July, 2008Page 1

Finding Non-targeted (Unknown) Pesticides

Using GC/MS and LC/Q-TOF

Chin-Kai Meng, Ph.D.Applications ChemistWilmington, Delaware

chin_meng@agilent.com302-633-8388

July, 2008Page 2

Outline - Workflow for Non-targeted Pesticides Analysis

• Business Issues and Challenges

• Sample Prep (QuEChERS) – Lehotay, Anastassiades and Mastovska

• GC/MSD

• (GC or LC)-QQQ

• LC/Q-TOF

July, 2008Page 3

Business Issues and Challenges

• Global trade increases the number of pesticides to monitor (> several hundreds)

• Regulation driving lower detection limits

• Speed: doing more analyses in a day

• Need for fast startup

Faster Data Review and Reporting

Total Solution Offering

Screen for Unknowns

July, 2008Page 4

GC-MSD Workflow

GC/MS (PTV) SIM/Scan– for known and unknown Final Report

Deconvolution

(+backflush)

Screen

Confirm

Quantify

July, 2008Page 5

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

TICs of Surface Water Extracts

How many pesticides (drugs, allergens etc.) are in these samples and how long does it take you to confirm?

July, 2008Page 6

17 Surface Water TICs (Scan): Pesticide Analysis Using DRS with Pesticide Database (927 entries)

*CDFA is the California Department of Food and Agriculture

CDFA* Agilent DRS

Targets Found (not counting

ISTD)37 Same 37

+99 more

False Positives 1 0

Processing Time ~8 hrs 32 min

Saving 7.5 hoursDRS: Deconvolution Reporting Software

July, 2008Page 7

What is Deconvolution?“…in the broad sense of extracting one signal from a complex mixture...” (From AMDIS)

There are four deconvoluted components under the white TIC.

July, 2008Page 8

AMDIS

Automatic Mass spectral Deconvolution and IdentificationSystem

• Developed by the National Institute of Standards and Technology (NIST)

• Developed to detect chemicals in violation of Chemical Weapons Convention (must minimize false positives and false negatives in reporting)

• Used to identify target compounds in complex matrices

How Does Deconvolution Work?

July, 2008Page 9

50

170 280

31075

185

160

Eliminate Ions Don’t Fit the Criteria

July, 2008Page 10

50

170 280

50170280

31075

75

185

185

310

160

160

Extracted Ion Chromatograms

(EIC)

Ion grouping criteria:

1. Same RetTime at apex

2. Same peak width

Spectrum is Deconvoluted/Cleaned

July, 2008Page 11

50

170 280

50170280

Related ions are grouped together as

a component.

A component in AMDIS

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Deconvolution

TIC of Spinach Extract

More than 370 peaks

found

Library Search

July, 2008Page 13

Carbaryl

PeachScan at 5.615 min

Deconvoluted/extracted spectrum

Library spectrum

A component in the scan above.

The power of deconvolution is appreciated while comparing the top two spectra (raw scan and the compound spectrum hidden in the raw scan).

July, 2008Page 14

Fenbuconazole

PeachScan at 10.776 min

Deconvoluted/extracted spectrum

Library spectrum

A component in the scan above.

July, 2008Page 15

DRS Report - Peach

July, 2008Page 16

DRS report from a SIM data file – for Additional Confirmation (4 ions for each target compound)

July, 2008Page 17

Can We Quant on Deconvoluted Ions?

• Yes!

• MSD ChemStation (G1701AA) E.02

• Deconvolution Reporting Software, DRS (G1716AA) A.04

• Pesticide RTL Library (G1672AA) – 927 entries

July, 2008Page 18

5 ion overlay

3 spectra

MSD & AMDIS areas & amounts

MSD ion AMDIS ion

“x” and “A”indicators

QEdit, p,p’-DDT selected

July, 2008Page 19

Graphics Report

MSD and AMDIS 5-ion overlay

Raw, deconvoluted and library spectra

MSD and AMDIS areas and amounts

July, 2008Page 20

Summary Quant Report optionally includes both MSD ChemStation and AMDIS amounts

partial report

July, 2008Page 21

DRS A.04 Report after importing AMDIS results QEdit reviewed with manual integrations

July, 2008Page 22

GC-MSD Workflow Summary

• GC/MS in full scan mode combined with deconvolution reporting software (DRS) enables unknown pesticide analysis at 10 µg/kg (ppb) in various food commodities from one injection

• Significant time saved on data analysis (screen and quant on 927pesticides), from hours to minutes

