1 The world leader in serving science Using Automated Software for Improved Results in GC Triple Quadrupole MS Pesticide Analysis Jason Cole and Paul Silcock Thermo Fisher Scientific
May 10, 2015
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The world leader in serving science
Using Automated Software for Improved Results in GC Triple Quadrupole MS Pesticide Analysis
Jason Cole and Paul Silcock
Thermo Fisher Scientific
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Triple Quadrupole GC-MS/MS
Fast becoming an essential tool in high-throughput, routine laboratories
• Especially true for laboratories performing pesticides analysis in food
• Becoming more so in environmental analysis
• Mainly driven by the selectivity advantages of MS/MS
Environmental
Food Safety
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http://books.google.com/ngrams 5.2 million books: ~4% of all books ever published
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GC-MS/MS – What’s So Special?
• Low detection limits
• Optimized sample preparation
• Consolidated analytical methods
• Faster, automated data processing
...it’s a high selectivity technique...
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Selectivity in a Method
McLafferty circa. 1980
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Method Performance Requirement
• Target compounds
• Matrices
• Sensitivity
Method performance requirement
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First - Sample Prep..
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Method performance requirement
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...then Instrument Detection...
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Total method selectivity
Method performance requirement
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...What about GC-MS/MS?...
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Total method selectivity
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...Use GC-MS/MS to Reduce Clean-Up...
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...Use GC-MS/MS to Consolidate Methods...
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Method 1performance requirement
Method 2performance requirement
Method 3performance requirement
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...Use GC-MS/MS to Consolidate Methods...
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Consolidated multi-residue method
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...so GC-MS/MS is Special as it Delivers...
High Selectivity
• Possibility to reduce selectivity in sample preparation
• Reduced sample prep steps create a more generic sample prep method – more compounds & matrices
• Consolidated GC-MS methods due to high performance – buffer against requirements
• Compressed chromatography possible
• Easy peak evaluation – auto-integrators
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...and often in Pesticides Analysis Leads to...
High Selectivity
• Possibility to reduce selectivity in sample preparation
• Reduced sample prep steps create a more generic sample prep method – more compounds & matrices
• Consolidated GC-MS methods due to high performance – buffer against requirements
• Compressed chromatography possible
• Easy peak evaluation – auto-integrators
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Why are we here today?
Discussion of the practical issues that arise in the lab due to the extra capability GC-MS/MS brings and nature of the technique
• Most people working with this instrumentation are looking for ways to easily create, optimize and manage in routine large multi-residue GC-MS/MS methods
• Also, improve analytical performance in pesticides analysis
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Practical Issues?
The power of the technique is great, but...
• We are consolidating more and more compounds into a single method
• We are having to develop 100s of Selected Reaction Monitoring (SRM) transitions
• We are having to maintain and manage large, high performance methods – in routine!
• All this, in a variety of complex matrices
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What’s Really Needed...
To really benefit from the productivity advantages of these multi-residue methodologies, we need to:
• Create complex methods - independent of where we begin
• Manage all the complex information associated with large complex methods
• Maintain these methods in routine
• Ensure we are not creating a new bottleneck!
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Demonstrate how we can use automated software
• Integrated into the set-up and operation of the GC-MS/MS system
• Remove the pain (and avoid the bottleneck)
• Method creation
• Method optimization
• Method management
• Method maintenance
• For improved results (and productivity) in pesticide analysis
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Enabling Technology
“To make the productivity advantages of high performance GC-MS/MS easy to achieve and available routinely; especially for high-throughput laboratories.”
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Anatomy of a Multi-Residue Pesticide Method
Peaks!
• Lots of them, too
• Multiple ions (SRM transitions)
• Multiple co-elutions
• Not much “clear baseline”
• Diverse chemistries
• Also chemistry similarities
• Large difference in response factors
• Different LOD requirements
• Different interference (matrix) pressures
• COMPLEXITY!!
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Complexity in Developing Multi-Residue MRM Methods
15. Run sequence
16. Examine each data compounds product ion spectrum in each run for each collision energy.
17. (Re-inject for any missed compounds)
18. Recording of best SRM transitions
19. Create MRM method to optimize collision energies
20. Create a pilot method(s) – to test selectivity of transitions in target matrices.
21. Choose final transitions
22. Segment method
23. Calculate appropriate dwell times (depending of number of overlapping transitions)
24. Test final method in matrix.
25. Check for “chopped” or missed peaks
26. Re-adjust method as necessary
1. List compounds (350 pesticides)
2. Arrange standard solutions into vial (s)
3. Set-up GC method
4. Run a full-scan
5. Examine data files to find compounds (extracting ions or using libraries)
6. Record retention times
7. Select and record appropriate pre-cursor ions
8. Create product ion scan methods
9. Segment these methods into windows based on chromatogram
10. Calculate appropriate scan times for good daughter ion experiments
11. Re-segment based on (10)
12. Set-up these methods for all collision energies to see progressive fragmentation
13. Decide on number of injections
14. Set-up sequence list
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Instrument & Data Processing Method Maintenance
• GC-MS/MS systems in routine pesticide analysis face high volumes of samples with high matrix load
• Cumulative deterioration of the GC column performance
• Backflushing set-up can help to mitigate
• Inevitably compound retention times drift and or GC columns need to be cut or replaced
• Need to locate compounds, update acquisition windows & update RT in data processing method
• Very laborious & time consuming- worse with a large method!
