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Pesticides e-Seminar Series -

Pesticide Analysis Workflow Using Hyphenated Mass Spectrometry Techniques

Date: 30 April 2009

Time : 10am Singapore Time

* Phone line needs to be IDD enabled. Not accessible from Mobile Phones

International Telephone Number for Singapore: +65-6622 1044 or Toll Free 8008 523 396International Telephone Number for Hong Kong: +852 3006 8101Toll Free International Numbers:Australia 1800 999 130 New Zealand 0800 450 755China 800 876 5011 Malaysia 1800 807 180Indonesia 0018 038 526 350 Taiwan 0080 185 5735India* 0008 008 521 136 South Korea 0079 885 214 717India (Backup)* 0008 001 006 542 Philippines 1800 185 50065Thailand* 0018 008 526 361 Vietnam 120 650 065

E-seminar, April 2009

Pesticide Analysis Workflow Using

Hyphenated Mass Spectrometry

Techniques

E-Seminar, April 30, 2009

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

[email protected]


MS Techniques Outline

• MS Analyzers Selection

• GC/MS – deconvolution

• QQQ (MS/MS)

• LC/Q-TOF

• Summary


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 –

– 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)


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 the COMPLEXITY of the MATRIX.


Basic Questions – Which MS Solution

Target analysis only?

Analysis of unknowns?

How much chemical noise from the matrix?

How much sample prep?

Scan or SIM or MS/MS

Scan MS with Quad, IT or TOF

Deconvolution or MS/MS

MS/MS


Representative Sample

Extraction

General Workflow: Screen , Confirm and Quantify

Clean-up

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)3

1

LC/QQQ MRM – for known targets S

C

Q

3 1

GC/QQQ MRM – for known targets S

C

Q

3 1

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

S

C

Q

Deconvolution

(+backflush)





• QQQ (MS/MS)

• LC/Q-TOF

• Summary


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?


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


50

170 280

31075

185

160

How Does Deconvolution Work?


50

170 280

50170280

31075

75

185

185

310

160

160

Eliminate Ions Don’t Fit the Criteria

Extracted Ion Chromatograms

(EIC)

Ion grouping criteria:

1. Same RetTime at apex

2. Same peak width


50

170 280

50170280

Spectrum is Deconvoluted/Cleaned

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



5 ion overlay

3 spectra

MSD & AMDIS areas & amounts

MSD ion AMDIS ion

“x” and “A”indicators

QEdit, p,p’-DDT selected


Pesticides in Red Wine - Fenpropathrin

250 ppb

1 ppm

5 ppm MF = 877Fenpropathrin

MF = 783Fenpropathrin

Not found

MF = 83

250 ppbRaw scan

Deconvoluted

Library entry

Found by AMDIS

MF = 65

100 ppbRaw scan

Deconvoluted

Library entry





• QQQ (MS/MS)

• LC/Q-TOF

• Summary


Analyzers for Trace Target Compounds

LC/QQQ MRM – for known targets S

C

Q

GC/QQQ MRM – for known targets S

C

Q

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 meet more demanding analytical requirements.


Advantages of a QQQ as a Chromatographic Detector - Multiple Reaction Monitoring (MRM)

170 210 250 290

210

222

268 280165

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

Spectrum with backgroundions (from EI)

Q1 lets onlytarget ion 210 pass through

190 210

210

Collision cell breaks ion 210 apart

150 170 190 210

210158

191

Q3 monitors onlycharacteristic fragments 158 and 191 from ion 210 for quant and qual.

160

158

190

191

no chemical background

Source


Why MS/MS?Greater Selectivity Than SIM

PrecursorIon

Product 1Product 3

Product 2

EI-SIMselectivity proportional to

spectral resolutionno selectivity against ions

with same m/z

EI-MS/MSPrecursor selectivity same as SIM

High probability that at least one product ion will be a unique dissociation product

of the precursor BUT not the interferenceinterference

analyte

interference

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

be the same as the SIM ion

unit mass resolution

Consistent MS/MS Results, Regardless of Matrices– Analysis of spiked p,p’-DDE at 10 ppb (All injections = 1 µL)

Apple

Orange

Spinach

Ginseng

Cabbage

SIM - EIC (246) MRM (2 transitions)

