Bruker Daltonics
Application Note # MT-103
FAST-SRM: A new Single Reaction Monitoring
Method for Fast, Targeted Drug Distribution Testing in
Tissue Sections
Targeted analysis of drug molecules and metabolites in
tissue sections by MALDI imaging is a powerful tool for
evaluating drug safety and pharmacology in general. To
achieve the necessary specificity against the low molecular
weight background (matrix, lipids, etc.), drug imaging
requires either the selection of a specific drug fragment in
an MS/MS experiment (single reaction monitoring, SRM) or
very high mass resolution and accuracy. MALDI-TOF/TOF
instruments have been used for SRM. However, measuring
a single fragment of a single precursor ion does not require
TOF/TOF ion optics. Here we report a new method that
enables SRM to be performed on a reflector-only MALDI-
TOF instrument. This technique is easy to set up and
makes fast reflector instruments such as the autoflex speed
perfectly suited for small molecule MALDI imaging, as
demonstrated here for the analysis of Erlotinib in mouse
liver.
Introduction
MALDI imaging of drug molecules and metabolites presents
the challenge of measuring the intensity of the targeted
drug signal against a complex background of matrix signals
and endogenous metabolites. The required specificity in
such analyses is usually achieved by measuring a specific
fragment of the targeted drug in MS/MS mode (single
reaction monitoring; SRM). If just one compound is to be
analyzed in a targeted fashion, a single fragment ion must
be monitored – just like SRM analysis on a QqQ. A variety of
instruments have been used for this kind of analysis, includ-
ing MALDI-ion traps, q-TOF, triple quad (QqQ) and TOF/
TOF instruments [1]. However, a TOF/TOF for full MS/MS
spectra recording is not an absolute requirement. Select-
ing the parent ions of interest in the precursor ion selector,
fragmenting the drug molecular ions and monitoring a single
fragment in a very narrow mass spectrum are sufficient.
Here we describe the new FAST-SRM assay [4], which is
based on MALDI-TOF technology and does not require a
TOF/TOF design (Fig. 1). In a TOF instrument, precursor ions
are selected using a pulsed ion gate, which only allows ions
to pass at a given time. On a TOF/TOF instrument, quadru-
pole 1(Q1) in the QqQ only allows ions to pass with a given
frequency. In both instruments the selected parent ions
fragment in a collision cell. In case of the TOF, fragmentation
may also occur as Laser Induced unimolecular Decomposi-
tion (LID) in the field-free region between ion source and
reflector. The ion reflector voltage is reduced so that only
a narrow mass range of fragment ions are reflected to the
detector, similar to the effect of Q3 in the QqQ.
For the selective detection of a single fragment, the reflector
voltage is reduced to permit the fragment ion of interest to
travel along the same ion trajectory through the reflector as
the parent ion at full reflector voltage.
 Single Reaction Monitoring (SRM)

Parent ion Fragmentation Fragment ion

selection selection

SRM on QqQ
Q1 CID: Q2 Q3 detector

SRM on TOF
pulsed ion gate CID cell / LID ion reflector

detector

Figure 1: The classical setup for SRM comprises a triple quadrupole for selection and fragmentation of the desired compound and the
detection of the characteristic fragment. Similarly the SRM mode on a tof uses a pulsed ion gate for the selection, CID or laser induced
decomposition for the fragmentation of the compound, and the reflector at lowered voltage to enable the detection of the desired fragment.
The CID cell is placed in this figure between the ion gate and the reflector for easier understanding of this method. Since the field-free
drift section in this TOF spreads between ion source and reflector, the fragmentation can in fact take place anywhere in this region with no
influence to the result.

As the reflector potential URf for SRM measurements is
reduced by the ratio of the fragment mass to the parent
mass, this is a simple calculation:
m/zSRM
UR f (SRM) = UR f (parent) •
m/zparent
In this particular configuration, ions with higher m/z ratios
travel through the ion reflector and lighter fragment ions
are reflected on trajectories that are not focused to the
detector. Using such settings means that there is no
trade-off in achieving a reasonable MS/MS performance
of the instrument over a larger mass range, and memory
consumption for SRM datasets is small compared to full
MS/MS or high-resolution images. The sensitivity of this
approach arises from the low number of background ions
that hit the detector. FAST-SRM provides exceptionally high
specificity and a precise m/z value of the targeted transition
is determined, even if there are several fragments present
with similar m/z values. This is very different from SRMs
on a QqQ. The user interface of flexImaging versions 2.1.26
and later includes the SRM mode. CompassFlex 1.3 adds
a convenient user interface for simple SRM acquisition
control. Users can switch between parent mode (with full
reflector voltage) and fragment mode, and run SRM with
collision gas either on or off. Here we applied the new
FAST-SRM mode for the analysis of mouse liver after
administration of Erlotinib. Erlotinib is a tyrosine kinase
inhibitor that acts on epidermal growth factor (EGFR)
and is used to treat pancreatic and non-small cell lung
cancers. MALDI imaging of animals dosed with Erlotinib
has been previously reported [2, 3]. The structure and
main fragmentation schema of Erlotinib in MALDI-TOF is
shown in Fig. 2A. Fragmentation of desmethyl Erlotinib – a
metabolite formed by demethylation – is shown in Fig. 2B.
 A Erlotinib
Fragmentation of Erlotinib MALDI imaging of Erlotinib
A
A 336.13 A
A Erlotinib

