Untargeted Metabolomics: Tandem LC-MSMS

Column and Flow Rate Selection

Insert Barnes table for flow rates and sensitivity gain.

Reverse Phase and Normal Phase Selections:

• Reverse Phase (C4, C8, C18, etc…)• Normal Phase (Silica, Amide, Amido, etc…)

Instrument Configuration

AB Sciex 5600 Triple Tof Mass Spectrometer (Q-Tof MS )

The Time of Flight (ToF) chamber allows for rapid detection and high mass accuracy as compared to traditional triple quadrupole instrumentation. All fragments are detected simultaneously instead of a precursor (parent ion) > fragment (daughter ion) pair.

High Resolution and Acquisition Speed

Precursor Scans collected over 100 msec;Fragmentation Spectra collected as low as 10 msec.

Typical Instrument Acquisition Method

•Precursor Ion Scan: 250 msec

•Product Ion Scan: 50 msec (X 20)

Selected ion for fragmentation

Total Cycle Time is 1.25 seconds

Example of MS/MS scanThe combination of precursor plus

fragmentation scans in one method allows the investigator to

simultaneously qualify and quantify potential compounds between

experimental groups.

Evaluation of Collected Data

1 2 3 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 29Time, min

0.0e0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e6

1.0e7

1.1e7

1.2e7

1.3e7

1.4e7

1.5e7

1.6e7

1.7e7

1.8e7

1.9e7

Inte

nsity

6.01

8.23

8.51

7.82

4.0625.5814.61

9.29

4.64

12.48

25.72

10.7719.15

5.07

19.6111.61 15.25 21.7715.7414.27 17.97

26.27

10.017.45

22.7120.73

6.3823.58

17.01

26.8724.14

TIC from PosNFc216-1.wiff (sample 1) - Pos NFc216-1TIC from PosNFc216-2.wiff (sample 1) - Pos NFc216-2TIC from PosNFc216-4.wiff (sample 1) - Pos NFc216-4TIC from PosVMP-1.wiff (sample 1) - Pos VMP-1TIC from PosVMP-2.wiff (sample 1) - Pos VMP-2TIC from PosVMP-4.wiff (sample 1) - PosVMP-4

• Chromatographic reproducibility

• Sample intensity comparisons

• Mass accuracies between samples

Evaluation of Collected Data continued

6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4Time, min

0.0e0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

Inte

nsity

8.245

XIC from PosNFc216-1.wiff (sample 1) - Pos NFc216-1, Experiment 1, +TOF MS (150 - 1000): 220.1174 +/- 0.0025 Da, Gaussian smoothedXIC from PosNFc216-2.wiff (sample 1) - Pos NFc216-2, Experiment 1, +TOF MS (150 - 1000): 220.1174 +/- 0.0025 Da, Gaussian smoothedXIC from PosNFc216-4.wiff (sample 1) - Pos NFc216-4, Experiment 1, +TOF MS (150 - 1000): 220.1174 +/- 0.0025 Da, Gaussian smoothedXIC from PosVMP-1.wiff (sample 1) - Pos VMP-1, Experiment 1, +TOF MS (150 - 1000): 220.1174 +/- 0.0025 Da, Gaussian smoothedXIC from PosVMP-2.wiff (sample 1) - Pos VMP-2, Experiment 1, +TOF MS (150 - 1000): 220.1174 +/- 0.0025 Da, Gaussian smoothedXIC from PosVMP-4.wiff (sample 1) - PosVMP-4, Experiment 1, +TOF MS (150 - 1000): 220.1174 +/- 0.0025 Da, Gaussian smoothed

• Chromatography tracings overlap

• Mass accuracy window set to 0.005 Daltons

Exporting Data to Statistical Programs

Programs like MS Convert can transform instrument raw files (.wiff, .dta, etc) in usable formats such as mzXML. Keep in mind when transforming the raw data, the size of the files

can increase significantly.

Data Analyses Software

There are a host of software platforms ranging from free

online resources to company specific programs.

Data Analyses Software continued

XCMS Online aligns chromatographyand outputs user friendly schematics

from uploaded data including PCA graphs, t-test, etc.