Research ArticleReceived: 29 October 2010    Revised: 2 December 2010     Accepted: 3 December 2010          Published onl...
S. L. Koeniger et al.         The ability to reliably and reproducibly deposit standards      parallel quantification of an...
A quantitation method for mass spectrometry imagingFigure 1. (a) Illustration of an integrated sectioning protocol for qua...
S. L. Koeniger et al.      low resolution mode, and the enhancement feature was set to        8 mg/kg were 2001 Æ 588 and ...
A quantitation method for mass spectrometry imaging Table 1. Statistical analysis of serial liver tissue sections analyzed...
S. L. Koeniger et al.      standard deviation for all three plates are shown and                  application techniques s...
A quantitation method for mass spectrometry imagingFigure 6. MSI images of olanzapine in liver for three serial sections f...
S. L. Koeniger et al.      [18] P. J. Trim, C. M. Henson, J. L. Avery, A. McEwen, M. F. Snel,    [33] K. Hattori, M. Kajim...
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Quantitative mass spec imaging


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Combining LC/MS quantitation with mass spec imaging for a "picograms per pixal" determination.

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Quantitative mass spec imaging

  1. 1. Research ArticleReceived: 29 October 2010 Revised: 2 December 2010 Accepted: 3 December 2010 Published online in Wiley Online Library: 2011Rapid Commun. Mass Spectrom. 2011, 25, 503–510( DOI: 10.1002/rcm.4891A quantitation method for mass spectrometry imagingStormy 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 Spanton11 Advanced Technology, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA2 Global Preclinical Safety, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA3 Exploratory Kinetics, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USAA 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 five10 mm tissue sections adjacent to those analyzed by MSI. Drug levels in tissue extracts were measured by liquidchromatography coupled to tandem mass spectrometry (LC/MS/MS). The integrated MSI response was correlated tothe LC/MS/MS drug concentrations to determine the amount of drug detected per MSI ion count. The study reportedhere evaluates olanzapine in liver tissue. Tissue samples containing a range of concentrations were created from liverharvested from rats administered a single dose of olanzapine at 0, 1, 4, 8, 16, 30, or 100 mg/kg. The liver samples werethen analyzed by MALDI-MSI and LC/MS/MS. The MALDI-MSI and LC/MS/MS correlation was determined fortissue 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 MSIresponses at the pixel level (100 mm). The details of the method, its importance in pharmaceutical analysis, andthe 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 ofmethod designed for mapping atomic or simple molecular pharmaceutical compound distributions. For MSI to reachspecies[1] to a family of techniques used to map biological its full potential, quantitative MSI information is needed tomolecules in complex tissues ranging from plant[2–4] and make it possible to extract safety and pharmacokineticanimal[5–8] samples to human biopsies from clinical trials.[9,10] data, to triage compounds in early drug discovery, and toAdvances 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-assisteddiversity of MSI applications.[11–14] With the success of these laser desorption/ionization (MALDI)[27] has become one ofadvancements, however, come new technological demands the most widely used ionization methods for MSI due toas MSI is extended to more challenging scientific questions. its high sensitivity, speed, broad molecular mass range (>300In the pharmaceutical industry, MSI provides an early Da up to 200 000 Da), and spatial resolution. Quantitativeavenue to determine the disposition of pharmaceutical MALDI assays developed for the high-speed analysis of smallcompounds in tissue during the discovery and candidate molecules have become well established and can provideselection phases of drug development when radiolabeled precision of equal to or greater quality (<5% relative standardcompounds 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 dynamicprovide 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 generallyin 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 directexogenous 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 isthat can serve as biological markers.[22,25,26] The relative possible to obtain quantitative data. The challenge associatedspatial 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: resolutions. 503Rapid Commun. Mass Spectrom. 2011, 25, 503–510 Copyright ß 2011 John Wiley & Sons, Ltd.
