Improved Pesticide Residue Analysis using Agilents GC ... Pestici… · Improved Pesticide Residue Analysis using Philip L. Wylie, Ph.D. Residue Analysis using Sr Applications Chemist

Improved Pesticide Residue Analysis using

Philip L. Wylie, Ph.D.Sr Applications Chemist forResidue Analysis using

Agilent’s GC/MS and GC/MS/MS Pesticide

Sr. Applications Chemist for Food SafetyAgilent Technologies

Analyzersg g

Chemical Analysis GroupSeptember 21, 2010

September 20, 20101

Outline

Tools for pesticide residue analysis

Why choose an analyzerWhy choose an analyzerEasy to order and install

Run actual samples right after installation

Development work already done for you

Pesticide analyzers available from Agilent

Review GC/MSD and GC/MS/MS pesticide solutions

Backflushing for rugged methods

Multimode Inlet for cold injections

Choosing a pesticide analyzer

September 20, 20102

Agilent TOTAL Portfolio for Pesticide AnalysisGC/Q

GC/QQQMRM

GC/QDRSGC

FPD/ECD

(semi)-volatile or non-polar

Gas phase or non polar

(moderate)-polar or thermally labile

pesticides

LC

TOF Q-TOFLC/QQQMRM

LC/Q

Routine Analysis Identify Unknowns

Exact Mass Database SearchLCUV, FLD

Liquid phase

Page 3

Routine Analysis Identify Unknownsphase

Agilent : GC/MS Tools for Pesticide Residue AnalysisAnalysis

7000B GC Triple Quad GC/MSD5975C / 7890GC

Automatically Target pesticideAutomatically search for 926 (or

430) pesticides after

Target pesticide analysis at very low

levelsGC/MS scan run

What you told us…

Not enough time to adopt new technologies

Help us reduce cost

Not enough skilled people

technologies

Help us improve our productivity!

Agilent has the technologies essential for higher productivity g p ybut…for many labs it’s asfor many labs it s as clear as alphabet soup!

Slide setAgilent Restricted

Date 2009Page 6

Typical Approach to New Method Implementation

O d ll th t Test and validate the new methodOrder all the parts …Test and validate the new method

Find or developthe method

Agilent Pesticide Analyzers – Arrive ready to use

You’ll be running real samples before the service engineer

leaves your laby

GC/MS(MS) Based Pesticide Analyzers Available from Agilentfrom Agilent

GC/MSD DRS P ti id A l GC/MS/MS P ti id A lGC/MSD DRS Pesticide Analyzer926 Pesticides & endocrine

disruptors21 minute Run, Constant

GC/MS/MS Pesticide Analyzer –Agilent Method555 Pesticides

42 minute Run, Constant21 minute Run, Constant Pressure, Post-column

Backflushing

42 minute Run, Constant Pressure, Post-column

Backflushing

GC/MSD Japanese Positive List GC/MS/MS Pesticide Analyzer –GC/MSD Japanese Positive List DRS Pesticide Analyzer

430 Pesticides31.5 minute Run, Constant Flow,

P t l B kfl hi

GC/MS/MS Pesticide Analyzer US Method

>230 Pesticides 19 minute Run, Constant Flow,

Mid l B kfl hi

September 20, 20109

Post-column Backflushing Mid-column Backflushing

Pesticide Analysis Using GC/MS with Deconvolution Reporting Software and a RetentionDeconvolution Reporting Software and a Retention Time Locked Pesticide Library

September 20, 201010

GC/MSD DRS Analyzers – What they can do.

From here…

…to here in 2 - 3 min Deconvolution Reporting SoftwareDeconvolution Reporting SoftwareGC/MSD in Scan ModeGC/MSD in Scan Mode

RTL Pesticide DatabasesRTL Pesticide Databases

gg

QEditQEditQuant on Normal or Quant on Normal or D l t dD l t d P kP kDeconvolutedDeconvoluted PeaksPeaks

