1. Research Article
Received: 29 October 2010 Revised: 2 December 2010 Accepted: 3 December 2010 Published online in Wiley Online Library: 2011
Rapid Commun. Mass Spectrom. 2011, 25, 503–510
(wileyonlinelibrary.com) DOI: 10.1002/rcm.4891
A quantitation method for mass spectrometry imaging
Stormy L. Koeniger1*, Nari Talaty1, Yanping Luo1, Damien Ready1, Martin Voorbach1,
Terese Seifert1, Steve Cepa1, Jane A. Fagerland2, Jennifer Bouska3, Wayne Buck1,
Robert W. Johnson1 and Stephen Spanton1
1
Advanced Technology, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA
2
Global Preclinical Safety, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA
3
Exploratory Kinetics, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA
A new quantitation method for mass spectrometry imaging (MSI) with matrix-assisted laser desorption/ionization
(MALDI) has been developed. In this method, drug concentrations were determined by tissue homogenization of five
10 mm tissue sections adjacent to those analyzed by MSI. Drug levels in tissue extracts were measured by liquid
chromatography coupled to tandem mass spectrometry (LC/MS/MS). The integrated MSI response was correlated to
the LC/MS/MS drug concentrations to determine the amount of drug detected per MSI ion count. The study reported
here evaluates olanzapine in liver tissue. Tissue samples containing a range of concentrations were created from liver
harvested from rats administered a single dose of olanzapine at 0, 1, 4, 8, 16, 30, or 100 mg/kg. The liver samples were
then analyzed by MALDI-MSI and LC/MS/MS. The MALDI-MSI and LC/MS/MS correlation was determined for
tissue concentrations of $300 to 60 000 ng/g and yielded a linear relationship over two orders of magnitude
(R2 ¼ 0.9792). From this correlation, a conversion factor of 6.3 W 0.23 fg/ion count was used to quantitate MSI
responses at the pixel level (100 mm). The details of the method, its importance in pharmaceutical analysis, and
the considerations necessary when implementing it are presented. Copyright ß 2011 John Wiley & Sons, Ltd.
Mass spectrometry imaging (MSI) has advanced from a always sufficient to interpret the biological implications of
method designed for mapping atomic or simple molecular pharmaceutical compound distributions. For MSI to reach
species[1] to a family of techniques used to map biological its full potential, quantitative MSI information is needed to
molecules in complex tissues ranging from plant[2–4] and make it possible to extract safety and pharmacokinetic
animal[5–8] samples to human biopsies from clinical trials.[9,10] data, to triage compounds in early drug discovery, and to
Advances in direct ionization methods and more recently understand the effective concentration at the site of action.
surface sampling techniques continue to enhance the With the advancement of modern lasers, matrix-assisted
diversity of MSI applications.[11–14] With the success of these laser desorption/ionization (MALDI)[27] has become one of
advancements, however, come new technological demands the most widely used ionization methods for MSI due to
as MSI is extended to more challenging scientific questions. its high sensitivity, speed, broad molecular mass range (>300
In the pharmaceutical industry, MSI provides an early Da up to 200 000 Da), and spatial resolution. Quantitative
avenue to determine the disposition of pharmaceutical MALDI assays developed for the high-speed analysis of small
compounds in tissue during the discovery and candidate molecules have become well established and can provide
selection phases of drug development when radiolabeled precision of equal to or greater quality (<5% relative standard
compounds are not typically available.[15–17] In the determi- deviation, RSD) than their electrospray ionization (ESI)
nation of tissue pharmacokinetics, MSI has been shown to counterparts.[28,29] In addition, MALDI has a linear dynamic
provide data analogous to whole body autoradiography range of 2 to 3 orders of magnitude,[29] and these positive
(WBA) with the advantages of high selectivity and multi- attributes should likewise extend to MALDI-MSI.
