA

Prepared by commercial vendors

Available for purchase

Analytical Strategy


Qualitative Screening Analysis

No standards required

Spectral Library Techniques

GC-MS (fullscan mode)

LC-MS/LIT (IDA-EPI)

Exact Mass Techniques (Exactive Orbitrap)

LC-HRMS

DART-HRMS

Analytical Strategy


LC-MS/MS Determination


Scope: >240 pesticides in 15 minutes

Chromatography: reverse phase

Columns: C18

100 x 2.1 mm, ~3 µm

50 cm x 4.6 mm, 1.9 µm

Mass Spectrometer parameters

Ionization: Positive electrospray

Detection: scheduled MRM (two transitions)

Detection Limit: < 10 ppb for most compounds



XIC of +MRM (358 pairs): 184.1/143.0 amu Expected RT: 3.5 ID: Acephate.1 from Sample 21 (A-J 200 pp... Max. 3.4e6 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.01 2 236 428 607 766 901 989 1096 1245 1445 1655 2416 3323

Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

7.3e6

Inte

ns

ity

, c

ps

3.43

NYK 200 ng/mL



Residues in strawberry sample

Pesticide PPB Pesticide PPB

Boscalid 128 Novaluron 9

Pyraclostrobin 54 Hexythiazox 5

Cyprodinil 47 Spiromesifen 2

Azoxystrobin 45 Propiconazole 1

Pyrimethanil 41 Acetamiprid 1

Chlorantranilaprole 23 Carbendazim 1

Fludioxinil 22 Methomyl 0.7

Fenhexamid 20 Thiophanate methyl 0.5

Bifenazate 9 Methoxyfenozide 0.3

Myclobutanil 9 Fenpyroximate 0.2



Trifloxystrobin @ 0.2 ppb in applesauce

GC-MS/MS Determination


Scope: > 200 pesticides in 20 minutes

GC

Retention time locking

Backflushing mid column

MS/MS detection

MRM (2-3 transitions/compound)

Detection Limit: 1 - 10 ppb for most analytes



Residues in strawberry sample

Pesticide PPB Pesticide PPB

THPI 237 Bifenthrin 7

Boscalid 128 Quinoxyfen 3

Pyraclostrobin 54 Spiromesifen 2

Cyprodinil 47 Propiconazole 1

Pyrimethanil 41 Fenpropathrin 0.7

Azoxystrobin 37 Biphenyl 0.3

Captan 27 Chlorpyrifos 0.1

Fludioxinil 22 Folpet 0.1

Fenhexamid 20 p,p’-DDE 0.1

Bifenazate 9 p,p’-DDT 0.04

Myclobutanil 9 o,p’-DDT 0.03

Malathion 8



o,p’-DDT in strawberry

Standard: 50 ng/mL

Incurred residue

@ 0.03 ppb

What’s New with the Pesticide Residue Program in the FDA?

Scope: > 900 compounds in 20 minutes

Screen for library matches - no standards

Agilent GC-MSD pesticide library

Identification:

Spectral matching of AMDIS deconvoluted

spectra

Retention time

GC-MS Fullscan Screen


GC-MS Fullscan ScreenCelery fullscan TIC


GC-MS Fullscan ScreenCelery deconvolution report

AMDIS NIST

R.T. Compound Name Match R.T. Diff Reverse

Match

Hit Num.

2.5859 Naphthalene-d8 99 -8.0 91 1

2.842 Carvone 75 -8.3 81 2

3.341 EPTC 94 -7.2 89 26.977 Anthracene 80 -5.4 88 5

7.9576 Diisobutyl phthalate 75 1.2 84 11

9.214 Di-n-butylphthalate 65 0.8 90 18

9.658 Parathion 82 2.5 76 2

11.925 Dieldrin 79 1.5 61 214.3189 Chrysene-d12 79 -12.1 91 1

14.8515 Bis(2-ethylhexyl)phthalate 96 3.3 85 7

15.7388 Permethrin I 60 6.5 63 2

15.833 Permethrin II 63 7.0 79

16.518 Cypermethrin II 43 11.7 57 1


Residues discovered and transferred to the LC-MS/MS and GC-MS/MS targeted analyses:

GC-MS Fullscan Screen

Fluopicolide

Spiromesifen

Spirodiclofen

Flonicamide

Chlorfenapyr

Etoxazole

Bifenazate

Etoxazole

Fenamidone

Famoxadone

Quinoxyfen

Sudan I*

*Food color prohibited in US - not added to target analysis


One instrument and Two Approaches

MRM by LC-MS/MS

MS library matching using LC-MS-Linear Ion

Trap Hybrid Mass Spectrometry

Targeted and library screening

Looking to the Future

LC-MS/LIT Mass Spectral Libraries

Kai Zhang1, Jon W. Wong1, Paul Yang2

1. Food and Drug Administration, College Park, MD, U.S.A.

2. Ontario Ministry of the Environment, Ontario, Canada




XIC of +MRM (244 pairs): Exp 1, 142.0/94.0 amu Expected RT: 0.8 ID: Methamidophos from Sample 1 (18 min S-25) of Data20100806 1... Max. 1.0e4 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

