Beruflich Dokumente
Kultur Dokumente
Chromatography, Workflows
& Data evaluation
Target Screening
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
Suspected-target
Target Screening Non-target Screening
Screening
Check if reference
substance is
Search for molecule Search for
available
in literature/ similarities with
databases or hydrophobicity,
compare with monoisotopic mass,
prediction program fragmentation
Validate the molecule behaviour, etc.
Produce a reference
substance
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
Target Screening
logD / logP Is the RT of the compound reasonable with regard to its logD/ logP (especially when
using the RP-HILIC coupling).
E.g. a very hydrophobic compound is unlikely to elute within the time span of HILIC
retention.
Standard/ reference Does the RT of the purchased standard match the RT of the target compound in the
substance sample?
Blank Is there no peak with the same m/z and RT present in the blank?
Isotopic pattern Is the isotopic pattern reasonable in terms of the molecular formula of the compound?
Does it match the pattern of the reference standard?
MS/MS Does the library spectrum match the fragmentation spectrum of the compound
contained in the sample?
MRM Same quantifier and qualifier fragments as well as same ratio as the reference
substance?
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
m/z 205.12339
whether or not a molecule with the same
m/z as the target compound is present
within the sample and to be able to
actually identify a particular compound,
further steps are necessary.
m/z 205.12339
Sample
m/z 205.12339
Thus, the next step of analysis is the
m/z 205.12339
comparison of the retention time of the
ibuprofen reference standard (bottom
picture) with the retention times of eluting
sample molecules (upper picture).
The reference substance was injected to
the same LC-MS system, using the same
column and the same chromatographic
method, which was also used for the
Same RT detection of the sample.
Reference substance
If a peak within the sample has the same
retention time and m/z as the reference
substance, the peak within the sample is
likely the target compound (red line).
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
Zero
abundance
Signal
RT
Unknown analyte
concentration in sample
labelled/ peak area labelled)
Deuterium-labeled diclofenac
Non-labeled
non-labelleddiclofenac
diclofenac
D D
302.08 Da
Triple quadrupole MS
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
Quantifier
Signal
Signal
Analyte Qualifier
RT m/z
Quantifier
Same Qualifier/
Isotope labeled
Signal
Signal
Qualifier Quantifier ratio but
analyte
different m/z
RT m/z
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
10 µM 7.5 µM 5 µM
.....
LC-MS/MS (MRM)
75
50
25
RT RT RT RT RT RT
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
75
50
25
RT RT RT RT RT RT
1
D
? µM
labelled
100
75
𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑛𝑜𝑛 − 𝑙𝑎𝑏𝑒𝑙𝑒𝑑
50 𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑙𝑎𝑏𝑒𝑙𝑒𝑑
25 25 000
= 0.25
10 000
RT RT
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
0.75
0.5
needs to be factored in once
again:
0.25
2.5 5 7.5 10
2,5 µM ∙ 0.5 = 5 µM
Non-labeled Diclofenac concentration [µM]
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
The correction with regard to the sample matrix is especially important. The
solvent used for the calibration curve is usually highly purified water.
However the sample to be investigated is likely highly complex, e.g. river
water. It is therefore distinctly different from the formulation of the calication
curve samples. Consequently the sample matrix may have effects on the
mass spectrometric detectability of the target compound.
Since the labeled and the non-labeled compounds have the same
physicochemical properties, the extent of a potential mass spectrometric
signal suppression will be the same for both. That allows the correction of
data and the precise quantification of an unknown target analyte
concentration.