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Pesticide AnalysisPesticide Analysis Workflow Using

Hyphenated Mass Spectrometry

Techniques Chinkai (Kai) Meng, Ph.D.Senior Applications ChemistWilmington, Delaware USA

chin meng@agilent [email protected]

February 12, 2009

E-seminar, Feb 2009

MS Techniques Outline

• MS Analyzers Selection

GC/MS deconvolution and backflush• GC/MS – deconvolution and backflush

• QQQ (MS/MS)

• LC/Q-TOF

• Summary

E-seminar, Feb 2009

Analytical Instrumentations

Chromatography (Separation – the best friend of any detector)• GC – volatile and semi-volatile non-polar compounds• LC – polar or moderately polar or thermally labile compounds

Detectors• GC (element selective, FPD, NPD, ECD) – high sensitivity, poor specificity • LC (UV, FLD) – general purpose, limited sensitivity• MS –MS

– SQ: high confidence from spectral confirmation, limited MDL– QQQ: low detection limits of target compounds in dirty matrices (MRM)– TOF/Q-TOF: always full spectrum and accurate mass for screening– TOF-TOF, Q-Trap, …and others (generally not for ROUTINE use).

E-seminar, Feb 2009

Guiding Principles in MS Analyzer Selection

The correct choice of analyzer depends on whether one is “LOOKING for UNKNOWNS” or whether one is “CONFIRMING & MEASURING KNOWNS”.

The correct choice of analyzer depends on theThe correct choice of analyzer depends on the COMPLEXITY of the MATRIX.

E-seminar, Feb 2009

Basic Questions – Which MS Solution

Target analysis only? Scan or SIM or MS/MS

Analysis of unknowns? Scan MS with Quad, IT or TOF

How much chemical noise from the matrix?

Deconvolution or MS/MS

How much sample prep? Backflush or MS/MS

E-seminar, Feb 2009

Representative

General Workflow: Screen , Confirm and Quantify S C Q

GC/MS (PTV) SIM/ScanRepresentative Sample

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

S

C

Deconvolution

QuEChERS LC/QQQ MRM – for known targets SGC/QQQ MRM – for known targets S1

Q(+backflush)

Extraction C

Q

C

Q

3 1

Cl

LC/QTOF or TOF Full Spectrum– for unknown compounds

Exact Mass Database Search

A th

3 1

Clean-up

C16 H19 N3 P ClC15 H25 O P S Cl

SMolecular Formula Generation

Q CAnother injection for MS/MS (QQQ or QTOF)3

1

E-seminar, Feb 2009

C15 H25 O P S ClC18 H21 O P Cl




• QQQ (MS/MS)

• LC/Q-TOF

• Summary

E-seminar, Feb 2009

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 40.00

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

E-seminar, Feb 2009

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

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

CDFA* Agilent DRSCDFA Agilent DRS

Targets Found (not counting

ISTD)37 Same 37

+99 moreISTD)

False Positives 1 0

Processing Time ~8 hrs 32 min

*CDFA i th C lif i D t t f F d d A i lt

Saving 7.5 hoursDRS: Deconvolution Reporting Software

E-seminar, Feb 2009

*CDFA is the California Department of Food and Agriculture

17 280

How Does Deconvolution Work?

50

170 0

3175

185

16 310

75 160

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17 280

Eliminate Ions Don’t Fit the Criteria

50

170 0

3175

185

16 310

75 160

Ion grouping criteria:

1 S R tTi t

5017

160

Extracted Ion

1. Same RetTime at apex

2. Same peak width17028018

Extracted Ion Chromatograms

(EIC)

E-seminar, Feb 2009

755310

17 280

Spectrum is Deconvoluted/Cleaned

50

170 0

A component in AMDIS

50 Related ions are170280

Related ions are grouped together as

a component.