DRS Report from 3 Integrated Processes

Total ion chromatogram

Targets are identified by comparison to locked R.T.s and 3 qualifier ion

Deconvoluted target spectra confirmed by AMDIS are searched

AMDIS deconvolutes component spectra and

searches target MS database using locked RT as a qualifier

Quant Results

qratios, then quantified using target ion area vs ISTD cal table

AMDIS are searched against NIST08 MS database

Confirmed NIST08 hits

Confirmed AMDIS hits

AMDIS Quant Result

Combined quantitative and qualitative HTML

Summary report

Quant Results Confirmed NIST08 hits

Summary report

AMDIS Deconvolution Pulls Out Individual Components and Their Spectra p p

TIC & Spectrum

TIC Sum of ions in Component 1

Deconvoluted peaks and spectra

Sum of ions in Component 1

Sum of ions in Component 2

Decoonvolut

Sum of ions in Component 3ion

MetalaxylMetalaxyl Identified by DRS in Mixed Vegetable Identified by DRS in Mixed Vegetable ((QuEChERSQuEChERS) Extract (AMDIS View)) Extract (AMDIS View)((QuEChERSQuEChERS) Extract (AMDIS View)) Extract (AMDIS View)

TICMetalaxyl buried in matrix

Spectrum before Deconvolution

Deconvoluted spectrum (white) and library spectrum (black)

DRS Report for Spinach Extract after importing AMDIS Results, QEdit reviewed with manual integrations

What are the Differences Between the Two DRS Pesticide Solutions?

Japanese Positive List Pesticide Database (SP1 P/N 7890-0457)

RTLPest3 (SP1 P/N 7890-0456)927 Compounds

430 Compounds

C t i ll GC bl ti id

Most GC-able pesticides

Many metabolites

Many endocrine disrupters Contains all GC-able pesticides discussed in the Japanese Positive List System or in Quarantine Station publications

Many endocrine disrupters

Important PCBs & PAHs

Some dyes (e.g., Sudan Red)No other endocrine disruptors

Locked RTs + Mass Spectra

Uses Japanese Ministry of Health Labor &

Synthetic musk compounds

Some OP fire retardants

Locked RTs + Mass Spectra Uses Japanese Ministry of Health Labor &Welfare constant flow GC/MS methodLocked RTs + Mass Spectra

Uses Agilent’s constant pressureGC/MS method

Targeted Pesticide Analysis Using GC/MS/MS

17

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

Quad Mass Filter (Q2)Quad Mass Filter (Q1) Collision CellSource

Spectrum with background

Q1 lets onlytarget ion 210

Collision cell breaks ion 210

Q2 monitors fragments 158

210

ions (from EI) pass through

210

apart and 191 for quant and qual.

170 210 250 290

222

268 280165

190 210 150 170 190 210

210158

191

160

158

190

191

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

Spinach Extract - Scan Chromatogram

Abundance

TIC:spinach incur1 scanD\datams

400000

450000

500000

TIC: spinach_incur1_scan.D\data.ms

250000

300000

350000

400000

100000

150000

200000

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

50000

Time-->

GC/MS/MS Virtually Eliminates Matrix InterferenceSpinach ExtractSpinach Extract

p,p’-DDE

Abundance

MRM

400000

450000

500000

Abundance

TIC: spinach_incur1_scan.D\data.ms

p,p’-DDE

200000

250000

300000

350000

400000 Scan

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

50000

100000

150000

200000


4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Time-->

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

MRM (2 transitions)

Apple

SIM - EIC (246)

S/N = 448Apple

Cabbage

S/N 448

S/N = 241

Ginseng S/N = 446

Orange S/N = 456

Spinach S/N = 260

April, 2009

Some Things are Common to All of the Pesticide AnalyzersAnalyzers

1) Backflushing

2) Multimode Inlet2) Multimode Inlet


Why do all GC/MS(MS) Pesticide Analyzers IncludePesticide Analyzers Include

Backflushing?gBecause you analyze dirty samples!y y y p


What Happens When You Don’t Backflush

9x103 Run time = 20 min

Dandelion root powder full scan (m/z 45-650) analysis without backflushing

1

2

9x10 Acetonitrile blank analysis after the dandelion root powder analysis without backflushing9x10

1

2

3Acetonitrile blank analysis after the dandelion root powder analysis without backflushingand run time of 20 min

Matrix peaks observed in two subsequent blank injections!

9x10

1

2

3 Run time = 35 min (additional 15 min at 290°C) At least additional 10 min at 290°C needed to elute the less volatile matrix

Counts vs. Acquisition Time (min)4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

components (e.g. sterols).

