Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt

Technische Universität München

Chromatography, Workflows
& Data evaluation

Non-target Screening

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Suspected-target
Target Screening Non-target Screening
Screening

Make a list of “known Screen for

Make a list of Screen for “Hidden “Unknown targets“
targets“ “expected targets“ targets“ (Known (Unknown
unknowns) unknowns)

Check if reference
substance is
Search for molecule Search for
available
in literature/ similarities with
databases or hydrophobicity,
compare with monoisotopic mass,
Validate and quantify prediction program fragmentation
the molecule behaviour, etc.

Tentatively identify Predict chemical

Identify and quantify the molecule
the molecule structure

Produce a reference
substance

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Non-target Screening
Known Unknowns (“Hidden Targets”):
Basically, “Hidden Targets” can be found in the literature or in databases (compare
“SuspectedTarget”) but are unknown to the investigator.
In contrast to a suspected-target screening, an accurate MS-system, like LC-QqToF and
Orbitrap-MS, scans a broad mass range in order to find molecules. Typical workflows in non-
target screening are:
- Automated peak detection by exact mass filtering
- Assignment of elemental formula to exact “mass of interest”
- Visualizing data points in so-called RT-MW-plots (retention time versus molecular weight)
to compare the results at various points in time.
- Retention time indexing (standardization method) and in silico fragmentation
prediction to support the characterisation process
- Spectral database search to confirm the identification of the molecule

Unknown Unknowns:
If the “mass of interest” cannot be identified by any of the aforementioned methods, it is
called a “unknown Unknown”. Consequently the only information about this compound is its
m/z and its retention time.
In this case, a comparative fingerprint analysis might help you to find the molecule
structure by comparing hydrophobicity, monoisotopic mass or fragmentation behaviour with
those of known substances.
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Peak detection
A batch of MS data is processed by an untargeted feature extraction algorithm that generates
a list of compounds with MW, RT, m/z. Mass deviation should be ≤ 5 ppm.

Check the peak shape

Check the isotopic pattern

and adducts

PD Dr. J. Graßmann; PD Dr. T. Letzel

 Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

RT-MW-plot

Sample 1 Sample 2
2500 2500
Example feature
2000 2000

Mass (Da)
Mass (Da)

1500 1500

1000 1000

500 500

0 0
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40
Retention time (min) Retention time (min)

RT-MW-plots allow you to compare different, but somehow related samples and hence
help to determine relevant compounds.

PD Dr. J. Graßmann; PD Dr. T. Letzel

 Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Special software may provide the TIC

possibility to calcutate sum formulas of
a compound of interest along with the
mass deviation from the mass
spectrometrically detected m/z.

Spectrum with
compound of interest,
i.e. detected m/z

Calculation of several
possible sum formulas

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

MS/MS fragment sectrum - example:

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Database search

Identifier
Deviation of Entry in the database
measured and “MassBank“ that contains
theoretical mass experimentally determined
MS spectra

Total score Comparison of

measured and
theoretical spectrum

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

RTI
RTI: „Retention time index“ = normalised retention time

As a prerequisite, a calibration curve needs to

be plotted. Therefore the retention times of
reference substances are measured with the
same LC-MS(/MS) method as the analyte.
Values on a linear scale between 50 and 150
are assigned to the results. Those so called RTI
values are correlated with the logD, provided
that logD = logP. By calulating the
corresponding RTI of unknown substances,
their logD value can subsequently be estimated.

PD Dr. J. Graßmann; PD Dr. T. Letzel

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

What you know

In comparison to suspected target screening, you may only know the m/z and the sum formula of
a compound. There may furthermore be indications about the compound´s hydrophobicity, which
is related to the captured retention time.
However you don´t know the logP or logD, which can only be calculated for compounds of which
you know the molecular structure.

Subsequent procedures
- Performing MS/MS measurements to learn about the chemical structure of a compound may
help with the identification. The obtained fragmentation pattern can be compared with
databases, which provide reference spectra of all kinds of compounds. This eventually may
result in the successful identification.
- Consulting scientific literature, i.e. someone may have detected the compound before and
already performed all the necessary steps for a successful identification, e.g. MS/MS
measurements. The provided information from literature (e.g. the detected m/z and retention
time or a proposed structural formula) can be further used for the comparison with your
experimental results, i.e. your retention time or MS/MS experiments. Retention time
prediction programs may then further verify your findings.

PD Dr. J. Graßmann; PD Dr. T. Letzel