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Demonstrate how we can use automated software
• Integrated into the set-up and operation of the GC-MS/MS system
• Remove the pain (and avoid the bottleneck)
• Method creation
• Method optimization
• Method management
• Method maintenance
• For improved results (and productivity) in pesticide analysis
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Software Overview
Automated SRM Development
Timed-SRM Instrument Method
Batch Acquisition, Data Review, and reporting software
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Complexity in Developing Multi-Residue MRM Methods
15. Run sequence
16. Examine each data compounds product ion spectrum in each run for each collision energy.
17. (Re-inject for any missed compounds)
18. Initial selection of best SRM transitions
19. Create a pilot method(s) – to test selectivity of transitions in target matrices.
20. Choose final transitions
21. Segment method
22. Calculate appropriate dwell times (depending of number of overlapping transitions)
23. Test final method in matrix.
24. Check for “chopped” or missed peaks
25. Re-adjust method as necessary
1. List compounds (350 pesticides)
2. Arrange standard solutions into vial (s)
3. Set-up GC method
4. Run a full-scan
5. Examine data files to find compounds (extracting ions or using libraries)
6. Record retention times
7. Select and record appropriate pre-cursor ions
8. Create product ion scan methods
9. Segment these methods into windows based on chromatogram
10. Calculate appropriate scans times for good daughter ion experiments
11. Re-segment based on (9)
12. Set-up these methods for all collision energies to see progressive fragmentation
13. Decide on number of injections
14. Set-up sequence list
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Surely, we have these pesticide transitions already!?
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Surely, we have these pesticide transitions already!?
Of course!
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Thermo Scientific TraceFinder Compound Data Store
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Compound-Based Method Creation
Selecting your compounds from CDS…
and processing methods
populates synced acquisition…
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Thoughts on Fishing and Triple Quadrupoles
"Give a man a fish; feed him for a day. Teach a man to fish; feed him for a lifetime“
Lao Tzu circa. 5th Century BC
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Thoughts on Fishing and Triple Quadrupoles
"Give a man a fish; feed him for a day. Teach a man to fish; feed him for a lifetime“
Lao Tzu circa. 5th Century BC
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AutoSRM Overview
1) Precursor ion selection
2) Product ion selection
3) Collision energy optimization
SR
M C
reation
Wo
rkflow
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Step 1 – Pick Your Precursor Ions
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Step 1 – Pick Your Precursor Ions
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Step 1 – Pick Your Precursor Ions
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Step 2 – Pick Your Product Ions
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Step 2 – Pick Your Product Ions
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Step 3 – Optimize Your Transitions
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Step 3 – Optimize Your Transitions
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AutoSRM Use Case
• Created and optimized > 250 transitions for > 80 compounds• Minimal user interaction over 24 hours
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Export from AutoSRM to Instrument Method
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Timed-SRM Method Overview
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Timed-SRM Method Overview
Acquisition windows centered around retention time
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Timed-SRM Method Overview
Acquisition windows allowed to overlap
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Timed-SRM Advantages
Segmented SRM
Timed SRM
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Timed-SRM Advantages
Acquisition Windows
Segmented SRM
Timed SRM
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Timed-SRM Advantages
• Removes wasted dwell time
• Allow higher overall dwell times
• Leads to higher sensitivity
Wasted Dwell Time
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Timed-SRM Advantages
• Peaks centered in acquisition window
• No peak elutes near acquisition break
• Allows for retention time shift (e.g. due to heavy matrix)
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Thermo Scientific TSQ 8000 GC-MS/MS Timed-SRM Case Study
• Previous method: Segmented SIM acquisition on single quad
• Required five injections for full list of pesticides
• Needed to analyze more pesticides (350 total), but current methodology took too long
• Wanted to consolidate to a single injection
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• Segmented SRM
• Closest compound to segment break:
5 seconds
• Average number of simultaneous transitions:
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• Timed SRM
• Closest compound to segment break:
15 seconds
• Average number of simultaneous transitions:
15 (4X higher dwell times)
TSQ™ 8000 GC-MS/MS Timed-SRM Case Study
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TSQ 8000 GC-MS/MS Timed-SRM Case Study
Tea Analysis: 4 pg on-column
Terbacil
Alachlor
Tolylfluanid
Pyridaben
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Export from Instrument Method to TraceFinder™ Software
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TraceFinder Software Overview
Batch Creation
Data Review
Report Generation
Routine Workflow
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Instrument & Data Processing Method Maintenance
• GC-MS/MS systems in routine pesticide analysis face high volumes of samples with high matrix load
• Cumulative deterioration of the GC column performance
• Backflushing set-up can help to mitigate
• Inevitably compound retention times drift and or GC columns need to be cut or replaced
• Need to locate compounds, update acquisition windows & update RT in data processing method
• Very laborious & time consuming- worse with a large method!
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TraceFinder Software Method Sync
• Links TraceFinder Software Method with instrument method
• Enables• Compound based acquisition setup
• Automated update of acquisition windows
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Method Sync – Automated RT Update
Updating retention times in data review…
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Method Sync – Automated RT Update
…updates both TraceFinder Software Method and Timed-SRM Method
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Summary
We can use integrated and automated software
• Easily adopt large multi-residue methods
• Remove the pain (and avoid bottlenecks)
• Method creation
• Method optimization
• Method management
• Method maintenance
• To create improved results (and productivity) in routine pesticide analysis
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Thank You for Your Attention!
Questions?
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