plexed detection.[16,18,19] MSI has demonstrated its value Imaging by mass spectrometry has been generally
in elucidating mechanisms of biotransformation[20–22] as well regarded as a qualitative method with some recent demon-
as drug transport in tumors.[23,24] Most notably, MSI provides strations of quantitative analyses for small molecules.[20,30–34]
the ability to simultaneously investigate the distribution of Fundamentally, the signal provided by MSI is a direct
exogenous molecules (e.g., pharmaceutical compounds) and measurement of analyte relative abundance and, therefore,
their localization with respect to endogenous molecules with the use of proper matrices and internal standards it is
that can serve as biological markers.[22,25,26] The relative possible to obtain quantitative data. The challenge associated
spatial distribution provided by MSI, however, is not with quantifying MALDI-MSI data has been in the determi-
nation of appropriate standards, as well as in the choice
and homogeneous deposition of an internal standard on the
* Correspondence to: S. L. Koeniger, Advanced Technology, tissue surface that can consistently reflect the changes in ion
GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA. extraction and ionization efficiency at micrometer scale
E-mail: stormy.koeniger@abbott.com resolutions.
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Rapid Commun. Mass Spectrom. 2011, 25, 503–510 Copyright ß 2011 John Wiley & Sons, Ltd.
2. S. L. Koeniger et al.
The ability to reliably and reproducibly deposit standards parallel quantification of analytes in adjacent tissue by
homogenously on a surface as internal standards for MS homogenization and LC/MS/MS quantitation. The overall
imaging is challenging to achieve. Currently, an internal aim of this work is to bridge these two quantitation methods.
standard is introduced onto the tissue via the MALDI It is demonstrated here that a sample pool, which includes
matrix application process. In some cases, it is possible to use tissue concentrations ranging over at least one order of
endogenous species[30,31] or matrix ions[35] as an internal magnitude, can be quantified by correlation of the tissue
standard to enable relative quantitation. With recent concentration determined by LC/MS/MS to the integrated
advancements in acoustic deposition devices, it may be MALDI-MSI response.
possible to quantitatively deposit an internal standard
over surfaces in 200 mm diameter spots which can then be
analyzed by MALDI-MSI. To simultaneously detect the EXPERIMENTAL
analyte of interest and the internal standard, methodologies
Materials and reagents
such as multiplexed imaging and dynamic pixel imaging
have been developed.[36,37] The combined application of All solvents unless otherwise specified were HPLC grade
acoustic deposition devices for internal standard deposition and purchased from Sigma Aldrich (St. Louis, MO, USA).
(on or under tissue samples) and multiplexed imaging has, Olanzapine was purchased from AK Scientific (Union City,
however, yet to be evaluated for MSI quantitation. CA, USA). Olanzapine-d3 was purchased from Toronto
Robust and absolute quantitative methods are now Research Chemicals (North York, Ontario, Canada) and
beginning to take form. Previous studies have shown that used as the internal standard for LC/MS/MS quantitation.
when a dilution series of an analyte is deposited on tissue a-Cyano-4-hydroxycinnamic acid (CHCA) was purchased
sections, a linear standard curve can be obtained.[15,16,20,34] from Sigma Aldrich and used without further purification
However, this standard curve may not adequately represent (99% purity).
the signals obtained from the sample. The analyte responses
observed by depositing compound either under or on top of
Animal dosing
the tissue can vary widely from each other depending on
the physicochemical properties of the analyte and the Male Sprague Dawley rats were purchased from Charles
solvent system employed for deposition of the compound. River Laboratories, Inc. (Wilmington, MA, USA) and housed
The analyte response, when it is deposited on top compared and treated under protocols approved by the Institutional
with under the tissue, is typically a factor of 2 to an order of Animal Care and Use Committee (IACUC) and according to
magnitude higher.[20,38] In 1987, Schweitzer et al.[39] devel- the Guide for the Care and Use of Laboratory Animals.[42]
oped the most widely accepted method for quantitative Each animal was administered a single oral dose of
whole body autoradiography (QWBA) by creating a dilution olanzapine in 0.2% hydroxypropyl methylcellulose at 0, 1,
series in blood which was then used to generate a standard 4, 8, 16, 30, or 100 mg/kg (N ¼ 3/group) and euthanized by
curve for quantitation. In an analogous fashion, methods to isoflurane anesthesia followed by exsanguination and
produce synthetic ’matrix-matched’ standards for MSI have cervical dislocation 1 h post-dosing. Liver was collected,
been developed and implemented by Becker and colleagues flash frozen in liquid nitrogen, and stored at –808C until
to analyze atomic species by laser ablation inductively further analysis.
