Finding Non-targeted

(Unknown) Pesticides
Using GC/MS and
LC/Q-TOF

Chin-Kai Meng, Ph.D.

Applications Chemist
Wilmington, Delaware
chin_meng@agilent.com
302-633-8388

Page 1

July, 2008

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

Page 2

July, 2008

Business Issues and Challenges

Global trade increases the number of pesticides to monitor
(> several hundreds) Screen for
Unknowns
Regulation driving lower detection limits
Speed: doing more analyses in a day Faster Data Review
and Reporting
Need for fast startup Total Solution
Offering

Page 3

July, 2008

GC-MSD Workflow
GC/MS (PTV) SIM/Scan
for known and unknown
Deconvolution

Final Report

Screen
Confirm

(+backflush)

Page 4

Quantify

July, 2008

TICs of Surface Water Extracts

How many pesticides
(drugs, allergens etc.) are in
these samples and how long
does it take you to confirm?
5.00

10.00

15.00

20.00

25.00

30.00

35.00

5.00

5.00

Page 5

10.00

15.00

20.00

25.00

30.00

35.00

40.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

40.00

July, 2008

17 Surface Water TICs (Scan): Pesticide Analysis

Using DRS with Pesticide Database (927 entries)
CDFA*

Agilent DRS

Targets Found
(not counting
ISTD)

37

Same 37
+99 more

False Positives

Processing Time

~8 hrs

32 min

DRS: Deconvolution Reporting Software

Saving 7.5 hours

*CDFA is the California Department of Food and Agriculture

Page 6

July, 2008

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.

Page 7

July, 2008

AMDIS

Automatic Mass spectral Deconvolution and Identification

System
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

Page 8

July, 2008

How Does Deconvolution Work?

280

170
185

50
75

Page 9

160

310

July, 2008

Eliminate Ions Dont Fit the Criteria

170

280

185

50
75

310

160

Ion grouping criteria:

1. Same RetTime at apex
2. Same peak width

Page 10

160
50
170 Extracted Ion
Chromatograms
280
(EIC)
185
75
310
July, 2008

Spectrum is Deconvoluted/Cleaned
170

280

50

A component in AMDIS

50
170

Related ions are

grouped together as
280
a component.

Page 11

July, 2008

More than
370 peaks
found

TIC of
Spinach
Extract
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Deconvolution

Library Search

Scan at 5.615 min

Peach

Deconvoluted/extracted
spectrum

A component in the
scan above.

Library spectrum

The power of deconvolution is

appreciated while comparing
the top two spectra (raw scan
and the compound spectrum
hidden in the raw scan).

Carbaryl

Page 13

July, 2008

Scan at 10.776 min

Peach

Deconvoluted/extracted
spectrum

A component in the
scan above.

Library spectrum

Fenbuconazole

Page 14

July, 2008

DRS Report - Peach

Page 15

July, 2008

DRS report from a SIM data file for Additional

Confirmation (4 ions for each target compound)

Page 16

July, 2008

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

Page 17

July, 2008

QEdit, p,p-DDT selected

MSD ion
5 ion
overlay

AMDIS ion

MSD & AMDIS

areas & amounts

3 spectra

Page 18

x and A
indicators

July, 2008

Graphics Report

MSD and AMDIS

5-ion overlay

Raw, deconvoluted
and library spectra

MSD and AMDIS

areas and amounts

Page 19

July, 2008

Summary Quant Report optionally includes

both MSD ChemStation and AMDIS amounts

partial report

Page 20

July, 2008

DRS A.04 Report after importing AMDIS results

QEdit reviewed with manual integrations

Page 21

July, 2008

GC-MSD Workflow Summary

GC/MS (PTV)
for known/unknown

SIM/Scan
Deconvolution

401

Final Report

S
C

83

256

Library Search

(+backflush)

GC/MS in full scan mode combined with deconvolution reporting

software (DRS) enables unknown pesticide analysis at 10 g/kg (ppb)
in various food commodities from one injection
Significant time saved on data analysis (screen and quant on 927
pesticides), 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

Page 22

July, 2008

LC/MS Workflow

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

Exact Mass
Database Search

Molecular
Formula Generation

Another
injection for
MS/MS
(QQQ or QTOF)

C16 H19 N3 P Cl
C15 H25 O P S Cl
C18 H21 O P Cl

Screen

Confirm

Quantify

Page 23

July, 2008

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

Page 24

July, 2008

Agilent Q-TOF Fundamentals

Exact Mass
Mass Accuracy (ppm)
Flight tube
Common with TOF
Ion Mirror

Collision cell
Common with QQQ
Ion optics
Common with Q & QQQ
Quad Mass Filter (Q1)

Octopole 2

Octopole 1

Detector

Ion Pulser
Lens 1 and 2

Rough
Pump

Page 25

Turbo

Turbo

Collision Cell

Turbo

DC Quad

Turbo

July, 2008

What does Exact Mass mean?

Element Atomic Number

0.2874 amu

Exact Mass

1.007825

12.000000

14.003074

15.994915

C6H6Cl6
C10H12N2O6S
C9H21O2PS3
C13H21O3PS
C15H17N4Cl
C12H21N2O4P
C11H20N4O3PS
C11H21N4O3P
C16H20N2O3

287.8600665
288.0416000
288.0441285
288.0949000
288.1141743
288.1238937
288.1256000
288.1351000
288.1473925

Lindane
Carbasulam
Terbufos
Iprobenfos
Myclobutanil
Diazoxon
Epronaz
Pirimetaphos
Imazamethabenz

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

Page 26

July, 2008

Mass Analysis for TOF

v = d/t
E = m v2 = 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.