Results provided by Dr Katarina Mastovska Covance Laboratories


Results provided by Dr. Katarina Mastovska, Covance Laboratories

Typical Backflush Configuration: Analysis ModeSplit/Splitless Z mL/min = very low flow

Vent

Aux EPC

Purged Ultimate

p pInjection Port

Z ml/min

y

Purged Ultimate Union

7000B GC MS/MS

Or

5975C MSD

7890A GC

Y+Z ml/min2-m x 0.25 mm

deactivated fused silica Y Z ml/min

P d

(Optional Retention Gap)

Y ml/min

Purged Union

15-m HP-5ms UI (0.25mm id x0.25um)

0.65-m fused silica (0.15 mm id)

Or Second column

July 2009

Purged Union: Backflush ModeSplit/Splitless

Vent

Aux EPC

7890A GC

p pInjection Port

Decrease inlet pressure during Purged Ultimate7890A GC pressure during backflush

2-m x 0.25 mm deactivated fused silica

7000B GC MS/MS

Or

5975C MSD

Purged Ultimate Union

(Optional Retention Gap)

15-m HP-5ms UI (0.25mm id x0.25um)

0.65-m fused silica (0.15 mm id)

Or Second column

July 2009

The Purged Ultimate Union (PUU) configurationsAux EPC Purged

PUU

S

Ultimate Union

Column 1 Column 2

MSD or

QQQ

Vocabularyinlet MS

(Turbo Only)

yPost-column

PUU

restrictorCapillary column

inletMid-columnPUU

Capillary columnCapillary columnMS(Turbo or Diff)

inletMid-columnPUU

Longer capillary columnShort MS(Turbo or Diff)

capillary column

Heavy Compounds May Be Left in Head of Column After Each InjectionAfter Each Injection

C lI l t

After Run 1 Carrier Flow

1 1 1 ColumnInlet After Run 2

1 1 1

1 1 12 2 2

After Run 31 1 12 2 23 3 3

These heavy materials build up and travel further into the column with each injection.

This buildup of heavy materials causes retention time shifts, peak distortion, p y , p ,higher bleed, ghost peaks, and loss of sensitivity

Page 28

Backflushing After Each InjectionEnd of Run Flow

B kfl h 20 Flow

ColumnInlet

End of Run Flow

Backflush 20 sec Flow

FlBackflush 30 sec Flow

Backflush 40 sec Flow

FlBackflush 60 sec Flow

Backflushing removes heavy materials after each injection

Page 29

Backflushing removes heavy materials after each injection.

Backflushing Eliminates Less Volatile Matrix Components (Using Mid-Column Backflush)( g )

Dandelion root powder full scan (m/z 45-650) analysis with backflushing

9x102x109x10 9x10

1.5

2

2.5

3 Last analyte RT = 18.45 minDeltamethrin (m/z 253>172)

2

3

4

5

69x10

1.5

2

2.5

3 Last analyte RT = 18.45 minDeltamethrin (m/z 253>172)

0.5

1

9x103

Run time = 20 min

Backflushing starts

0

1

Counts vs. Acquisition Time (min)18.2 18.3 18.4 18.5 18.6

0.5

1

9x103

Run time = 20 min

Backflushing starts

1

1.5

2

2.5

Acetonitrile blank analysis (no backflushing, additional 15 min at 290°C)after the dandelion root powder analysis with backflushing

(after deltamethrin safely elutes from the first column)

1

1.5

2

2.5

Acetonitrile blank analysis with extended runNo matrix peaks from the previous injection observed!

Results provided by Dr Katarina Mastovska Covance Laboratories

0.5


No matrix peaks from the previous injection observed!0.5


No matrix peaks from the previous injection observed!

30


Long-term System PerformanceOverlays of GC-MS/MS chromatograms for selected analytes in spiked samples obtained within the sequence of 125 matrix injections