336.13 0 mg/kg 200 mg/kg 50 mg/kg

B
336.13 [M+H+] = 394.18 Da B 394 → 336

336.13 [M+H+] = 394.18 Da

B C
C 380 → 336
B des-Methyl-Erlotinib

322.12
B des-Methyl-Erlotinib
D
D 380 → 322
322.12

336.13 [M+H+] = 380.16 Da

Figure 2: 336.13 [M+H+] = 380.16 Da Figure 3:

A: Structure of Erlotinib with m/z values of the major fragments.
B: One of two possible structures of desmethyl Erlotinib, with m/z
values of the major fragments produced when either side chain is
cleaved. In the alternative desmethyl Erlotinib structure, the upper
side chain is demethylated. Both structures give rise to fragments of
the same mass that cannot be differentiated in an ms2 experiment.
A: Optical image of mouse liver sections on a glass slide. From left to
right: Control (0 mg/kg), 200 mg/kg and 50 mg/kg.
B: FAST-SRM measurement of Erlotinib, observed transient 394.18 Da
→ 336.13 Da
C: FAST-SRM measurement of desmethyl Erlotinib, observed transient
380.16 Da → 336.13 Da
D: FAST-SRM measurement of desmethyl Erlotinib, observed transient
380.16 Da → 322.12 Da. In figures B, C, and D, areas with intensities
≥50% of the most intense signal result in maximum pixel intensity.

Experimental
To evaluate the sensitivity of FAST-SRM, three mice set up and evaluated using flexImaging 2.1 software. One
were dosed with 0 (control), 50 and 200 mg Erlotinib/ MS and three SRM datasets – 394 → 336, 380 → 336 and
kg body weight. Mice were sacrificed two hours post 380 → 322 (based on the fragment ions of Erlotinib and
dose and livers were frozen in liquid nitrogen. The animal desmethyl Erlotinib) – were acquired from each sample. For
experiments were conducted in accordance with German the statistical plots, the intensity values were exported from
federal animal protection laws and approved by the local flexImaging into a text file and processed with R [5].
Institutional Animal Care and Use Committee. 10 μm
cryosections of the livers were cut on a cryomicrotome
and transferred onto Indium-Tin-Oxide coated glass
slides (Bruker Part #237001) and dried. α-cyano-4-
hydroxycinnamic acid (CHCA) matrix was applied to the
glass slide with the ImagePrep™ station (Bruker) using
the standard preparation method with the maximum
wetness setting. For MALDI imaging, 200 laser shots
were accumulated per pixel, with a raster width of 300
μm and a pixel diameter of 150 μm (laser spot size ~100
μm). This allowed acquisition of multiple SRM datasets
in consecutive runs from the same sample area using
a grid offset of 150 μm. The imaging experiments were
 Metabolization of Erlotinib – SRM enables quantitation
A B C
394->336 380->336 380->322
30

30

30
1.0

0.45
25

25

25
0.9

0.40
20

20

20
0.8

0.35
[a.u]

[a.u]

[a.u]
15

15

15
0.7

0.30
0.6
10

10

10

0.25
0.5
5

5
0 50 100 150 200 0 50 100 150 200
0

0
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200

Dose mg/kg Dose mg/kg Dose mg/kg

r2= 0.9995 r2= 0.9955 r2= 0.9957

Figure 4: Mean and 95% confidence intervals (CI) of the intensity over all pixels of the respective section plotted against dose
(FAST-SRM mode).
A: Erlotinib, observed transition 394.18 Da → 336.13 Da. (CI is too small to be seen on this scale)
B: Desmethyl Erlotinib, observed transition 380.16 Da → 336.13 Da
C: Desmethyl Erlotinib, observed transition 380.16 Da → 322.12 Da