In

te

ns

ity

, c

ps

+EPI (229.10) Charge (+0) CE (35) CES (15) FT (250): Exp 2, 6.808 min from Sample 1 (18 min S-25) of Data20100806 10 18 21 25 30 ... Max. 3.8e6 cps.

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z, Da

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

3.2e6

3.4e6

3.6e6

3.8e6

Inte

ns

ity

, c

ps

172.0

229.0

116.0

157.1

142.189.0

61.974.0

XIC of +MRM (244 pairs): Exp 1, 229.1/172.4 amu Expected RT: 6.8 ID: Tebuthiuron from Sample 1 (18 min S-25) of Data20100806 10 1... Max. 2.4e5 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

Inte

ns

ity

, c

ps

LC

MRM

transition

Intensitythreshold

[M+H]+

Enhanced

product ion

spectrum

Library

search

Survey

scan Quantitation

Targeted screening: information dependent acquisition-enhanced

product ion (IDA-EPI) method




Carbaryl

202.1>145

Fit: 93

RevFit: 94

Purity: 89

202.1>127

Carbaryl

202.1>145

Carbaryl confirmed in peach sample




XIC of +MRM (557 pairs): 200.0/107.0 amu Expected RT: 8.6 ID: Pyrimethanil 1 from Sample 46 (1.67 ppb Qginseng) of Data20100416... Max. 6600.0 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0

1000

2000

3000

4000

5000

6000

6600

In

te

ns

it

y,

c

ps

8.38

XIC of +MRM (557 pairs): 200.0/82.0 amu Expected RT: 8.6 ID: Pyrimethanil 2 from Sample 46 (1.67 ppb Qginseng) of Data20100416 ... Max. 3800.0 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0

500

1000

1500

2000

2500

3000

3500

3800

In

te

ns

it

y,

c

ps

8.40

8.278.609.09

XIC of +MRM (557 pairs): 200.0/107.0 amu Expected RT: 8.6 ID: Pyrimethanil 1 from Sample 53 (Qginseng Inc1) of Data20100416 ms... Max. 7900.0 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

7900

In

te

ns

it

y,

c

ps

8.38

XIC of +MRM (557 pairs): 200.0/82.0 amu Expected RT: 8.6 ID: Pyrimethanil 2 from Sample 53 (Qginseng Inc1) of Data20100416 msm... Max. 7600.0 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

7600

In

te

ns

it

y,

c

ps

8.42

False Positive: Pyrimethanil?

•RT match

•Two Pyrimethanil MRMs

• 200 → 107

• 200 → 82

•Ion Ratios (200→107/200→82)

•Requirement: ± 20 %

•Sample/Standard: ~ 60 %

Sample

Standard

200→107

200→107

200→82

200→82




Unknown

Reference

Pyrimethanil

Fit: 35

RevFit: 30

Purity 22

200> 107

EPI spectrum




Currently building MS libraries for targeted

compounds

Plan to increase screening capability by

adding other non-targeted compounds



Resolution

100,000 @ 1 scan per second

10,000 at 10 scans per second

Mass accuracy: <1 ppm

Scan speed: → 10 per second

Scan range: 50 – 4000 m/z

Detection Limit: < 10 ng/ml (100 ppb) most compounds

Sample introduction

LC (shown here)

DART

Exactive Orbitrap® High Resolution MS



Exactive Orbitrap® High Resolution MS

•


Screen > 500 pesticides

Sample introduction by reverse phase uHPLC

Column: C18, 100 x 2.1 mm, 1.9 µm particles

Identification

Retention time

HRMS Resolution of 100,000

Limit of Detection : < 10 ng/ml (100 ppb)

Plan to expand to other chemical contaminants:

mycotoxins, plant toxins, veterinary drugs, dyes,

emerging organic pollutants, etc…


LC-HRMS Pesticide Screening Analysis




RT: 0.00 - 10.53

0 1 2 3 4 5 6 7 8 9 10

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

8.94

9.30

8.64

7.546.94 7.56

7.49

6.84

7.861.02 9.698.12

0.951.12

10.436.615.67 6.545.525.231.24 1.46 4.910.07 1.90 2.550.42 4.223.05 3.43

NL:1.03E9

m/z= 100.00000-1200.00000 MS 20100425_027_DSOrangeInc3

QuEChERS extract of a Peach Sample analyzed by

uHPLC-single stage Orbitrap MS (full scan analysis)