  2. 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 together504 Copyright ß 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 503–510
  3. 3. A quantitation method for mass spectrometry imagingFigure 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/MSquantitation, five sections are collected before (Q1) and after (Q2) sections analyzed by MSI. (b) Illustration of a simplifiedsectioning 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 offive 10 mm tissue sections were collected into a pre-weighed the 10 mM tissue homogenate stock with blank tissue500 uL Eppendorf (VWR, Radnor, PA, USA) vial (Q1); (2) homogenate. An identical dilution series was created inthree 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) wasreproducibility; and (3) five 10 mm sections were collected added in a 1:2 homogenate/acetonitrile ratio, vortexedinto pre-weighed Eppendorf tubes for duplicate analysis for 15 s, and centrifuged at 13 000 rpm for 10 min. Theof olanzapine concentrations by LC/MS/MS (Q2). Tissue supernatant was collected and diluted to a 30% acetonitrilesections for LC/MS/MS quantitation were weighed at room concentration prior to injection onto a Thermo Scientifictemperature and stored at –808C until further analysis. Tissue (West Palm Beach, FL, USA) BETASIL Cyano columnsections for MSI analysis were stored at room temperature (50 Â 3 mm; 5 m particles). The analytes were eluted atin 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 atlayer chromatography (TLC) sprayer (Sigma Aldrich) just 90% aqueous with 0.1% formic acid and electrosprayed intobefore analysis by manually spraying 10 mg/mL CHCA in a triple-quadrupole linear ion trap mass spectrometer50% 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 modehumidity. A total of $5 mL of matrix solution was used in the multiple reaction monitoring (MRM) was used for theprocess. 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 productLC/MS/MS quantitation ions were produced with the ’high’ buffer gas setting atTwo dilution series of olanzapine were prepared to collision energies of 34 and 47 eV for olanzapine anddetermine matrix effects on ionization and compound olanzapine-d3, respectively.extraction from tissue À one in tissue homogenate and theother in 50% aqueous acetonitrile. To create a tissue Mass spectrometry imaginghomogenate stock solution, 500 mL of a standard solutioncontaining 10 mM of olanzapine in 95% saline and 5% Mass spectrometry imaging was performed on a Qstar XLacetonitrile was added to 50 mg of liver tissue obtained Elite (AB Sciex, Concord, Ontario, Canada) equipped with anfrom rats dosed with vehicle (vehicle liver tissue). Blank AB Sciex oMALDITM source consisting of a 355 nm solid-statetissue homogenate was generated from 200 mg of vehicle laser with an elliptical spot size of 100 Â 150 mm. MSI analysesliver tissue containing 1 mg of tissue per 10 mL of saline. were performed at a spatial resolution of 100 Â 150 mm withTissue 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 productof liver per 10 uL saline. All solutions were sonicated for ion scan mode to obtain MS/MS spectra of the [MþH]þ ion of45 min in an ice bath. An 11-point standard curve ranging olanzapine at m/z 313.1. The quadrupole was operated in 505Rapid Commun. Mass Spectrom. 2011, 25, 503–510 Copyright ß 2011 John Wiley & Sons, Ltd.
  4. 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.506 Copyright ß 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 503–510
  5. 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 tissuesfor each plate. MALDI signals corresponding to the product revealed an average ion count per pixel of 2 Æ 1, showingion at m/z 256 were integrated over the entire tissue section the advantage of MS/MS in reducing background chemicalto obtain the integrated response for each image. A summary noise. For each animal, the average tissue response andFigure 3. MSI response as a function of dose (mg/kg) in $10 mm tissue sections of liver. Each data point represents the averageof three serial sections from a single animal analyzed on different MALDI target plates. (Inset) Linear regression of MSIresponses for individual tissue sections for each MALDI target plate. NA ¼ data not available. 507Rapid Commun. Mass Spectrom. 2011, 25, 503–510 Copyright ß 2011 John Wiley & Sons, Ltd.