E-seminar, Feb 2009

TIC of Spinach

More than 370 peaks

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Extract found

Deconvolution

Library Search

QEdit, p,p’-DDT selected

5 ionMSD ion AMDIS ion

5 ion overlay

MSD & AMDIS areas & amounts

3 t“x” and “A”

E-seminar, Feb 2009

3 spectra indicators

Pesticides in Red Wine - FenpropathrinFound by AMDIS

250 ppb Not found250 ppbRaw scan

Found by AMDIS

100 ppbRaw scanRaw scanRaw scan

1 ppm MF = 783Fenpropathrin

MF = 83DeconvolutedMF = 65Deconvoluted

5 ppm MF = 877Library entryLibrary entry

Fenpropathrin

E-seminar, Feb 2009

Without Backflush: Increased Chemical Background (Spectral Noise) and Changes in Retention Time

4.6e+07

Increased baseline and spectral noise due to carryover of matrix

3e+07

3.4e+07

3.8e+07

4.2e+07carryover of matrix

Highly retained matrix is altering the column

1.8e+07

2.2e+07

2.6e+07

3e+07B

A

altering the column selectivity and changing the retention time

6000000

1e+07

1.4e+07

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0002000000

Overlay of two chromatograms of a blank extract injected BEFORE (A) and

E-seminar, Feb 2009

AFTER (B) three injections without backflush

With Backflush: No Increased Chemical Background (Spectral Noise) and No Change in Retention Time

Stable retention times and baseline . . . less chemical noise- less maintenance4.2e+07

4.6e+07

- better results

3e+07

3.4e+07

3.8e+07

1 8e+07

2.2e+07

2.6e+07

3e+07

6000000

1e+07

1.4e+07

1.8e+07

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.000

2000000

6000000

E-seminar, Feb 2009

Overlay of three chromatograms of lettuce extract run with 2 min of backflush




• QQQ (MS/MS)

• LC/Q-TOF

• Summary

E-seminar, Feb 2009

Analyzers for Trace Target Compounds

Who should use QQQ (MS/MS)?

User doing Selected Ion Monitoring (SIM) for target compound analysis in traditional markets, needing additional sensitivity and selectivity with less sample prep to meetsensitivity and selectivity with less sample prep, to meet more demanding analytical requirements.

LC/QQQ MRM – for known targets S

C

GC/QQQ MRM – for known targets S

C C

Q

C

Q

E-seminar, Feb 2009

Multiple Reaction Monitoring (MRM)

Quad Mass Filter (Q3)Quad Mass Filter (Q1) Collision Cell

Spectrum with background

Q1 lets onlytarget ion 210

Collision cell breaks ion 210

Q3 monitors onlycharacteristic

210

ions (from EI) pass through

210

apart fragments 158 and 191 from ion 210 for quant and qual.

170 210 250 290

222

268 280165

190 210 150 170 190 210

210158

191

qual.

160

158

190

191

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170 210 250 290 190 210 150 170 190 210 160 190no chemical background

Why MS/MS?Greater Selectivity Than SIMGreater Selectivity Than SIM

EI-SIM EI-MS/MSEI SIMselectivity proportional to

spectral resolutionno selectivity against ions

Precursor selectivity same as SIMHigh probability that at least one product ion will be a unique dissociation product of the precursor BUT not the interference

interference

Product 2

no selectivity against ions with same m/z

of the precursor BUT not the interference

analyte

Precursor

Product 1Product 3

interference

unit mass resolutionIon

The precursor ion should NOT be used for ion ratios or quantitation since the interferences will

E-seminar, Feb 2009

ratios or quantitation since the interferences will be the same as the SIM ion

SIM vs MRM for HCB100 fg HCB in Clean Matrix100 fg HCB in Clean Matrix

M 2

83.8

Injecting 100 fg of

e M

S: S

IM S/N=26:1 RMS

Injecting 100 fg of Hexaclorobenzene (HCB)and analyzing in SIM.

Sing

le9 I j ti 100 f f

83.8

:213

.9

S/N=37:1 RMS

Injecting 100 fg of Hexaclorobenzene (HCB)and analyzing in MRM.

MS/

MS:

28 RMS y g

You obtain the same S/N!

