The pesticide residue changes during food processing and ...
Improved Pesticide Residue Analysis using Agilents GC ... Pestici… · Improved Pesticide Residue...
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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
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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
September 20, 201020
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
September 20, 201022
Why do all GC/MS(MS) Pesticide Analyzers IncludePesticide Analyzers Include
Backflushing?gBecause you analyze dirty samples!y y y p
September 20, 201023
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
September 20, 201024
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
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
No matrix peaks from the previous injection observed!0.5
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
No matrix peaks from the previous injection observed!
30
Results provided by Dr. Katarina Mastovska, Covance Laboratories
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
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Saw Palmetto Berry
3x10
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Counts vs. Acquisition Time (min)4 4.05 4.1 4.15 4.2
3x10
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Counts vs. Acquisition Time (min)9.9 10 10.1 10.2 10.3 10.4
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Counts vs. Acquisition Time (min)15.3 15.4 15.5 15.6 15.7
2x10
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Counts vs. Acquisition Time (min)18.1 18.2 18.3 18.4 18.5 18.6
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Counts vs. Acquisition Time (min)4 4.05 4.1 4.15 4.2
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Counts vs. Acquisition Time (min)15.3 15.4 15.5 15.6 15.7
2x10
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Counts vs. Acquisition Time (min)18.1 18.2 18.3 18.4 18.5 18.6
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Berry Powder
Scutellaria
2x10
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Counts vs. Acquisition Time (min)9.9 10 10.1 10.2 10.3 10.4
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0
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Counts vs. Acquisition Time (min)18.1 18.2 18.3 18.4 18.5 18.6
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Counts vs. Acquisition Time (min)4 4.05 4.1 4.15 4.2
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Counts vs. Acquisition Time (min)9.9 10 10.1 10.2 10.3 10.4
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31
Results provided by Dr. Katarina Mastovska, Covance Laboratories
Why are all Pesticide Analyzers Configured with a
M lti d I l t?Multimode Inlet?Because you analyze labile pesticides!
September 20, 201032
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
September 20, 201033
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)
September 20, 201034
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
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, 201042
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
September 20, 201043
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
430 Pesticides31.5 minute Run, Constant Flow,
September 20, 201044
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
42 minute Run, Constant Pressure, Post-column
US Method>230 Pesticides
19 minute Run, Constant Flow, Mid-column Backflushing
orBackflushing Mid column Backflushing
But which one?
September 20, 201045
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
GC/MS/MS Pesticide Analyzer –Agilent Method555 Pesticides
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
September 20, 201049
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