Application Note 5989-7670EN: Replacing Multiple 50-Minute GC and GC-MS/SIM Analyses with One 15-Minute Full-Scan GC-MS Analysis for Non-targeted Pesticides Screening and >10x Productivity Gain

SIM/ScanGC/MS (PTV)– for known/unknown

256

83

401 Final Report S

C

Q

Deconvolution

Library Search(+backflush)

July, 2008Page 23

LC/MS Workflow

LC/Q-TOF or TOF Full Spectrum – for unknown compounds Another

injection for MS/MS (QQQ or QTOF)

Confirm

Molecular Formula GenerationC16 H19 N3 P ClC15 H25 O P S ClC18 H21 O P Cl

Exact Mass Database Search

Screen

Quantify

July, 2008Page 24

Why Use (Q-)TOF?

QQQ• Routine target compound screening/quantitation in a single injection

• MRM detection limit meets all regulatory requirements

TOF/Q-TOF• Always full spectrum• Un-limited number of routine target and non-target compound screening

(using exact mass databases)

Q-TOF• Full scan accurate MS/MS spectra for added confidence for total

unknowns

All systems share SAME software platform, ion source, and collision cell – 100% workflow portability

July, 2008Page 25

Agilent Q-TOF Fundamentals

Ion opticsCommon with Q & QQQ

Flight tubeCommon with TOF

Octopole 1

DC Quad

Collision cellCommon with QQQ

Rough Pump

Turbo Turbo Turbo

Quad Mass Filter (Q1)

Collision CellLens 1 and 2

Octopole 2

Ion Pulser

Ion Mirror

Detector

Turbo

Exact Mass

Mass Accuracy (ppm)

July, 2008Page 26

What does “Exact Mass” mean?Element Atomic Number Exact Mass

H 1 1.007825C 6 12.000000N 7 14.003074O 8 15.994915

C6H6Cl6 287.8600665 LindaneC10H12N2O6S 288.0416000 CarbasulamC9H21O2PS3 288.0441285 TerbufosC13H21O3PS 288.0949000 IprobenfosC15H17N4Cl 288.1141743 MyclobutanilC12H21N2O4P 288.1238937 Diazoxon C11H20N4O3PS 288.1256000 EpronazC11H21N4O3P 288.1351000 PirimetaphosC16H20N2O3 288.1473925 Imazamethabenz

0.2874 amu

Uses accurate mass on TOF/Q-TOF to identify all of them.

July, 2008Page 27

v = d/t

E = ½ m v2 = ½ m (d/t)2

m = (2E/d2) t2

Energy (E) and Distance (d) are fixed

The measured mass is proportional to the flight time (time-of-flight).

The Key for getting useful TOF results is good mass accuracy.

Mass Analysis for TOF

July, 2008Page 28

Calculation of Exact Mass and Errorin Measured Mass (MH+) of Reserpine

Error if the electron was not omitted!

NH

N

O OO

O

OO

O OO

CH3

CH3

CH3

CH3

CH3

CH3

H

HH

Reserpine (C33H40N2O9)

Atom Mass of Atom # of Atoms SumHydrogen 1.00783 40 40.31300Carbon 12.00000 33 396.00000Nitrogen 14.00307 2 28.00615Oxygen 15.99492 9 143.95424Total 608.27338Plus H 1.00783 1 1.00783Total 609.28121Minus e- 0.00055 1 0.00055

609.28066

Calculated = exact

(Measured - Calculated)Calculated

0.9027038 ppm

X 1,000,000 = ppm

July, 2008Page 29

How Much Accuracy is Needed?

Reserpine (C33H40N2O9) has a protonated ion at 609.28066 (MH+)

Single quad reports mass to +/- 0.1 = 165 ppm

(0.1/609.28 x 10e6 = 164.128 ppm)

Number of possible formulas using only C, H, O & N:

Accuracy Mass Difference Number of Formulas165 ppm 0.1 amu (609.18 - 609.38) 20910 ppm 0.061 amu 135 ppm 0.030 amu (609.25 - 609.31) 73 ppm 0.0018 amu 42 ppm 0.0012 amu (609.2795 - 609.2819) 2

Measured mass accuracy can narrow the number of hits in a TOF database search, therefore reduce risk of investing effort on the wrong molecule.

July, 2008Page 30

Automatic Internal Referencing(Easy-to-use)

Reference Sprayer

Analytical Sprayer

Dual Spray ESI source andCalibration Delivery System (CDS) automates the introduction of reference masses

AutoTune

July, 2008Page 31

TOF/Q-TOF Unlimited Number of Compound Screening

5556.D (Grape)MS1 Full Spectrum

15 out of 510 compounds had hits from database search.