coupled plasma ionization (LA-ICP).[40] In these methods,
tissue homogenates rather than whole blood are used to
Tissue sectioning and processing
generate the analyte dilution series, refrozen, then sectioned,
and analyzed with the samples of interest by MSI. These Pieces of liver were mounted with minimal amounts of
methods are well suited to LA-ICPMS for the analysis of Optimum Cutting Temperature (OCT) medium and cut into
atomic species; however, they have not been established for cylinders using a biopsy punch (diameter $8 mm) to provide
MALDI-MSI analysis. Recently, multi-isotope imaging mass approximately the same tissue surface area for all samples.
spectrometry (MIMS) has been developed to obtain direct The tissues were sectioned on a cryostat (Microm HM500 M,
quantitative analysis of isotopes within subcellular compart- Lukas Microscope Services Inc., Skokie, IL, USA) at –178C at
ments using secondary ion mass spectrometry (SIMS), but 10 mm thickness. For advanced studies that require multiple
these methods are also unique to the capabilities of SIMS techniques, a sectioning protocol has been developed in
instrumentation and can not be readily translated to which serial sections are collected and processed according
MALDI-MSI methods.[41] to individual protocols for each technique. To incorporate
The standard methods of liquid chromatography/tandem histology, MSI, MS quantitation, and immunohistochemistry
mass spectrometry (LC/MS/MS) are becoming increasingly (IHC), sections are collected in the following order: (1) five
valuable to complement MSI studies for the quantitation sections for MS quantitation (Q1); (2) two sections for
and confirmation of molecular species in histological tissue hematoxylin and eosin (H&E) staining; (3) two to three
sections. Drug concentrations determined by LC/MS/MS in sections for MSI; (4) two sections for H&E staining; (5) three
tissue sections serial or near to those imaged by MSI have sections for IHC; (6) two sections for H&E staining; and (7)
been shown to be proportional to the MALDI-MSI response five sections for quantitation (Q2). This protocol is illustrated
in several studies.[15,17,20,33] The two primary methods of in Fig. 1(a) and can be modified according to the experimental
quantifying MALDI-MSI data that are evolving in the field needs of the study. For example, in this study where
are: (1) creation of ’matrix-matched’[40] standards that are quantitation is the primary focus, histology and immuno-
sectioned and imaged with the samples of interest in a histochemistry were not required, allowing for MS quanti-
method analogous to that employed in QWBA,[39] and (2) tation samples (Q1 and Q2) to be collected closer together
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wileyonlinelibrary.com/journal/rcm Copyright ß 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 503–510
3. A quantitation method for mass spectrometry imaging
Figure 1. (a) Illustration of an integrated sectioning protocol for quantitation (Q), histology (H), MSI (M), and immunohis-
tochemistry (I). Each $10 mm section is processed according to specific protocols for each analysis. For LC/MS/MS
quantitation, five sections are collected before (Q1) and after (Q2) sections analyzed by MSI. (b) Illustration of a simplified
sectioning protocol used in this study for evaluating MSI quantitation.
(see Fig. 1(b)). Tissue sections were collected as follows: (1) from 0.2 to 1000 nM was created using serial dilution of
five 10 mm tissue sections were collected into a pre-weighed the 10 mM tissue homogenate stock with blank tissue
500 uL Eppendorf (VWR, Radnor, PA, USA) vial (Q1); (2) homogenate. An identical dilution series was created in
three adjacent sections for MSI were each thaw mounted onto 50% aqueous acetonitrile.