Deltamethrinm/z 253>174

3x10

Dichlorvosm/z 185>93

Malathionm/z 173>99

Ethionm/z 231>129

3x10

Phosalonem/z 367>182

3x10 2x10 2x10

Deltamethrinm/z 253>174

3x10

Dichlorvosm/z 185>93

Malathionm/z 173>99

Ethionm/z 231>129

3x10

Phosalonem/z 367>182

3x10 2x10 2x10

GinsengRootPowder

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4

3x10

0

0.5

1

1.5

2

2.5

3

3x10

0.5

1

1.5

2

2.5

3

3.5

4

2x10

0

1

2

3

4

5

6

7

2x10

0

0.5

1

1.5

2

2.5

3

3.5GinsengRootPowder

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4

3x10

0

0.5

1

1.5

2

2.5

3

3x10

0.5

1

1.5

2

2.5

3

3.5

4

2x10

0

1

2

3

4

5

6

7

2x10

0

0.5

1

1.5

2

2.5

3

3.5

Saw Palmetto Berry

3x10

1.5

2

2.5

3

3.5

0

Counts vs. Acquisition Time (min)4 4.05 4.1 4.15 4.2

3x10

1

1.5

2

2.5

0

Counts vs. Acquisition Time (min)9.9 10 10.1 10.2 10.3 10.4

3x10

1 5

2

2.5

3

3.5


0


2x10

4

5

6

7

8

0

Counts vs. Acquisition Time (min)18.1 18.2 18.3 18.4 18.5 18.6

2x10

2

2.5

3

3.5

4Saw Palmetto Berry

3x10

1.5

2

2.5

3

3.5

0


3x10

1

1.5

2

2.5

0


3x10

1 5

2

2.5

3

3.5


0


2x10

4

5

6

7

8

0


2x10

2

2.5

3

3.5

4

Berry Powder

Scutellaria

2x10

3 5

4

4.5

0

0.5

1


3x10

4

5

0

0.5

1


3x10

2

2.5

2x10

6

7

0

0.5

1

1.5


3x10

2.5

3

0

1

2

3


0

0.5

1

1.5


Berry Powder

Scutellaria

2x10

3 5

4

4.5

0

0.5

1


3x10

4

5

0

0.5

1


3x10

2

2.5

2x10

6

7

0

0.5

1

1.5


3x10

2.5

3

0

1

2

3


0

0.5

1

1.5


ScutellariaPowderedExtract

0

0.5

1

1.5

2

2.5

3

3.5


0

1

2

3

4


0.5

1

1.5

2


0

1

2

3

4

5

6


0.5

1

1.5

2

2.5


ScutellariaPowderedExtract

0

0.5

1

1.5

2

2.5

3

3.5


0

1

2

3

4


0.5

1

1.5

2


0

1

2

3

4

5

6


0.5

1

1.5

2

2.5


31


Why are all Pesticide Analyzers Configured with a

M lti d I l t?Multimode Inlet?Because you analyze labile pesticides!


What’s a Multimode Inlet?

• Same features as a Split/Splitless InletSame features as a Split/Splitless Inlet• Uses same liners & septa• Temperature programmable – Two cooling options

• Liquid N2 or Air coolingCO2 li• CO2 cooling

• Cold split & splitless injections• Cold pulsed split & splitless injections• PTV solvent-vent injections for large volume injectionj g j• Twist top for easy liner exchange• New 2 mm dimpled liner for cold injections


Liner Choice – More Important than you Think!

Agilent 2 mm i.d. dimpled deactivated liner (P/N 5190-2296)

D i d f di t l• Designed for dirty samples• Designed for labile pesticides• Dimples block line of sight between syringe & column• Dimples spread matrix over large surface for longer p p g glife & less matrix-caused injection problems• Keeps column clean• 2 mm i.d. for good peak shape• Only for cold injections• Only for cold injections

• Liner volume is too small for hot splitless

After >125 5-µL dietary supplement injections (QuEChERS Extracts)


1uL cold splitless vs 5uL solvent vent

1 L C ld litl1 uL Cold splitless

5 uL Solvent Vent5 uL Solvent Vent

5uL vs 10uL Solvent Vent with MMI

5 uL Solvent Vent

10 uL Solvent Vent

Setting up a large volumeSetting up a large volume injectioninjection

using the Multi-Mode Inletand the Solvent Vent

Calculator WizardCalculator Wizard

MMI Solvent Elimination Wizard for LVI

Click on the Solvent Elimination Calculator button on the GC Inlet Parameters tabon the GC Inlet Parameters tab

Injection Rate and Vent Time are Calculated Interactively Based on the Input ValuesInteractively Based on the Input Values

Optimized Values are Generated by the Wizard

Confused? Which Analyzer do you Need?

GC/MSD DRS P ti id A l GC/MS/MS P ti id A lGC/MSD DRS Pesticide Analyzer926 Pesticides & endocrine

disruptors21 minute Run, Constant


42 minute Run, Constant21 minute Run, Constant Pressure, Post-column

Backflushing


Backflushing

GC/MSD Japanese Positive List GC/MS/MS Pesticide Analyzer –GC/MSD Japanese Positive List DRS Pesticide Analyzer


P t l B kfl hi

GC/MS/MS Pesticide Analyzer US Method

>230 Pesticides 19 minute Run, Constant Flow,

Mid l B kfl hi


Post-column Backflushing Mid-column Backflushing

If you need to…S f l t f d i 25 i /Screen for a lot of compounds in ~25 min/run

Save hours of data review time

N d t b thi i i dNeed to be sure nothing is missed

Look for banned or miss-used pesticides

See most pesticides in the 10 100 ppb rangeSee most pesticides in the 10 – 100 ppb range

