Automated Software for Improved Results in Triple Quadrupole Gas Chromatography-Mass Spectrometry Pesticide Analysis

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

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

3

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

4

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

Selectivity in a Method

McLafferty circa. 1980

7

Method Performance Requirement

• Target compounds

• Matrices

• Sensitivity

Method performance requirement

8

First - Sample Prep..

Ste

p 1

– e

xtr

ac

tio

n

Ste

p 2

- c

lea

n u

p (

1s

t)

Ste

p 3

- c

lea

n u

p (

2n

d)

Method performance requirement

9

...then Instrument Detection...

Ste

p 1

– e

xtr

ac

tio

n

Ste

p 2

- c

lea

n u

p (

1s

t)

Ste

p 3

- c

lea

n u

p (

2n

d)

Ste

p 4

- G

C-M

S d

etec

tio

n (

sin

gle

qu

ad S

IM)

Total method selectivity

Method performance requirement

10

...What about GC-MS/MS?...

Ste

p 1

– e

xtr

ac

tio

n

Ste

p 2

- c

lea

n u

p (

1s

t)

Ste

p 3

- c

lea

n u

p (

2n

d)

Total method selectivity

Ste

p 4

- G

C-M

S/M

S d

etec

tio

n (

Trip

le q

ua

d S

RM

)

Method performance requirement

11

...Use GC-MS/MS to Reduce Clean-Up...

Ste

p 1

– e

xtr

ac

tio

n

Total method selectivity

Ste

p 2

- G

C-M

S/M

S d

etec

tio

n (

Trip

le q

ua

d S

RM

)

Method performance requirement

12

...Use GC-MS/MS to Consolidate Methods...

Ste

p 1

– e

xtr

ac

tio

n

Total method selectivity

Ste

p 2

- G

C-M

S/M

S d

etec

tio

n (

Trip

le q

ua

d S

RM

)

Method 1performance requirement

Method 2performance requirement

Method 3performance requirement

13

...Use GC-MS/MS to Consolidate Methods...

Ste

p 1

– e

xtr

ac

tio

n

Total method selectivity

Ste

p 2

- G

C-M

S/M

S d

etec

tio

n (

Trip

le q

ua

d S

RM

)

Consolidated multi-residue method

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

...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

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

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

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

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

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

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

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

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

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

Software Overview

Automated SRM Development

Timed-SRM Instrument Method

Batch Acquisition, Data Review, and reporting software

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

Surely, we have these pesticide transitions already!?

29

Surely, we have these pesticide transitions already!?

Of course!

30

Thermo Scientific TraceFinder Compound Data Store

31

Compound-Based Method Creation

Selecting your compounds from CDS…

and processing methods

populates synced acquisition…

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

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

AutoSRM Overview

1) Precursor ion selection

2) Product ion selection

3) Collision energy optimization

SR

M C

reation

Wo

rkflow

35

Step 1 – Pick Your Precursor Ions

36

Step 1 – Pick Your Precursor Ions

37

Step 1 – Pick Your Precursor Ions

38

Step 2 – Pick Your Product Ions

39

Step 2 – Pick Your Product Ions

40

Step 3 – Optimize Your Transitions

41

Step 3 – Optimize Your Transitions

42

AutoSRM Use Case

• Created and optimized > 250 transitions for > 80 compounds• Minimal user interaction over 24 hours

43

Export from AutoSRM to Instrument Method

44

Timed-SRM Method Overview

45

Timed-SRM Method Overview

Acquisition windows centered around retention time

46

Timed-SRM Method Overview

Acquisition windows allowed to overlap

47

Timed-SRM Advantages

Segmented SRM

Timed SRM

48

Timed-SRM Advantages

Acquisition Windows

Segmented SRM

Timed SRM

49

Timed-SRM Advantages

• Removes wasted dwell time

• Allow higher overall dwell times

• Leads to higher sensitivity

Wasted Dwell Time

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

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

• 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

TSQ 8000 GC-MS/MS Timed-SRM Case Study

Tea Analysis: 4 pg on-column

Terbacil

Alachlor

Tolylfluanid

Pyridaben

54

Export from Instrument Method to TraceFinder™ Software

55

TraceFinder Software Overview

Batch Creation

Data Review

Report Generation

Routine Workflow

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

TraceFinder Software Method Sync

• Links TraceFinder Software Method with instrument method

• Enables• Compound based acquisition setup

• Automated update of acquisition windows

58

Method Sync – Automated RT Update

Updating retention times in data review…

59

Method Sync – Automated RT Update

…updates both TraceFinder Software Method and Timed-SRM Method

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

Thank You for Your Attention!

Questions?

Stay connected with us

Twitter @ChromSolutions

Chromatography Solutions Blog http://chromblog.thermoscientific.com/blog

YouTubehttp://www.youtube.com/ChromSolutions

Facebookhttp://www.facebook.com/ChromatographySolutions

Pinteresthttp://pinterest.com/chromsolutions/