A c q u is it io n T im e (m in )1 1 .8 1 1 .9 1 2 1 2 .1 1 2 .2 1 2 .3

Cou

nts 4x1 0

-0 .1

0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

1

1 .1

2 4 6 .0 -> 1 7 6 .1 , 2 4 6 .0 -> 1 7 5 .1

R a t io = 2 3 .5

A c q u isitio n T im e (m in )1 1 .8 1 1 .9 1 2 1 2 .1 1 2 .2 1 2 .3

Cou

nts 3x1 0

0

1

2

3

4

5

6

7

8

2 4 6 .0 -> 1 7 6 .1 , 2 4 6 .0 -> 1 7 5 .1

R a t io = 2 3 .6


Cou

nts 4x 1 0

-0 .1

0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

1

1 .1

1 .2

2 4 6 .0 -> 1 7 6 .1 , 2 4 6 .0 -> 1 7 5 .1

R a t io = 2 4 .0


Cou

nts 3x1 0

0

1

2

3

4

5

6

7

8

2 4 6 .0 -> 1 7 6 .1 , 2 4 6 .0 -> 1 7 5 .1

R a t io = 2 2 .9

Acquisition Time (min)11.8 11.9 12 12.1 12.2 12.3

Cou

nts 4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

246.0 -> 176.1 , 246.0 -> 175.1

Ratio=23.3

S/N = 448

S/N = 456

S/N = 260

S/N = 446

S/N = 241

AppNote: 5990-3578; 175 pesticides



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

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

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

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

* = Below Calibration Level


5 µLColdSL ScanEIC

5 µLColdSL SIMEIC

Acquisition Time (min)11.8 11.9 12 12.1 12.2 12.3

Cou

nts 5x10

-0.10

0.10.2

0.30.4

0.50.6

0.70.8

0.91

1.11.2

1.3

246.0 -> 176.1 , 246.0 -> 175.1

Ratio=23.8

1 µLHotSL QQQMRM

S/N = 35

S/N = 375 S/N = 434

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

Same as left, after deconvolution


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

The Limitations of Converting an LC/MS/MS to GC/MS/MSESI, APCI and APPI - Atmospheric pressure sources

– Designed to separate ions from molecules before entering the vacuum manifold– Higher m/z metal rods are typically ‘temperature compensated’ for 100ºC

operation (some only 50ºC) – no problem since only ions enter the analyzer

EI, PCI and NCI - Vacuum sources

– All neutral molecules (carrier gas, analytes and matrix) enter the vacuummanifold BEFORE ionization

– Most molecules are NOT ionized and must be pumped away– High boiling molecules common to GC will contaminate all cooler surfaces

• Contamination causes loss of sensitivity• Cool (< 100ºC) metal quadrupoles are easily contaminated under GC/MS



Why Heated Quartz “Gold” Quads?

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

• Low coefficient for thermal expansion for quartz– 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


MS Technique Outline



• QQQ (MS/MS)

• LC/Q-TOF

• Summary


September, 2008Page 28

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

Two key concepts for TOF:

1. Exact Mass

2. Mass Error


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).

Mass Analysis for TOF



What does “Exact Mass” mean?Element 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 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.

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



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

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

Error if the electron was not omitted!0.9027038 ppm

X 1,000,000 = ppm



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.

Terbuconazole


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

125.0158

MS1 Full Spectrum

MS/MS Full Spectrum

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

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


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.


MS Technique Outline



• QQQ (MS/MS)

• LC/Q-TOF

• Summary


Summary

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

– 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

• 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)


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 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, 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 of Incorporating GC/QQQ into Pesticide Analysis Methods


Thank you for your attention. Questions?

Jan. in Amsterdam


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

and the 6410A LC/QQQ:

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


Identify Dichlofenthion in MSD ChemStation- Red wine

MF = 630/670Dichlofenthion

No match

No match250 ppb

500 ppb

1 ppm

hidden


Identify Dichlofenthion in AMDIS

MF = 630/670Dichlofenthion

No match

No match250 ppb

500 ppb

1 ppm

MF = 82

250 ppbRaw scan

Deconvoluted

Library entry

hidden


Agilent Has the TOTAL Portfolio for PesticideAnalysis

(semi)-volatile or non-polar

(moderate)-polar or thermally labile

GCFPD/ECD

LCUV, FLD

GC/QDRS

LC/QLC/QQQMRM

GC/QQQMRM

Q-TOFTOF

GAS

LIQUID

Routine Analysis Identify Unknowns

phase

phase

Exact Mass Database Search

hidden

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