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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt


Technische Universität München

Chromatography, Workflows
& Data evaluation

Target Screening
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,
prediction program fragmentation
Validate the molecule behaviour, etc.

Tentatively identify Predict chemical


Identify and quantify the molecule
the molecule structure

Produce a reference
substance
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Target Screening

Detection as well as quantification of a known substance can be


achieved by target screening.
For this purpose an isotope labeled reference material/ standard is
measured under the same conditions as the sample. Then the
presence or absence of each substance on the target list is established
individually. Consequently, the exact mass needs to be filtered using
extracted ion chromatograms and afterwards measured RT and MS/MS
fragmentation can be matched with those of the reference standard
(compare the following slides).
For target analysis hybrid instruments involving a triple quadropole,
such as LC-QqQ-MS, are used. Operated in MRM mode, this set-up is
ideal for sensitive and specific quantification in complex matrices.
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Checklist for the successful identification of a target compound

m/z Is the m/z of the target compound contained in the sample?

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

Internal standard (1)

A sample might however contain not only


two chemically very distinguishable (i.e.
m/z 205.12339 Sample ibuprofen and 4-Hydroxyphenyl hexyl
ketone), but also chemically very alike
m/z 205.12339 molecules e.g. isomers. To clarify

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.

Since the target compound is known (=


target screening), the next step would be
to buy a reference substance, if
available. For a common compound e.g.
like ibuprofen, it will be easy to find a
supplier.
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Internal standard (2)

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

Peak from the sample solvent


Target compound or the mobile phase
Blank All of the measurements, i.e. detection of
the sample and injection of the reference
substance have to be conducted at least
three times to validate the results.

Furthermore a blank has to be measured.


The blank only contains the solvent in
which the sample and the reference
Sample substance are prepared. By means of a
blank injection (upper picture), signals
from the solvent and the mobile phase can
be distinguished from those originating
from the sample.
The peak, which is marked with the blue
line is present in the blank and the sample.
Although it might have the same m/z as
Reference substance the target compound, it can be excluded to
originate from the sample. It is therefore
disregarded during the further steps of
analysis.
The red marked peak is not contained
in the blank.
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification methods – Overview

External in sample matrix Effects of the sample matrix like


quantification e.g. signal suppression can be
factored in
not in sample matrix Matrix effects are not considered
Internal addition of isotope Matrix effects are included in the
quantification labeled analyte quantification  gold standard
method
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification – preparation of a calibration curve using LC-


MS - external quantification (1)
General procedure:
It is possible to quantify an unknown concentration of the target compound. For this
purpose a calibration curve has to be prepared: The injection of increasing amounts
of the reference standard to the LC-MS system results in increasing mass
spectrometric EIC peak area. Plotting the area under the curve against the injected
concentration results in a calibration curve. Additionally, a constant amount of an
isotope-labeled standard needs to be added to each sample. It has the same
physio-chemical properties and the same retention time as the reference standard.

labelled/ peak area labelled)


Ratio (peak area non-

Target analyte concentration [µM]


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification – preparation of a calibration curve


using LC-MS – external quantification (2)

Determination of recovery, i.e. matrix effects

Sample matrix includes all compounds contained in a sample


except for the compound of interest. The matrix may be very
complex (e.g. wastewater samples, urine samples, etc.) and can
therefore have distinct effects on the detection of the compound
of interest and the overall quality of the analysis. The sample
matrix may e.g. cause signal suppression. For this reason, it is
important to determine its effect on the detectability of a
compound of interest.
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification – preparation of a calibration curve using


LC-MS – external quantification (2)

Determination of recovery, i.e. matrix effects

Increasing concentrations of the analyte are spiked to a no-matrix sample


(e.g. pure water) or to a sample containing a matrix, which is comparable to
the complex matrix of the sample to be analyzed.
1µM or 1µM or
2µM or 2µM or
5µM or 5µM or
10µM 10µM
etc etc

No-matrix, Matrix comparable to Real sample containing


e.g. pure water real sample. Compound an unknown concentration
of interest not contained of the compound of interest
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification – preparation of a calibration curve using LC-


MS – external quantification (2)
Calibration curves – spiked sample without complex matrix and spiked
sample with complex matrix (1)
1µM or No-matrix sample spiked with increasing concentrations of the
2µM or
analyte of interest.
5µM or
10µM  Peak areas without e.g. signal suppression effects due to
etc complex matrix

Injection 1(0µM) 2(1µM) 3(2µM) 4(5µM) 5(10µM) etc.


Zero
abundance

Peak areas 0 100 200 500 1000


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification – preparation of a calibration curve using LC-


MS – external quantification (2)
Calibration curves – spiked sample without complex matrix and spiked
sample with complex matrix (2)
1µM or Complex matrix sample spiked with increasing concentrations
2µM or
of the analyte of interest.
5µM or
10µM  Peak areas with e.g. signal suppression effects due to
etc complex matrix

Injection 1(0µM) 2(1µM) 3(2µM) 4(5µM) 5(10µM) etc.