Results
Figure 3A shows optical images of the selected
mouse liver sections and the results of the FAST-
SRM measurements of Erlotinib (B) and its metabolite
desmethyl Erlotinib (C, D). The distribution of Erlotinib
was determined from the SRM dataset 394 → 336,
and shows an unambiguous linear correlation of signal
intensity and administered dose (r² = 0.9995) (Fig. 4).
The low intensity of the metabolite signals indicates
that 2 hours post-dose, very little of the Erlotinib had
been metabolized. At a dose of 200 mg/ kg, the relative
response of desmethyl Erlotinib compared to Erlotinib
indicates that 5% of the drug had been metabolized
(Fig. 4). Due to the low concentration of the metabolite,
variations across the images are quite large (CVs ~40 %).
However, due to the large number of spectra that were
analyzed, a good linear correlation of SRM response of
drug and metabolite to the dose of Erlotinib was observed
and the mean intensities for the different doses could still
be quantified.
using the FAST-SRM approach. Comparing the analyses of
Erlotinib and desmethyl Erlotinib in MS and SRM modes
shows a clear advantage of the SRM approach. While the
394.18 Da signal for Erlotinib in the MS mode shows a
linear relationship with the dose, a similar relationship was
not observed for the 380.16 Da desmethyl Erlotinib signal
in MS-mode (Fig. 5). A major reason for this is interference
from a matrix signal (CHCH: 2MH++1). In contrast, all
signals measured using the FAST-SRM mode showed a
linear intensity:dose relationship (Fig. 4).
Table 1: Coefficients of variation (CV) for the measured intensities of
the Erlotinib signal in MS-mode and FAST-SRM mode (calculated over
all pixels of the respective liver sections). In the control sample, the
CV value is significantly higher in MS mode than in FAST-SRM mode.
This suggests higher specificity and a lower influence of chemical
noise in the FAST-SRM measurement.

In addition to the three SRM transitions, we also acquired

an MS image dataset to monitor drug and metabolite 0 mg/kg 50 mg/kg 200 mg/kg
distributions (images not shown). At high concentrations,
the MS readout provided similar CVs of approx. 40 % MS (m/z 394.18) 167 % 42 % 39 %
(Tab. 1). However, in the control sample (0 mg/kg), a FAST-SRM
44 % 36 % 45 %
dramatic increase in CV in the MS image indicated a (m/z 394.18 → mz 336.13)
high level of background noise compared to that seen
 Erlotinib and des-Methyl-Erlotinib in MS-mode
A B

ms mode 394 ms mode 380

[a.u]

[a.u]
0 50 100 150 200 0 50 100 150 200

Dose mg/kg Dose mg/kg

R2= 0.9915 R2= 0.9129

Figure 5: Mean and 95% confidence intervals (CI) of the intensity over all pixels of the respective section plotted against dose (MS mode).
A: 394.18 Da (CI too small to be seen on this scale)
B: 380.16 Da

Conclusion
The new FAST-SRM mode described here is suitable for
highly specific and robust analysis of drugs and metabolites
in tissue with low background interference (see also [4]).
We have shown in this proof-of-concept study that the
FAST-SRM mode provides a significantly lower detection
limit than the MS mode, although an absolute number
requires more detailed study. Using FAST-SRM turns fast
reflector MALDI-TOF instruments (such as the Autoflex
speed with its 1 kHz operation) into universal imaging
instruments.
Authors
Isabel Winkelmann1, Axel Walch1, Barbara Maria Grüner 2 und
Jens Siveke2, Arne Fütterer 3, Detlev Suckau3, Michael Becker 3,
Sören Deininger 3, Cesar Barahona3, Armin Holle3
[1] Institute for Pathology, Helmholtz-Centre Munich, Germany
[2] 2nd Department of Internal Medicine, Technical University of Environment for Statistical Computing, R Foundation for
Munich, Germany
[3] Bruker Daltonik GmbH, Bremen, Germany
References
[1] Walch A, Rauser S, Deininger SO, Höfler H. MALDI imaging
mass spectrometry for direct tissue analysis: a new frontier for
molecular histology. Histochem Cell Biol.
2008 Sep; 130(3):421-34.
[2] Signor L, Varesio E, Staack RF, Starke V, Richter WF, Hopfgartner
G. (2007)
Analysis of erlotinib and its metabolites in rat tissue sections by
MALDI quadrupole time-of-flight mass spectrometry.
J Mass Spectrom. 42(7):900-9
[3] Laukien S, Creedon E, Kowalski JM, Kowalski P, Kellersberger K,
Agar N.
Small Molecule Drug Imaging of Mouse Tissue by MALDI-
TOF/TOF Mass Spectrometry and FTMS
Bruker Daltonics Application note MT-93/FTMS38.
[4] Marshall PS; Toteu-Djomte V, Biggadike K.
Percutaneous Absorption of drug compounds into skin measured
by a new MALDI-TOF SRM mode ASMS 2010, Session ThP16,
Poster 397
[5] R Development Core Team (2010) R: A Language and
Statistical Computing, ISBN 3-900051-07-0
http://www.R-project.org

For research use only. Not for use in diagnostic procedures. Keywords Instrumentation & Software
Tissue imaging autoflex speed
drug distribution flexImaging
ImagePrep
 to change specifications without notice. © Bruker Daltonics 09-2010, MT-103 #270415
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