different stainless steel MALDI target plates (AB Sciex, Foster To extract compound from samples and standards,
City, CA, USA) to assess plate-to-plate and section-to-section acetonitrile containing the internal standard (250 nM) was
reproducibility; and (3) five 10 mm sections were collected added in a 1:2 homogenate/acetonitrile ratio, vortexed
into pre-weighed Eppendorf tubes for duplicate analysis for 15 s, and centrifuged at 13 000 rpm for 10 min. The
of olanzapine concentrations by LC/MS/MS (Q2). Tissue supernatant was collected and diluted to a 30% acetonitrile
sections for LC/MS/MS quantitation were weighed at room concentration prior to injection onto a Thermo Scientific
temperature and stored at –808C until further analysis. Tissue (West Palm Beach, FL, USA) BETASIL Cyano column
sections for MSI analysis were stored at room temperature (50 Â 3 mm; 5 m particles). The analytes were eluted at
in a vacuum desiccator until processing for MSI analysis. 500 mL/min with a 10–90% gradient of acetonitrile with
The matrix for MSI analysis was deposited with a thin 0.1% formic acid over 3 min after an initial 2 min hold at
layer chromatography (TLC) sprayer (Sigma Aldrich) just 90% aqueous with 0.1% formic acid and electrosprayed into
before analysis by manually spraying 10 mg/mL CHCA in a triple-quadrupole linear ion trap mass spectrometer
50% acetonitrile and 0.1% trifluoroacetic acid using 20–25 (QqQLIT, QTRAP 5500, AB Sciex, Concord, Ontario, Canada)
passes over 20 min in a chemical fume hood at $40% relative for analysis with duplicate injections. Positive ion mode
humidity. A total of $5 mL of matrix solution was used in the multiple reaction monitoring (MRM) was used for the
process. analysis of olanzapine (m/z 313!256) and the internal
standard olanzapine-d3 (m/z 316!256). Precursor and
product ions were transmitted at unit resolution, and product
LC/MS/MS quantitation
ions were produced with the ’high’ buffer gas setting at
Two dilution series of olanzapine were prepared to collision energies of 34 and 47 eV for olanzapine and
determine matrix effects on ionization and compound olanzapine-d3, respectively.
extraction from tissue À one in tissue homogenate and the
other in 50% aqueous acetonitrile. To create a tissue
Mass spectrometry imaging
homogenate stock solution, 500 mL of a standard solution
containing 10 mM of olanzapine in 95% saline and 5% Mass spectrometry imaging was performed on a Qstar XL
acetonitrile was added to 50 mg of liver tissue obtained Elite (AB Sciex, Concord, Ontario, Canada) equipped with an
from rats dosed with vehicle (vehicle liver tissue). Blank AB Sciex oMALDITM source consisting of a 355 nm solid-state
tissue homogenate was generated from 200 mg of vehicle laser with an elliptical spot size of 100 Â 150 mm. MSI analyses
liver tissue containing 1 mg of tissue per 10 mL of saline. were performed at a spatial resolution of 100 Â 150 mm with
Tissue samples consisting of five 10 mm tissue sections a laser fluence of 5.9 mJ (1 kHz). Imaging acquisitions
(average total weight ¼ 3 mg) were also prepared using 1 mg were performed in positive ion mode using the product
of liver per 10 uL saline. All solutions were sonicated for ion scan mode to obtain MS/MS spectra of the [MþH]þ ion of
45 min in an ice bath. An 11-point standard curve ranging olanzapine at m/z 313.1. The quadrupole was operated in
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Rapid Commun. Mass Spectrom. 2011, 25, 503–510 Copyright ß 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm
4. S. L. Koeniger et al.
low resolution mode, and the enhancement feature was set to 8 mg/kg were 2001 Æ 588 and 3454 Æ 624 ng/g, respectively
enhance the product ion at m/z 256.0. MSI images were (see Table 1). These values are in agreement with published
created from the conversion of raw data files to image files results.[44]
(.img) via the oMALDITM 5.1 server and further processed In an analysis of the changes in bulk liver tissue
and visualized through in-house software developed in concentrations, the olanzapine concentrations determined
IDL 7.1[43] which allows batch processing, statistical analysis from 5, 10, or 20 tissue sections (10 mm) varied by 12%.
of images, and region of interest (ROI) integration. Duplicate adjacent samples of 5, 10, or 20 tissue sections
varied by 5, 12, and 16%, respectively. This trend indicates
that the changes in bulk tissue concentrations become
important as the number of tissue sections analyzed
RESULTS AND DISCUSSION increases; thus, five tissue sections were collected and
analyzed for quantifying the MALDI-MSI data. The RSD
Quantitation of olanzapine in tissue sections by
in the olanzapine concentration for samples Q1 and Q2 for
LC/MS/MS
each animal (see Fig. 1) ranged from 1 to 24% with an average
Olanzapine was chosen for this study because its pharma- of 7%. Thus, there was no significant difference in the bulk
cokinetic properties are well characterized,[44] and it has been tissue concentration of olanzapine across the liver regions
analyzed previously in MSI studies.[45] The homogenization analyzed.