Zero
abundance

Peak areas 0 50 100 250 500


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification – preparation of a calibration curve using LC-


MS – external quantification (2)
Calibration curves – spiked sample without complex matrix and spiked
sample with complex matrix (3)

Peak areas 0 100 200 500 1000

Peak areas 0 50 100 250 500

 Comparison of peak areas of spiked non-matrix and spiked matrix samples


shows a halving of the peak areas in samples, which contain a complex matrix
 Recovery in complex samples is 50% compared to the sample containing no
complex matrix
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification – preparation of a calibration curve using LC-


MS – external quantification (2)
Calibration curves – spiked sample without complex matrix and spiked
sample with complex matrix (4)
Analyte is spiked in increasing concentration to the complex matrix, which is
comparable to the matrix of the actual sample with unknown analyte concentration.
Based on this procedure a calibration curve can be prepared.

labelled/ peak area labelled)


Ratio (peak area non-

Target analyte concentration [µM]


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification – preparation of a calibration curve using LC-


MS – external quantification (2)
Referring to the prepared calibration curve, it is possible to calculate the unknown quantity of
analyte contained in the complex matrix sample.

Injection 1(0µM) 2(1µM) 3(2µM) 4(5µM) 5(10µM) etc.

Zero
abundance
Signal

RT

Unknown analyte
concentration in sample
labelled/ peak area labelled)

with complex matrix


• Ratio of the peak area of the non-labelled
Ratio (peak area non-

compound to the peak area of the


labelled compound in sample = y
• Unknown analyte concentration is
calculated by solving the equation for x
 x = 7.5 µM
• Result is multiplicated with the recovery
y = m*x + t rate of 50 %
 x = 15.0 µM
Target analyte concentration [µM]
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification by means of multiple reaction monitoring (MRM) (1)

By means of MRM the concentration of analyte can be determined. To be


able to quantify the target compound, the same compound but synthetically
manufactured and isotope labeled is used as internal standard (IS). The
target compound and the labeled compound are identical with regard to their
physicochemical properties, but differ in mass. The concentration of the
unlabelled compound can be quantified by means of preparing a calibration
curve. The calibration curve is done by measuring increasing concentrations
of the unlabeled target compound in the presence of a consistent
concentration of the isotope labeled compound, which is also added to the
sample.
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification by means of multiple reaction monitoring (MRM) (2)

Deuterium-labeled diclofenac

Non-labeled
non-labelleddiclofenac
diclofenac

D D

302.08 Da

13C labeled diclofenac


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification by means of multiple reaction monitoring (MRM) (3)

By means of utilizing e.g. a triple quadrupole mass spectrometer, two stages of


mass filtration can be applied (see also Level 1 slides „Mass spectrometry_mass
analyzer“). A target compound is selected in quadrupole 1 (Q1), whereupon it is
fragmented in Q2. In Q3 analyte fragments are sorted and usually two are selected
for detection (quantifier and qualifier). For the quantification however merely the
peak areas of the quantifiers of the non-labeled as well as the label compound are
used.

Triple quadrupole MS
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification by means of multiple reaction monitoring (MRM) (4)

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

Quantification by means of multiple reaction monitoring (MRM)


Calibration curve (1) – external quantification
Increasing concentrations of diclofenac + constant concentration of isotope labeled diclofenac
Always adding 10 µM of IS to each reference standard

10 µM 7.5 µM 5 µM
.....

LC-MS/MS (MRM)

Quantification by means of the quantifier peak areas


10µM 7.5µM 5µM
labeled labeled labeled
100

75

50

25

RT RT RT RT RT RT
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification by means of multiple reation monitoring (MRM)


Calibration curve (2) – external quantification
10µM 7.5µM 5µM
labeled labeled labeled
100

75

50

25

RT RT RT RT RT RT

𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑛𝑜𝑛 − 𝑙𝑎𝑏𝑒𝑙𝑒𝑑


𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑙𝑎𝑏𝑒𝑙𝑒𝑑

10 000 7 500 5000


=1 = 0.75 = 0.5
10 000 10 000 10 000
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification by means of multiple reaction monitoring (MRM)


Calibration curve (3) – external quantification
Diclofenac 10µM 7.5µM 5µM

10 000 7 500 5000


=1 = 0.75 = 0.5
10 000 10 000 10 000
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantification by means of multiple reaction monitoring (MRM)


Determination of unknown concentration (1) – external quant.
Adding
2.5µM diclofenac in sample

Ratio (peak area non-labelled/


10µM of IS to sample

1
D

peak area labelled)


0.75
? µM
0.5
D D
LC-MS/MS 0.25
D
(MRM)
2.5 5 7.5 10
Non-labelled Diclofenac concentration [µM]

? 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

Quantification by means of multiple reaction monitoring (MRM)


Determination of unknown concentration (2) – external quant.

2.5µM diclofenac in sample


Ratio (peak area non-labeled/

To determine the final


1 concentration of Diclofenac in
the sample, the recovery rate
peak area labeled)

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

Quantification by means of multiple reaction monitoring (MRM)


Correction of MS signal inconstancies by means of the internal standard (IS)
- Internal quantification

By means of the addition of the labeled IS at constant concentration to all


samples, one willl be able to correct the data for all kind of signal
inconstancies, e.g. mass spectrometric signal loss or matrix effects.

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.