Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt

Technische Universität München

Chromatography, Workflows
& Data evaluation

Suspected Target Screening

PD Dr. J. Graßmann; PD Dr. T. Letzel; S. Große

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; S. Große

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Suspected Target Screening

For a suspected target approach, additional information about the sample is

used to create a list of expected or theoretically predicted compounds. This
information can be the origin of the sample, the applied treatment processes,
related substances or possible degradation pathways.

Besides the isotopic pattern and other physicochemical properties (logP/ logD),
specific databases (compare following slide) usually provide the appropriate
chemical formula of the expected substance. That makes it easy to calculate
the exact m/z plus or minus potential adducts. That in turn, can be extracted
from a full scan chromatogram of the sample.

PD Dr. J. Graßmann; PD Dr. T. Letzel; S. Große

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Databases
• Chemical information: ChemSpider (@), Chemicalize (@)
• Compounds: FOR-IDENT (compare picture below)(@) → environmentally
relevant pharmaceuticals, pesticides and biocides from peer-reviewed
scientific literature, REACH-registered chemicals of emerging concern
• Analytical information: DAIOS (@) → water-relevant molecules with
additional data like transformation trees, MS-MS-fragmentation data

Search for name,

CAS number, sum Information on
formula, InChi-Key, structure, names,
IUPAC name, ion category, logP, logD
species, at different pH-
monoisotopic mass, values
logP, halogens

PD Dr. J. Graßmann; PD Dr. T. Letzel; S. Große

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Example:

You may want to analyze a river water sample for the presence of pharmaceuticals
like ibuprofen or diclofenac as well as their known or theoretically predicted
transformation products (= suspected targets). Therefore, you start by making a list
of the expected targets:

Compound Sum formula Average molecular Monoisotopic mass

weight (for more information
see slides „Mass
spectrometry Basics“
Diclofenac C14H11Cl2NO2 296.148 g/mol 295.01667
Ibuprofen C13H18O2 206.28 g/mol 206.13067

Known degr. product of C7H7NO 121.14 g/mol 121.05276

diclofenac from literature *
Etc. ... ... ...

* Coelho et al., Science of The Total Environment 407(11):3572-3578 · May 2009

PD Dr. J. Graßmann; PD Dr. T. Letzel; S. Große

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

After injecting the sample of river water to

a LC-MS system, the experimental
EIC of 295.01667
outcome can be analyzed for the presence
of the suspected targets (compare table on
previous slide).
By extracting the ion chromatograms of the
respective suspected target m/z (EICs),
compounds eluting from the HPLC-column
EIC of 206.13067 can be displayed.

In this particular example, m/z identical to

all suspected targets (diclofenac, ibuprofen
and the degr. product) can be found in the
river water sample.
However, the occurrence of a particular
m/z, which matches the analyte m/z, does
EIC of 121.05276 not necessarily confirm the presence of the
suspected analyte within the sample
(compare „Target screening“ slides). A
sample may contain other compounds with
the same m/z as the analyte( next slide).
PD Dr. J. Graßmann; PD Dr. T. Letzel; S. Große
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

More than one compound present within the sample may have the same molecular
weight, which is reflected by several peaks within the EIC of the m/z of interest.
Comparable to the compounds shown below, which possess the same m/z.
Although they have the same m/z (i.e. same elemental composition), they likely
differ greatly for instance with regard to their chemical structure, hydrophobicity,
their functional groups etc.. Thus they are differently retained on the HPLC column,
which results in different retention times for each individual compound.
To be able to eventually ascribe one peak to the suspected target analyte,
considering further properties, e.g. like water solubility, can be helpful ( logP or
logD, compare slides „Polarity and Solubility“)

EIC of m/z 205.12339 [M-H]-, i.e. negative ionization mode

(compare „Mass spectrometry_Basics“)
?

?
? which peak is ibuprofen?

PD Dr. J. Graßmann; PD Dr. T. Letzel; S. Große

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

In the example below, a water sample is screened for the presence of ibuprofen.
Unfortunatley, the EIC of the according m/z value shows three peaks (see below). Now
information about the polarity of compounds can be helpful, because this property may allow
conclusions to be drawn about retention behavior.
In the case of ibuprofen, an isomeric compound named 4-hydroxyphenyl hexyl ketone is
known. 4-hydroxyphenyl hexyl ketone has a logD of 3.56 (at pH 7.4), whereas ibuprofen
has a logD of 1.34 (at pH 7.4). Thus ibuprofen has (although still rather hydrophobic) a
better water solubility than 4-hydroxyphenyl hexyl ketone. Furthermore at pH 7.4 a much
higher percentage of ibuprofen will be negatively charged compared to 4-hydroxyphenyl
hexyl ketone. Consequently the interaction of 4-hydroxyphenyl hexyl ketone with the
stationary phase of a RP-column can be assumed to be much stronger, which would result
in a later elution (compare graph below, red arrow). So in this particular example and
provided that both compounds are present within the sample, the peak with the highest
retention time is probably 4-Hydroxyphenyl hexyl ketone.

EIC of m/z 205.12339 [M-H]-, i.e. negative ionization mode

(compare „Mass spectrometry_Basics“)

PD Dr. J. Graßmann; PD Dr. T. Letzel; S. Große

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Tentative molecule identification

Molecule search resulted in:
- Suspected target m/z
- Suspected target logP or logD
- Retention times of compound(s) which match the m/z of the suspected target
compound

To find out, which peak is the analyte of interest, you would usually inject a purchasable
reference standard of said compound into the LC-MS system. By measuring the standard
with the same chromatographic method you learn about the retention time, which allows
you to compare it with the observed retention time of the previously analyzed e.g. river
water sample. However in case no reference standard is available, you will have to verify
its presence in another way.

How to verify the results:

- Detection of the sample with a liquid chromatographic system comprised of the
coupling of a HILIC and a reversed phase column may provide information about the
hydrophobicity of the eluting compounds (compare slides in“Basics“).
- Check the isotopic patterns for plausibility
- Performing MS/MS measurements to learn about the chemical structure of a
compound may help with the identification.
- Consulting databases, which offer retention time prediction. The predicted RT can
then be compared with the obtained results.
PD Dr. J. Graßmann; PD Dr. T. Letzel; S. Große