Residues extracted from peaches

Carbendazim

0 1 2 3 4 5 6 7 8 9 10 11 12

100 6.02011

191.95 192.00 192.05 192.10 192.15 192.20 192.25

100 192.07675

0 1 2 3 4 5 6 7 8 9 10 11 12

100 7.99044

201.95 202.05 202.15 202.25 202.35

100202.08608

0 1 2 3 4 5 6 7 8 9 10 11 12

100 8.56261

317.90 318.00 318.10 318.200

100318.00192

0 1 2 3 4 5 6 7 8 9 10 11 12

100 9.12773

336.80 336.90 337.00 337.10 337.20 337.30 337.40

100337.12151

0 1 2 3 4 5 6 7 8 9 10 11 12

1009.41904

341.70 341.90 342.10 342.30 342.50 342.70

100342.07704

0 1 2 3 4 5 6 7 8 9 10 11 12Time (min)

((min)

100 10.62230

365.05 365.15 365.25 365.35m/z

100365.14511

(B)

Carbaryl

Phosmet

Fenbuconazole

Propiconazole

Pyridaben

192.07675

202.08626

337.12145

342.07706

C11H12NO4PS2

C9H9N3O2

C12H11NO2

C19H17ClN4

C15H17Cl2N3O2

C19H25ClN2OS

δM = 0 ppm

δM = -0.89

318.00181

δM = 0.36

δM = 0.18

δM = -0.06

365.14489

δM = 3.34

Sample introduction by Direct Analysis in

Real Time (DART)

HRMS: Resolution = 100,000

Scope: > 500 pesticides and toxins

Analysis time: 7.5 minutes/sample



Screening using DART-HRMS

Method:

Surface of raw agricultural commodities are

moistened with solvent and swabbed with

foam

No sample preparation or extraction!

Swabs are directly analyzed using a DART

ionization source combined with high

resolution mass spectrometry

No chromatography!







Custom foam rail autosampler



He(23S) + nH2O → [(H2O)n-1 + H]+ + OH·- He(11S)[(H2O)n + H]+ + M → [M + H]+ + nH2O




Diazinon

202.08627 m/z

Carbaryl

305.10831 m/z

Diazinon

Carbaryl

Findings

Up to 10 pesticides per sample

Average 2-3 pesticides per sample

Publications

Rapid Commun. Mass Spectrom. 2011, 25, 127–139 Sara. E. Edison1, Lora A. Lin1, Bryan M. Gamble1, Jon Wong2 and Kai Zhang2

1USFDA, Forensic Chemistry Center, Cincinnati, OH, USA

2USFDA, Center for Food Safety and Applied Nutrition, College Park, MD, USA

Practical considerations for rapid screening for pesticides using ambient pressure desorption ionization with high-resolution mass spectrometry, Sara E. Edison1*, Lora A. Lin1, Lenin Parrales2 (Submitted to Food Additives and Contaminants)

1Food and Drug Administration, Forensic Chemistry Center, Cincinnati, OH

2Food and Drug Administration, San Francisco District Office, Alameda, CA






Location: University of Maryland

College Park, MD, USA

By invitation only: Latin American countries

Scope: Focus on fresh fruits and vegetables

Hands-on laboratory training

Instrumental Techniques: LC-MS/MS

Certificate of Accomplishment of Pesticide

Residue Analysis in Fresh Fruit and Produce

June 6-10, 2011



Morning -Lecture Afternoon -Laboratory

Monday Pesticide compliance in LA

Sample preparation

Laboratory SOPs

Sample preparation

Tuesday Principles of GC and LC

Detection: Element and Mass

LC-MS

Group A: LC-MS training

Group B: Calibration and other

sample preparation

Wed Quantitative analysis

Identification and confirmation

QC/QA

Group A: Calibration and other

sample preparation

Group B: LC-MS training

Thursday Method Development

Validation of single lab method

Regulations

Sample analysis

Results interpretation

Friday Review and evaluation

Agenda



Course Instructors



Registration

contact Dr. Janie Dubois at:

[email protected]

Acknowledgements


David Eide, Michael Smoker (USFDA-Kansas City)

Jon Wong, Ronald Roy, Kai Zhang (USFDA-CFSAN)

Greg Mercer, Barb Neuhaus (USFDA-Seattle)

Luis Suguiyama, US Environmental Protection Agency

US Department of Agriculture - Foreign Agriculture Service

fast screening of over 1000 pesticides and toxins … a. sack.pdflc-ms/ms determination fast...

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