and extraction of olanzapine in low milligram quantities of
tissue were found to be robust and reproducible with an
MALDI tissue imaging
average RSD of 7%. A linear response curve between
0.2 and 1000 nM was established for the olanzapine standard Liver is one of the most commonly imaged tissues
in 50% acetonitrile (R2 ¼ 0.9955) and tissue homogenate for pharmaceutical compounds by MSI during method
(R2 ¼ 0.9975). The extraction efficiency for olanzapine was development as it is easily harvested from animals, easy to
98% as determined from the two calibration curves. The limit section, and typically provides high concentrations of analyte
of detection (LOD, 3s) and limit of quantitation (LOQ, 10s)[46] to evaluate sensitivity. The molecular distribution of both
were determined to be 2.4 and 8.1 nM. The response curve in exogenous and endogenous species in the liver as measured
tissue homogenate was used to calculate the concentration of by WBA and MSI is also relatively homogeneous compared
olanzapine in tissue extracts (nM), which was converted into with other organs.[47] For this reason, liver was chosen as an
the amount of olanzapine extracted from five tissue sections ideal specimen to develop MSI quantitation.
per weight of tissue (ng/g) and the amount of olanzapine The reproducibility, linearity, and quantitation of MALDI-
per tissue section (pg per tissue section), assuming that all MSI analyses in liver tissue were investigated by creating
five tissue sections were of equal weight. A summary of three identical MALDI plates prepared with adjacent liver
the amount of olanzapine per tissue section measured as a tissue sections from animals in each dosing group. These
function of administered dose for each animal is shown in tissue sections were collected between the two sets of five
Fig. 2. Linear regression of these data resulted in a coefficient tissue sections analyzed for drug concentrations by LC/MS/MS
of determination of 0.891 (see Fig. 2, inset). The average as reported above. At 100 mm spatial resolution, each
olanzapine concentrations in liver for animals dosed at 4 and liver section required approximately 120 min to image.
Figure 2. LC/MS/MS concentration of olanzapine in a liver tissue section plotted as a function of dose (mg/kg). Liver sections
($10 mm) were collected adjacent to tissue sections analyzed by MSI. Each data point is determined from the average from
samples Q1 and Q2 (see Fig. 1(b)) from a single animal. Inset: Linear regression of data.
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5. A quantitation method for mass spectrometry imaging
Table 1. Statistical analysis of serial liver tissue sections analyzed by MSI
Olanzapine tissue MSI inter-tissue MSI intra-tissue
concentrationa statisticsb statisticsc
Integrated Integrated Ion count Ion count Ion count Ion count
Dose ng/g, response, response, per pixel, per pixel, per pixel, per pixel,
mg/kg average %RSD average % RSD average % RSD average SDd
1 321 8 27720 12 9 18 9 7
10 9
7 7
4 1406 <1 168375 11 49 2 50 29
50 31
63 44
8 3690 10 284013 12 89 11 86 58
82 47
100 90
16 8208 3 911789 7 285 13 - -
258 159
312 201
30 12550 5 1626083 5 520 9 495 315
494 279
572 337
100 32811 2 4030463 2 1300 7 1275 687
1230 644
1394 760
a
Concentration measured by LC/MS/MS from duplicate injection of samples Q1 and Q2 (see Fig. 1) from one animal at each
dose.
b
Value corresponding to two or three serial liver tissue sections analyzed on separate MALDI target plates for animal
reported in a.
c
Values reported for tissue sections reported in b.
d
SD ¼ standard deviation.
The signal was observed to be stable over the entire run time of these data is shown in Fig. 3. Analysis of vehicle tissues
for each plate. MALDI signals corresponding to the product revealed an average ion count per pixel of 2 Æ 1, showing
ion at m/z 256 were integrated over the entire tissue section the advantage of MS/MS in reducing background chemical
to obtain the integrated response for each image. A summary noise. For each animal, the average tissue response and
Figure 3. MSI response as a function of dose (mg/kg) in $10 mm tissue sections of liver. Each data point represents the average
of three serial sections from a single animal analyzed on different MALDI target plates. (Inset) Linear regression of MSI
responses for individual tissue sections for each MALDI target plate. NA ¼ data not available.
