1) The document discusses methods for predicting retention times (RT) in untargeted metabolomics to help identify compounds. 2) It compares existing approaches like prediction from molecular structure (QSRR) and projection from isocratic runs, and presents a new method called PredRet that allows sharing of RT data across chromatographic systems. 3) PredRet builds models to map RTs between systems accurately, which can help distinguish isomers and limit the number of candidate identifications from mass spectral matches alone.

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Jan Stanstrup†, Steffen Neumann‡, and Urška Vrhovšek†
†Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach
(FEM), San Michele all’Adige (TN), Italy
‡Department of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry, Halle, Germany
The Role of Retention Time
in Untargeted Metabolomics
Caparica-Lisbon, 21st September 2015

2. Research and Innovation Centre
From feature to identified compound
Match to authentic standard MSI LEVEL I
Match to public MS library MSI LEVEL II
No orthogonal data: only spectra
Compound not in any MS library MSI LEVEL III/IV
Orthogonal data: e.g. spectra + retention time

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From feature to identified compound
Match to authentic standard MSI LEVEL I
Match to public MS library MSI LEVEL II
No orthogonal data: only spectra
Compound not in any MS library MSI LEVEL III/IV
Orthogonal data: e.g. spectra + retention time

4. Research and Innovation Centre
From feature to identified compound
Match to authentic standard MSI LEVEL I
Match to public MS library MSI LEVEL II
No orthogonal data: only spectra
Compound not in any MS library MSI LEVEL III/IV
Orthogonal data: e.g. spectra + retention time

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From feature to identified compound
Match to authentic standard MSI LEVEL I
Match to public MS library MSI LEVEL II
No orthogonal data: only spectra
Compound not in any MS library MSI LEVEL III/IV
Orthogonal data: e.g. spectra + retention time

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MSI LEVEL II: match to public db
But are you sure that the spectra of your unknown
couldn’t match other isomers equally well?
You got your match to a database spectra.

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1) Examine mass spectra  No way to discriminate

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1) Examine mass spectra  No way to discriminate
2) Check spectra libraries  Still cannot be sure

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"Piled Higher and Deeper" by
Jorge Cham www.phdcomics.com
1) Examine mass spectra  No way to discriminate
2) Check spectra libraries  Still cannot be sure

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"Piled Higher and Deeper" by
Jorge Cham www.phdcomics.com
1) Examine mass spectra  No way to discriminate
2) Check spectra libraries  Still cannot be sure
3) Buy all standards (if available)  Expensive!

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"Piled Higher and Deeper" by
Jorge Cham www.phdcomics.com
1) Examine mass spectra  No way to discriminate
2) Check spectra libraries  Still cannot be sure
3) Buy all standards (if available)  Expensive!

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"Piled Higher and Deeper" by
Jorge Cham www.phdcomics.com
1) Examine mass spectra  No way to discriminate
2) Check spectra libraries  Still cannot be sure
3) Buy all standards (if available)  Expensive!
So can we at least limit the number of isomers
by using the information we already have?

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Why retention time information is
usually neglected
Retention time is specific to the chromatographic
system and there are no RT references
No coordinated efforts to share and exploit RT info.

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aAbate-Pella D, et al. (2015). Retention Projection Enables Accurate Calculation of Liquid Chromatographic Retention Times Across
Labs and Methods. J Chromatogr.
Prediction from structure (QSRR) Projection from
isocratic runsa
PredRetb
Accuracy Low Very high High to medium
Universality (any structure) High Low Medium
Additional lab work None Lots None
Model complexity High Low Low
Current approaches to retention time prediction
and their characteristics

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Retentiontime(min)
1
2
3
4
Predicted logD
-6 -4 -2 0 2 4
Stanstrup, J., Gerlich, M., Dragsted, L. O., & Neumann, S. (2013). Metabolite profiling and beyond: Approaches for the rapid
processing and annotation of human blood serum mass spectrometry data Metabolomics and Metabolite Profiling. Analytical and
Bioanalytical Chemistry, 405(15), 5037–5048.
Simplest QSRR model

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Candidates after filtering as a
function of prediction accuracy

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Median
Candidates after filtering as a
function of prediction accuracy

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Median
Max
Candidates after filtering as a
function of prediction accuracy

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aAbate-Pella D, et al. (2015). Retention Projection Enables Accurate Calculation of Liquid Chromatographic Retention Times Across
Labs and Methods. J Chromatogr.
Prediction from structure (QSRR) Projection from
isocratic runsa
PredRetb
Accuracy Low Very high Medium to high
Universality (any structure) High Low Low to medium
Additional lab work None Lots None
Model complexity High Low Low
Previous approaches to
retention time prediction

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aAbate-Pella D, et al. (2015). Retention Projection Enables Accurate Calculation of Liquid Chromatographic Retention Times Across
Labs and Methods. J Chromatogr.
bStanstrup, J., Neumann, S., & Vrhovsek, U. (2015). PredRet: Prediction of Retention Time by Direct Mapping between Multiple
Chromatographic Systems. Analytical Chemistry.
Prediction from structure (QSRR) Projection from
isocratic runsa
PredRetb
Accuracy Low Very high Medium to high
Universality (any structure) High Low Low to medium
Additional lab work None Lots None
Model complexity High Low Low
Previous approaches to
retention time prediction

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PredRet models

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Compound overlap between
systems in PredRet

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"Piled Higher and Deeper" by
Jorge Cham www.phdcomics.com

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Accuracy of PredRet predictions

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Candidates after filtering as a
function of prediction accuracy

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Median
Max
Candidates after filtering as a
function of prediction accuracy

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Median
Max
Median
Max
Candidates after filtering as a
function of prediction accuracy

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Conclusions
● PredRet allows sharing of retention time information and
across chromatographic systems

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Conclusions
● PredRet allows sharing of retention time information and
across chromatographic systems
● The accuracy of PredRet can allow differentiation of isomers

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Conclusions
● PredRet allows sharing of retention time information and
across chromatographic systems
● The accuracy of PredRet can allow differentiation of isomers
● Compound need to be in the database (someone need to
record it)

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Conclusions
● PredRet allows sharing of retention time information and
across chromatographic systems
● The accuracy of PredRet can allow differentiation of isomers
● Compound need to be in the database (someone need to
record it)
● Chromatography needs to be similar (e.g. acidic C18-based)

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Conclusions
● PredRet allows sharing of retention time information and
across chromatographic systems
● The accuracy of PredRet can allow differentiation of isomers
● Compound need to be in the database (someone need to
record it)
● Chromatography needs to be similar (e.g. acidic C18-based)
● When people use PredRet the database expands

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Thank you for your attention
Share the RTs!