Avoid being the lead story on the evening news

GC/MSD DRS Pesticide Analyzer

926 Pesticides & endocrine926 Pesticides & endocrine disruptors

21 minute Run, Constant Pressure, Post-column Backflushing


Post column Backflushing

If your focus is on Japanese import/export market and you need to…Screen for a lot of compounds

Save hours of data review time

Need to be sure nothing is missed

Look for banned or miss-used pesticidesLook for banned or miss used pesticides

See most pesticides in the 10 – 100 ppb range

A id b i th l d t th iAvoid being the lead story on the evening newsGC/MSD Japanese Positive List

DRS Pesticide Analyzery



Post-column Backflushing

If you need to…Achieve the lowest detection limits (low ppb to ppt range)Achieve the lowest detection limits (low ppb to ppt range)

Minimize matrix interference

Analyze for a list of target pesticides

Do less sample cleanup

Want to replace old GC/MS or selective detector methodsGC/MS/MS Pesticide Analyzer –

Agilent Method GC/MS/MS Pesticide Analyzer –Agilent Method555 Pesticides


US Method>230 Pesticides

19 minute Run, Constant Flow, Mid-column Backflushing

orBackflushing Mid column Backflushing

But which one?


Which One of the configuration to order?-- Not that critical: They are interchangeable!Not that critical: They are interchangeable!

EPCConstant PressurePost column Backflush

Agilent (EU based) 500+ P ti id

SP1 7890-0501 Setup

Inlet QQQor a splitter

EPC Post-column Backflush 500+ Pesticides

flexibility to add GC detectors and easily scaled for shorter runtime

EPCConstant FlowMid-column Backflush

USA targeted 200+ Pesticides

SP1 7890-0502 Setup

Inlet QQQ

provides ultimate performance and shortest cycle time

46

p p y

Backflush Configurations for the GC/QQQ Pesticide Analyzersy

CONFIGURATION POST COLUMN MID-COLUMN

Best Features Easy to understand /Fl ibili Minimal activity /VersatilityBest Features Flexibility Minimal activity /Versatility

Important Limitations Turbo Only / Signal dilution

Setup / Concurrent BF is more

ComplexRTL and Method scalingRTL and Method scaling (speedup method) Easy to do Manual and difficult

Constant Pressure (CP) or Constant Flow (CF) CP CF

Signal Loss / Peak dilution Moderate Least (BEST)

Post-Run or Concurrent Post-Run ONLY Post-Run OR Concurrent

E f P t REase of Use Easy

Easy for Post‐Run Concurrent harder

Changing column Easy (1 column, no venting)

Change Col 1, easyChange both cols, must vent

MS systems Turbo ONLY Turbo & Diff

Fast Track GC/MS(MS) Pesticide Analyzers… no on-site break-in needed, ready for injections

At User’s LabAt FactoryInstall Purged Ultimate Union Plug S t I t tInstall Purged Ultimate Union for BackflushingInstall ColumnsComplete Checkout in factory

Plug and Play

Set up InstrumentCheck RTLRun Pesticide StandardAnalyze Real Samplesp y

Run Pesticide Checkout SampleProvide Complete Pesticide Analysis Method

Ready for Ready for the 1the 1stst runrun

y p

Analysis Method

How to order

GC/MSD DRS Pesticide Analyzer926 Pesticides & endocrine

disruptors


Order SP1 P/N 7890-0456 Order SP1 P/N 7890-0501

GC/MSD Japanese Positive List GC/MS/MS Pesticide Analyzer –GC/ S Japa ese os t e stDRS Pesticide Analyzer

430 Pesticides

O d SP1 P/N 7890 0457

GC/ S/ S est c de a y eUS Method

>230 Pesticides

O d SP1 P/N 7890 0502


Order SP1 P/N 7890-0457 Order SP1 P/N 7890-0502

Thank YouThank You

Agilent - Making sense out of the alphabet soup

Portrait with Vegetables (The Greengrocer) Artist: Giuseppe Arcimboldoppb. 1527 d. 1593, Milan, Italy

Improved Pesticide Residue Analysis using Agilents GC ... Pestici… · Improved Pesticide Residue Analysis using Philip L. Wylie, Ph.D. Residue Analysis using Sr Applications Chemist

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