E-seminar, Feb 2009

M

SIM vs MRM for HCB100 fg HCB in Clean Matrix 300 fg HCB in Diesel100 fg HCB in Clean Matrix 300 fg HCB in Diesel

M 2

83.8

e M

S: S

IM S/N=6:1 RMSS/N=26:1 RMS

Sing

le9

83.8

:213

.9

S/N=86:1 RMS

S/N=37:1 RMS

MS/

MS:

28 RMSRMS

E-seminar, Feb 2009

M

Comparing GC/Q SIM to GC/QQQ MRM in Various Matrices – Analysis of p,p’-DDE at 10 ppb (All injections = 1 µL)(All injections = 1 µL)

Apple

SIM - EIC (246) MRM (246->176,175)

S/N = 448pp

Cabbage S/N = 241

Ginseng S/N = 446

Orange S/N = 456

Spinach S/N = 260

E-seminar, Feb 2009

Carrot Extract - 1 µL with GC/QQQ is Much Better than 5 µL GC/Q

GC/Q GC/QQQ5 µL (Multi-mode Inlet) 1 µLµ ( ) µ

Pesticide Cold SL Scan + DRS Cold SL SIM Hot SL (ppb)

Diclobenil 0.38*Pentachlorobenzene 0 75*Pentachlorobenzene 0.75Trifluralin 2.3*Tefluthrin 0.53*4,4'-Dichlorobenzophenone 1.2*Chlorpyrifos 24 7Chlorpyrifos 24.7o,p'-DDE 3.7p,p'-DDE X X 240o,p'-DDD 9p p' DDD Xp,p'-DDD X Sum = 45o,p'-DDT Xp,p'-DDT X X 130Fenazaquin X Not in Method

E-seminar, Feb 2009

* = Below Calibration Level

p,p’-DDE in Carrot Extract (240 ppb) – 5 µL GC/Q vs. 1 µL GC/QQQ

5 µLCold

S/N = 355 µLCold

S/N = 375

Co dSL ScanEIC

ColdSL SIMEICEIC EIC

5x10

1 3

246.0 -> 176.1 , 246.0 -> 175.1

Ratio=23.8 S/N = 434

0.6

0.70.8

0.91

1.11.2

1.3

1 µLHotSL

-0.10

0.10.2

0.30.4

0.5SL QQQMRM

E-seminar, Feb 2009

Acquisition Time (min)11.8 11.9 12 12.1 12.2 12.3

Why a GC/MS/MS System?

• Allows for the selective quantitation of target compounds in high chemical background samples

• Better S/N in complex matrices than can be achieved by single quadrupole scan or SIM approaches.

• Newer regulations in some markets specify analytical power commensurate with GC/MS/MS

E-seminar, Feb 2009

Technology

Agilent took the best technologies from their industry leading 597X Series GC/MSD:

– Heated monolithic gold plated quartz quadrupole– Proven reliable high performance source design

AUTOTUNE– AUTOTUNE

and the 6410A LC/QQQ:

– Linear acceleration enhanced Collision Cell– Wide Mass-Bandwidth QQQ ion optics

M H t ft– MassHunter software

E-seminar, Feb 2009

Why Heated Quartz “Gold” Quads?

Unlike LC/MS, many high boiling neutral molecules enter the source and manifold of a GC/MS or GC/MS/MS

• Higher temperature reduces potential for contamination– 200C max – Virtually eliminates the need to clean quads

• Low coefficient for thermal expansion for quartzp q– Stable structure during maintenance cycles (hot-cool-hot) for source

or detector

More stable tunes and methods over a longer period of time in real world sample environments

E-seminar, Feb 2009

MS Technique Outline



• QQQ (MS/MS)

• LC/Q-TOF

• Summary

E-seminar, Feb 2009

Agilent Q-TOF Fundamentals

Flight tubeCommon with TOF

Two key concepts for TOF:

1. Exact Mass

Collision cell

Ion Mirror2. Mass Error

Ion opticsCommon with Q & QQQ

Collision cellCommon with QQQ

Octopole 1Quad Mass Filter (Q1) Octopole 2

Detector

DC Quad

Rough Pump

Turbo Turbo Turbo

Collision CellLens 1 and 2Ion Pulser

Turbo

E-seminar, Feb 2009

September, 2008Page 31

p

Mass Analysis for TOF

v = d/t

E ½ m v2 ½ m (d/t)2E = 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 timeproportional to the flight time (time-of-flight).