Page 27

July, 2008

Calculation of Exact Mass and Error

in Measured Mass (MH+) of Reserpine
Atom
Hydrogen
Carbon
Nitrogen
Oxygen
Total
Plus H
Total
Minus e-

Mass of Atom
1.00783
12.00000
14.00307
15.99492
1.00783
0.00055

# of Atoms
Sum
40
40.31300
33
396.00000
2
28.00615
9
143.95424
608.27338
1.00783
1
609.28121
0.00055
1
609.28066

Calculated = exact

CH3

N
O

(Measured - Calculated) X 1,000,000 = ppm

Calculated

CH3
N
H

O
CH3

CH3
H
O
O

0.9027038 ppm

Error if the electron was not omitted!

Page 28

O
CH3

CH3

Reserpine (C33H40N2O9)
July, 2008

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 Formulas

165 ppm

0.1 amu (609.18 - 609.38)

209

10 ppm

0.061 amu

13

5 ppm

0.030 amu (609.25 - 609.31)

3 ppm

0.0018 amu

2 ppm

0.0012 amu (609.2795 - 609.2819)

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

Page 29

July, 2008

Automatic Internal Referencing

(Easy-to-use)
Analytical Sprayer
Reference Sprayer

Dual Spray ESI source and

Calibration Delivery System
(CDS) automates the
introduction of reference masses
AutoTune

Page 30

July, 2008

TOF/Q-TOF Unlimited Number of Compound Screening

Molecular Feature
Extractor

15 out of 510 compounds had

hits from database search.

5556.D (Grape)
MS1 Full Spectrum

Page 31

July, 2008

Run Another Analysis in MS/MS (Targeted) Mode

to Confirm Hits Found in MS1 Mode

Page 32

Precursor Mass

Compound

RT

Spiroxamine

10.884

298.27406

Terbuconazole

14.139

308.15242

Tebufenoxide

15.492

353.22236

(MH)+

July, 2008

MS/MS data
collected

Next Step:
Find
compounds by
Targeted
MS/MS

Page 33

July, 2008

Spiroxamine

CH3

CH3

H3C

H3C

N
CH3

143.22668

x10 4 Cpd 4:+ Scan (10.654-11.177 min, 25 scans) 5556.d

100.18206

121.05092

7
6

MS1 Full Spectrum

5
4
3
2

298.27442
C18H36NO2

The compound name Spiroxamine

came up in the MS1 mode.
Use formula results and MS/MS
results to confirm the hit.

0
x10 3 Cpd 4:+ Scan (10.654-11.177 min, 25 scans) 5556.d

x10 3 Cpd 4:+ Product Ion (10.972 min) (298.27405[z=1] -> **) 5556.d
144.14176
C8H18NO

298.27442
C18H36NO2

1.5
1.25

MS/MS Full Spectrum

1
0.75
299.27649

0.5

100.11532
C6H14N

0
97.5

1
0
80

Page 34

300.28402

0.25

298.27944

100

120

140

160 180 200 220 240 260

Counts vs. Mass-to-Charge (m/z)

280

300

320

298

298.5 299 299.5 300 300.5

Counts vs. Mass-to-Charge (m/z)

301

Boxes represent theoretical

isotope ratios
July, 2008

Cl

Terbuconazole

125.57554

The compound name Terbuconazole came

up in the MS1 mode.
Use formula results and MS/MS results to
confirm the hit.
151.61282

x10 4 Cpd 11:+ Scan (13.974-14.248 min, 5 scans) 5556.d

121.05086

7
6

H3C

OH

MS1 Full Spectrum

CH3
CH3

4
3

308.15216
C16H23ClN3O

2
1

x10 4 Cpd 11:+ Scan (13.974-14.248 min, 5 scans) 5556.d

0
x10 4

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

308.15216
C16H23ClN3O

70.04052
C4H6O

0.8
0.6

MS/MS Full Spectrum

4
3

310.14951

0.4
311.15253

0.2
0

307

125.01448
C7H6Cl

308.15199

0
60

Page 35

80

100

120

140 160 180 200 220 240

Counts vs. Mass-to-Charge (m/z)

260

280

300

320

340

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

Boxes represent theoretical

isotope ratios
July, 2008

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.

Page 36

July, 2008

Pesticide Workflow: Screen

, Confirm

GC/MS (PTV) SIM/Scan

for known and unknown

Representative
Sample

and Quantify

Deconvolution
Final Report

S
C
Q

(+backflush)

QuEChERS
Extraction

GC/QQQ MRM for known targets

1
3

1
3

LC/QTOF or TOF Full Spectrum

for unknown compounds

LC/QQQ MRM for known targets

Exact Mass
Database Search

Clean-up
1
3

Molecular
Formula Generation

Another
injection for
MS/MS
(QQQ or QTOF)

C16 H19 N3 P Cl
C15 H25 O P S Cl
C18 H21 O P Cl

Page 37

July, 2008

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 38

July, 2008

Wrap-up E-Seminar Questions

Thank you for attending Agilent e-Seminars.
Our e-Seminar schedule is expanding every
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www.agilent.com/chem/education
Or register for
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