Molecular Feature Extractor

July, 2008Page 32

Run Another Analysis in MS/MS (Targeted) Mode to Confirm Hits Found in MS1 Mode

Compound RTPrecursor Mass

(MH)+

Spiroxamine 10.884 298.27406

Terbuconazole 14.139 308.15242

Tebufenoxide 15.492 353.22236

July, 2008Page 33

Next Step: Find

compounds by Targeted MS/MS

MS/MS data collected

July, 2008Page 34

4x10

0

1

2

3

4

5

6

7

Cpd 4:+ Scan (10.654-11.177 min, 25 scans) 5556.d

121.05092

298.27442C18H36NO2

3x10

0

1

2

3

4

5

Cpd 4:+ Product Ion (10.972 min) (298.27405[z=1] -> **) 5556.d

144.14176C8H18NO

298.27944100.11532C6H14N

Counts vs. Mass-to-Charge (m/z)80 100 120 140 160 180 200 220 240 260 280 300 320

O

O

NCH3

CH3

CH3 CH3

CH3

100.18206143.22668

Spiroxamine

MS1 Full Spectrum

MS/MS Full Spectrum

The compound name Spiroxamine came up in the MS1 mode.

Use formula results and MS/MS results to confirm the hit.

3x10

0

0.25

0.5

0.75

1

1.25

1.5

Cpd 4:+ Scan (10.654-11.177 min, 25 scans) 5556.d

298.27442C18H36NO2

299.27649

300.28402

Counts vs. Mass-to-Charge (m/z)97.5 298 298.5 299 299.5 300 300.5 301

Boxes represent theoretical isotope ratios

July, 2008Page 35

NN

N

OH

Cl

CH3

CH3

CH3

4x10

0

1

2

3

4

5

6

7

Cpd 11:+ Scan (13.974-14.248 min, 5 scans) 5556.d

121.05086

308.15216C16H23ClN3O

4x10

0

1

2

3

4

5

6

Cpd 11:+ Product Ion (14.121 min) (308.15240[z=1] -> **) 5556.d

70.04052C4H6O

125.01448C7H6Cl 308.15199

Counts vs. Mass-to-Charge (m/z)60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

125.57554Terbuconazole

MS1 Full Spectrum

MS/MS Full Spectrum

The compound name Terbuconazole came up in the MS1 mode.

Use formula results and MS/MS results to confirm the hit.

4x10

0

0.2

0.4

0.6

0.8

1

Cpd 11:+ Scan (13.974-14.248 min, 5 scans) 5556.d

308.15216C16H23ClN3O

310.14951

311.15253

Counts vs. Mass-to-Charge (m/z)307 308 309 310 311

Boxes represent theoretical isotope ratios

151.61282

July, 2008Page 36

Screen Pesticides with LC-TOF/Q-TOF

• Accurate Mass provides added compound selectivity

• Higher resolution provides added interference selectivity

• Always full spectral data

• Unlimited number of compounds can be screened (search exact mass compound database for identification)

• Sensitivity is the same regardless of number of compounds screened*

• MS/MS (Q-TOF) assists compound confirmation

*Triple quadrupole (QQQ) mass spectrometer can be more sensitive up to a limited number of compounds. That limit has not been definitively determined.

July, 2008Page 37

Representative Sample

Extraction

Pesticide Workflow: Screen , Confirm and Quantify

Clean-up

LC/QQQ MRM – for known targets S

C

Q

QuEChERS

S C Q

C16 H19 N3 P ClC15 H25 O P S ClC18 H21 O P Cl

LC/QTOF or TOF Full Spectrum– for unknown compounds

S

Exact Mass Database Search

Molecular Formula Generation

Q CAnother injection for MS/MS (QQQ or QTOF)

GC/MS (PTV) SIM/Scan– for known and unknown Final Report

S

C

Q

Deconvolution

(+backflush)

3 1

3 1

3 1

GC/QQQ MRM – for known targets S

C

Q

July, 2008Page 38

Summary

• GC/MS and Deconvolution to Screen, Confirm and Quantify

• QQQ for Targeted analysis in complex matrix – Sensitivity unsurpassed– Complex matrix with less clean-up

• TOF/Q-TOF for Targeted and Unknown Screening– Sensitive full scan analysis– Searching exact mass database leads to identification– Quantitative– Accurate mass MS/MS for identification of fragments and structure

elucidation

Page 39

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