  6. 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.508 Copyright ß 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 503–510
  7. 7. A quantitation method for mass spectrometry imagingFigure 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 ofMALDI matrix (with a TLC sprayer).that the pixel intensities represent the true relative abundance Acknowledgementsof olanzapine throughout the tissue and that they are nothighly influenced by inhomogeneity of matrix crystallization. The authors gratefully acknowledge Erin Seeley, MichelleTo more accurately determine the influence of matrix Reyzer, Richard Caprioli, Margery Stark Altman, Philippedeposition 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 acousticdeposition device. REFERENCESCONCLUSIONS [1] R. Castaing, G. J. Slodzian. Microscopy 1962, 154, 395. [2] A. K. Mullen, M. R. Clench, S. Crosland, K. R. Sharples.This study methodically correlates tissue drug concentrations Rapid Commun. Mass Spectrom. 2005, 19, MALDI-MSI responses to quantitate two-dimensional [3] Y. Li, B. Shrestha, A. Vertes. Anal. Chem. 2008, 80, 407.molecular distributions obtained in MSI analyses of histo- [4] Z. Li, L. Chu, J. V. Sweedler, P. W. Bohn. Anal. Chem. 2010, 82, 2608.logical tissue sections. This method provides a direct and [5] R. M. Caprioli, T. B. Farmer, J. Gile. Anal. Chem. 1997, 69,relatively simple method for quantifying MALDI-MSI data studies that consist of a sample pool containing at least [6] M. Stoeckli, P. Chaurand, D. E. Hallahan, R. M. order of magnitude difference in analyte concentration. Nat. Med. 2001, 7, 493.This effectively eliminates the need for standards that may [7] A. S. Woods, S. N. Jackson. AAPS J. 2006, 8, E391.not always be representative of the sample. The MALDI-MSI [8] T. A. Zimmerman, E. B. Monroe, K. R. Tucker, S. S. Ruba- khin, J. V. Sweedler. Methods Cell Biol. 2008, 89, 361.responses were demonstrated to be linear over 2 orders of [9] E. H. Seeley, R. M. Caprioli. Proteom. Clin. Appl. 2008, 2,magnitude with an average precision of 14%. These analytical 1435.figures of merit are respectable in comparison with those [10] L. H. Cazares, D. Troyer, S. Mendrinos, R. A. Lance, J. O.from well-established LC/MS/MS and MALDI-MS methods. Nyalwidhe, H. A. Beydoun, M. A. Clements, Clements. R. R. Quantifying MSI data is an ongoing technological chal- Clements, O. J. Semmes. Clin. Cancer Res. 2009, 15,lenge due to the lack of appropriate calibration and internal 5541.standards. Current efforts in our laboratory are focused on [11] R. G. Cooks, Z. Ouyang, Z. Takats, J. M. Wiseman. Science 2006, 311, 1566.addressing these issues with acoustic deposition of internal [12] J. S. Becker, M. Zoriy, V. L. Dressler, B. Wu, J. S. Becker. Purestandards and continued evaluation of this method on Appl. Chem. 2008, 80, 2643.multiple organs/tissue types for a variety of pharmaceutical [13] G. J. Van Berkel, S. P. Pasilis, O. Ovchinnikova. J. Masscompounds. Despite these challenges, it has been demon- Spectrom. 2008, 43, 1161.strated here that MSI provides a relative measure of the [14] V. Kertesz, G. J. Van Berkel. J. Mass Spectrom. 2010, 45, 252.molecular distributions that can be quantified by correlation [15] M. L. Reyzer, Y. Hsieh, K. Ng, W. A. Korfmacher, R. M. Caprioli. J. Mass Spectrom. 2003, 38, 1081.with LC/MS/MS concentrations. Successful implementation [16] Y. Hsieh, R. Casale, E. Fukuda, J. Chen, I. Knemeyer, J.of MSI quantitation for both endogenous and exogenous Wingate, R. Morrison, W. A. Korfmacher. Rapid Commun.molecular species of all classes will have widespread impact Mass Spectrom. 2006, 20, 965.on drug discovery programs. [17] R. J. A. Goodwin, A. R. Pitt. Bioanalysis 2010, 2, 279. 509Rapid Commun. Mass Spectrom. 2011, 25, 503–510 Copyright ß 2011 John Wiley & Sons, Ltd.
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