Key Learning Objectives - Learn how the use of automated software can make SRM development faster and more highly optimized. - Learn how the use of a compound data store can further simplify method creation. - Learn how the use of retention time-based SRM acquisition can increase MS/MS sensitivity and make method maintenance easier. Event Overview: In recent years, Gas Chromatography-triple quadrupole mass spectrometry has increased in popularity due to its ability to offer lower detection limits in complex matrices, simplified sample prep requirements, and faster analysis times. Of course, new instrument technology presents the need for the acquiring of new skills to harness the advantages offered by its adoption into current workflows. In this webinar, a strategy for addressing both of these challenges is discussed in the context of new software designed to automate common method development and method maintenance tasks. Also, in addition to making the triple quadrupole easier to use, this strategy can increase sensitivity of the analysis, which will be demonstrated using a complex SRM pesticide method as an example. For more information: www.thermoscientific.com/tsq8000

1. 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

2. 2
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

3. 3
http://books.google.com/ngrams 5.2 million books: ~4% of
all books ever published

4. 4

5. 5
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...

6. 6
Selectivity in a Method
McLafferty circa. 1980

7. 7
Method Performance Requirement
• Target compounds
• Matrices
• Sensitivity
Method
performance
requirement

8. 8
First - Sample Prep..
Step1–extraction
Step2-cleanup(1st)
Step3-cleanup(2nd)
Method
performance
requirement

9. 9
...then Instrument Detection...
Step1–extraction
Step2-cleanup(1st)
Step3-cleanup(2nd)
Step4-GC-MSdetection(singlequadSIM)
Total method selectivity
Method
performance
requirement

10. 10
...What about GC-MS/MS?...
Step1–extraction
Step2-cleanup(1st)
Step3-cleanup(2nd)
Total method selectivity
Step4-GC-MS/MSdetection(TriplequadSRM)
Method
performance
requirement

11. 11
...Use GC-MS/MS to Reduce Clean-Up...
Step1–extraction
Total method selectivity
Step2-GC-MS/MSdetection(TriplequadSRM)
Method
performance
requirement

12. 12
...Use GC-MS/MS to Consolidate Methods...
Step1–extraction
Total method selectivity
Step2-GC-MS/MSdetection(TriplequadSRM)
Method 1
performance
requirement
Method 2
performance
requirement
Method 3
performance
requirement

13. 13
...Use GC-MS/MS to Consolidate Methods...
Step1–extraction
Total method selectivity
Step2-GC-MS/MSdetection(TriplequadSRM)
Consolidated
multi-residue
method

14. 14
...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

15. 15
...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

16. 16
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

17. 17
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

18. 18
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!

19. 19
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

20. 20
Enabling Technology
“To make the productivity
advantages of high performance
GC-MS/MS easy to achieve and
available routinely; especially for
high-throughput laboratories.”

21. 21

22. 22
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!!

23. 23
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

24. 24
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!

25. 25
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

26. 26
Software Overview
Automated SRM Development
Timed-SRM Instrument Method
Batch Acquisition, Data Review, and
reporting software

27. 27
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

28. 28
Surely, we have these pesticide transitions already!?

29. 29
Surely, we have these pesticide transitions already!?
Of course!

30. 30
Thermo Scientific TraceFinder Compound Data
Store

31. 31
Compound-Based Method Creation
Selecting your compounds
from CDS…
and processing methods
populates synced acquisition…

32. 32
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

33. 33
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

34. 34
AutoSRM Overview
1) Precursor ion selection
2) Product ion
selection
3) Collision
energy
optimization
SRMCreationWorkflow

35. 35
Step 1 – Pick Your Precursor Ions

36. 36
Step 1 – Pick Your Precursor Ions

37. 37
Step 1 – Pick Your Precursor Ions

38. 38
Step 2 – Pick Your Product Ions

39. 39
Step 2 – Pick Your Product Ions

40. 40
Step 3 – Optimize Your Transitions

41. 41
Step 3 – Optimize Your Transitions

42. 42
AutoSRM Use Case
• Created and optimized > 250 transitions for > 80 compounds
• Minimal user interaction over 24 hours

43. 43
Export from AutoSRM to Instrument Method

44. 44
Timed-SRM Method Overview

45. 45
Timed-SRM Method Overview
Acquisition
windows centered
around retention
time

46. 46
Timed-SRM Method Overview
Acquisition
windows allowed
to overlap

47. 47
Timed-SRM Advantages
Segmented SRM
Timed SRM

48. 48
Timed-SRM Advantages
Acquisition Windows
Segmented SRM
Timed SRM

49. 49
Timed-SRM Advantages
• Removes wasted dwell time
• Allow higher overall dwell times
• Leads to higher sensitivity
Wasted Dwell Time

50. 50
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)

51. 51
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

52. 52
• Segmented SRM
• Closest compound to segment
break:
5 seconds
• Average number of simultaneous
transitions:
55
• 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

53. 53
TSQ 8000 GC-MS/MS Timed-SRM Case Study
Tea Analysis: 4 pg on-column
Terbacil
Alachlor
Tolylfluanid
Pyridaben

54. 54
Export from Instrument Method to TraceFinder™
Software

55. 55
TraceFinder Software Overview
Batch Creation
Data Review
Report Generation
Routine Workflow

56. 56
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!

57. 57
TraceFinder Software Method Sync
• Links TraceFinder Software Method with instrument
method
• Enables
• Compound based acquisition setup
• Automated update of acquisition windows

58. 58
Method Sync – Automated RT Update
Updating retention times in data review…

59. 59
Method Sync – Automated RT Update
…updates both TraceFinder Software Method and
Timed-SRM Method

60. 60
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

61. 61
Thank You for Your Attention!
Questions?
Stay connected with us
Twitter
@ChromSolutions
Chromatography Solutions Blog
http://chromblog.thermoscientific.com/blog
YouTube
http://www.youtube.com/ChromSolutions
Facebook
http://www.facebook.com/Chromatography
Solutions
Pinterest
http://pinterest.com/chromsolutions/