E-seminar, Feb 2009

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

H 1 1.007825

C 6 12.000000

N 7 14.003074

O 8 15.994915

C6H6Cl6 287.8600665 LindaneC10H12N2O6S 288.0416000 CarbasulamC9H21O2PS3 288 0441285 TerbufosC9H21O2PS3 288.0441285 TerbufosC13H21O3PS 288.0949000 IprobenfosC15H17N4Cl 288.1141743 MyclobutanilC12H21N2O4P 288.1238937 Diazoxon

0.2874 amu

The Key for getting useful TOFC11H20N4O3PS 288.1256000 EpronazC11H21N4O3P 288.1351000 PirimetaphosC16H20N2O3 288.1473925 Imazamethabenz

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

E-seminar, Feb 2009


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

Calculation of Error in Measured Mass (MH+) of Reserpine(MH ) of Reserpine

Atom Mass of Atom # of Atoms SumHydrogen 1.00783 40 40.31300Carbon 12 00000 33 396 00000Carbon 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

NH

N

O

O OO

CH3CH3

CH3HH

609.28066

Calculated = exact

(Measured - Calculated) X 1 000 000O O

O

OOCH3CH3

CH3

H

R i (C H N O )

(Measured Calculated)Calculated

0.9027038 ppm

X 1,000,000 = ppm

E-seminar, Feb 2009


Reserpine (C33H40N2O9)Error if the electron was not omitted!

TOF/Q-TOF Unlimited Number of Compound Screening

5556.D (Grape) MS1 Full Spectrum

• Molecular Feature Extractor found 510 compounds in the TIC.

• 15 out of 510 compounds had hits from EXACT MASS database search, i.e., these 15 matched entries in the database within 3 ppm mass accuracy.

The three highlighted compounds were further confirmed by MS/MS

E-seminar, Feb 2009


MS/MS.

Cl125.57554Terbuconazole

The compound name Terbuconazole came

CH3 CH3

The compound name Terbuconazole came up in the MS1 mode search.

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

NN

OHCH34x10

5

6

7

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

121.05086

MS1 Full Spectrum

confirm the hit.151.61282

N

N

1

2

3

4

5

308.15216C16H23ClN3O

04x10

4

5

6

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

70.04052C4H6O

MS/MS Full Spectrum

1

2

3

4

125.01448C7H6Cl 308.15199

MS/MS Full Spectrum

Boxes represent theoretical

E-seminar, Feb 2009

0

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

pisotope ratios

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

• Sensitivity is the same regardless of number of compounds y g pscreened*

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

*Triple quadrupole (QQQ) mass spectrometer can be more sensitive up to a limited

E-seminar, Feb 2009

( )number of compounds. That limit has not been definitively determined.

MS Technique Outline



• QQQ (MS/MS)

• LC/Q-TOF

• Summary

E-seminar, Feb 2009

Summary

• GC/MS and Deconvolution to Screen, Confirm and Quantify • QQQ for routine Targeted trace analysis in complex matrix g y p

– MRM Sensitivity unsurpassed (up to a few hundred compounds)– 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– Searching exact mass database leads to identification• Unlimited number of compounds• Sensitivity is the same regardless of number of compounds screened

– Quantitative– Accurate mass MS/MS for identification of fragments and structure

elucidation (hotlink available to search on-line databases)

E-seminar, Feb 2009

( )

References

Application Note 5989-7670: 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 Gaintargeted Pesticides Screening and >10x Productivity Gain

Pesticide Brochure 5989-8652

GC-QQQ Brochure 5990-3152

Current Trends in Mass Spectrometry, pp 33-38, Nov. 2008 (Supplement to LC/GC North America): Comprehensive Screening Confirmation andto LC/GC North America): Comprehensive Screening, Confirmation, and Quantification of Organic Pesticides in Foods by GC-MS and LC-MS

The Applications Book, LC/GC Europe, pp 9-11, Dec. 2008: The Benefits f I ti GC/QQQ i t P ti id A l i M th dof Incorporating GC/QQQ into Pesticide Analysis Methods

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Thank you for your attention. Questions?

January in Amsterdam

E-seminar, Feb 2009

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