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6. S. L. Koeniger et al.
standard deviation for all three plates are shown and application techniques such as acoustic droplet devices
these can be directly compared with the data provided in which mimic traditional MALDI dried-droplet methods
Fig. 2. The integrated MSI response was found to be linear as a have been shown to increase sensitivity and reproducibility
function of administered dose with high coefficients of in MALDI-MSI analyses.[21,49]
determination (R2 > 0.9) for all three plates (see inset in Fig. 3). As demonstrated above, a linear and reproducible
The reproducibility of triplicate analyses as determined by response can be obtained by MALDI-MSI. In order to
the integrated MSI response was on average 14%. MSI quantitate this response, LC/MS/MS tissue concentrations
analysis of tissue sections having small folds exhibited higher were used to determine the relationship between the amounts
variance in replicate analyses. Tissue thickness has been of analyte per tissue section and the integrated MSI response,
shown to negatively affect analyte response[48] and, thus, as demonstrated in Fig. 4. Here the amount of olanzapine
when performing quantitative MSI studies across multiple measured by LC/MS/MS is plotted on the ordinate (pg/
samples it is important to minimize tissue folding. A tissue section) and the MSI response is plotted on the
statistical analysis for a single animal at each dose is provided abscissa (ion counts/tissue section) such that the slope of the
in Table 1. From this analysis, it is shown that RSDs are on plot yields an amount of compound per MSI count or pg/
average $30% higher in the MSI measurements than in the ion count. For the system studied here, the slope yielded a
LC/MS/MS analysis. This precision for the integrated conversion factor of 6.3 Æ 0.23 fg/ion count. The intercept
MSI response is not as high as that observed from traditional in Fig. 4 is a measure in the discrepancy between the detection
MALDI; however, there is still room for improvement limits of the two analytical methods; however, in this
considering that all the samples were prepared by manual system the intercept is relatively small (41 pg) such that
matrix application (with a TLC sprayer). Automated matrix the difference in the detection limits for the two analytical
methods for olanzapine is within the experimental error for
this measurement. It is important to note that error in the
MSI quantitation method is low when the signals from
the majority of pixels within the MSI image are above the
detection limit and are therefore defined.
The data demonstrate the linearity and reproducibility of
the integrated MALDI-MSI response over large surface areas
(50 mm2), but the question remains as to the limitations in the
reproducibility and linearity of the data, and thus the ability
to quantitate at the resolution of the measurement (100 mm).
Representative MSI images of olanzapine in liver from each
dosing group are shown in Fig. 5. The linear color scale has
been converted from MSI ion counts into pg/pixel through
the relationship shown in Fig. 4. It is apparent in these
images that the distribution of olanzapine in the liver is not
homogeneous and that it varies widely from pixel to pixel
Figure 4. The linear relationship between the amount of (see Table 1 under ’MSI intra-tissue statistics’). The major
olanzapine in a single tissue section of rat liver as measured features observed in the olanzapine liver distributions are
by LC/MS/MS and the integrated MSI response in an adja-
reproducible (Fig. 6). From these results, it can be inferred
cent tissue section.
Figure 5. MSI images of olanzapine in liver obtained from animals dosed at 1, 4, 8, 16, 30 and 100 mg/kg.
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7. A quantitation method for mass spectrometry imaging
Figure 6. MSI images of olanzapine in liver for three serial sections from two animals (A, B) dosed at 4 mg/kg of olanzapine.
Serial sections for each animal were analyzed on different MALDI target plates and prepared separately by manual spraying of
MALDI matrix (with a TLC sprayer).
that the pixel intensities represent the true relative abundance Acknowledgements
of olanzapine throughout the tissue and that they are not
highly influenced by inhomogeneity of matrix crystallization. The authors gratefully acknowledge Erin Seeley, Michelle
To more accurately determine the influence of matrix Reyzer, Richard Caprioli, Margery Stark Altman, Philippe
deposition and tissue properties on the signal response in Lesuisse and Sheryl Ferger for their guidance and contri-
a single pixel, future work in our laboratory will evaluate butions to this work.
the deposition of an internal standard with an acoustic
deposition device.
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