Mark Bokhart (Presenter)
North Carolina State University
Bio: Mark Bokhart is a PhD candidate in analytical chemistry at North Carolina State University in David Muddiman’s laboratory. He received a BS in Chemistry from Michigan State University (MSU) in May 2013. While at MSU, he worked at the Diagnostic Center for Population and Animal Health (DCPAH), a full service veterinary diagnostic laboratory, where he performed method development and validation along with routine analysis of samples submitted to the toxicology section. Current research interest and projects are centered on using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Recent efforts have been focused on quantitative mass spectrometry imaging of xenobiotics in thin tissue sections for the quantification of therapeutics in tissue microenvironments.
Authorship: Mark T. Bokhart(1), Andrew Lucas(2), Allison Schorzman(2), William Zamboni(2), David C. Muddiman(1)
(1) Department of Chemistry, North Carolina State University, Raleigh, NC (2)Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC
| Short Abstract Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) is a powerful analytical platform for the visualization of analyte distributions within tissue sections. Recent method development and optimization extended the analytical capabilities of IR-MALDESI MSI to provide quantitative images of xenobiotics in tissue sections. In this work, we present quantitative MSI for novel poly-2-oxazoline (POx) polymeric micelle formulations of paclitaxel (PTX) in a mouse model. The concentration and distribution of PTX in tumor, spleen and liver was determined using MSI with concentrations validated with LC-MS/MS analysis of serial sections. |
Long Abstract
Introduction
Mass spectrometry imaging (MSI) has proven itself as an important method for determining the spatial distribution of analytes in a research setting. Quantitative mass spectrometry results are difficult to achieve due to heterogeneous tissue microenvironments, thus limiting MSI application for quantitative data needed for PK/PD tissue studies. However, the incorporation of a normalization compounds, utilized by several groups including our own, accounts for tissue-specific ionization efficiency and can provide quantitative information with the inclusion of a matrix-matched calibration curve.(1,2)
Methods
Infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) is performed using a mid-IR laser (2.94 um) which resonantly excites water present within the biological tissue and an exogenous ice matrix, causing the complete ablation of all material in the focal volume of the laser, effectively sampling a voxel of tissue. The laser ablation plume is ejected normal to the sample surface, where it intersects with an orthogonal electrospray plume. The ablated neutral material partitions into the electrospray plume where analytes ionization in an ESI-like fashion. A thermally-assisted pneumatic sprayer was used to evenly apply an internal standard, a structural analogue of paclitaxel, onto the microscope slide prior to thaw-mounting the analyzed tissue for the normalization of analyte on a per-voxel. A calibration curve was created in solution and spotted on top of non-dosed, tissue-matched sections and analyzed simultaneously with the dosed tissue section. The IR-MALDESI source is coupled to a Q Exactive Plus mass analyzer, which provides high resolving power (RP=140,000 at m/z 200), high mass measurement accuracy data (<5 ppm) for the accurate identification of analytes in MS and MS2 experiments. Mice were dosed according to one of three treatment arms comparing FDA approved formulation Taxol® with poly-2-oxazoline (POx) polymeric micelle formulations of paclitaxel (PTX); 20 mg/kg Taxol, 150 mg/kg 50:20 POx:PTX, or 150 mg/kg 50:40 POx:PTX at 1 hour post-dose time point. MSI data was interpreted and visualized using MSiReader, where concentrations corresponding to regions of interest for calibration curve and dosed tissue can be calculated.
Results
The quantitative abilities of IR-MALDESI MSI were evaluated using conventional analytical figures of merit. Calibration curves were created with natural isotope standards of the analyte spotted on blank tissue adjacent to the quantified tissue. Increased linearity and reduced per-voxel variability of analyte demonstrate the internal standards ability to account for matrix effects and ionization suppression resulting from the complex molecular environment of a biological tissue.
Absolute ion abundance and reproducibility are dependent on instrument ion transfer parameter S-lens RF level. S-lens radio frequency (RF) level was optimized and providing absolute quantification of xenobiotics in tissue microenvironments through reduced per-voxel variability. MSI revealed nearly homogenous distribution of PTX within spleen and liver at higher concentration than within the tumor tissue section. Concentration of PTX was reduced in tumor regions for all treatment arms, with higher concentration of PTX located within the bursal membrane for the polymeric POx formulations. LC-MS/MS analysis of serial sections were used to validate concentrations determined with quantitative MSI. Experiments are being performed to further reduce variability of instrumental response and increase sensitivity on a per-voxel basis for IR-MALDESI MSI.
Conclusions
Mass spectrometry imaging of paclitaxel for several formulations reveled higher concentration of paclitaxel in tumor, particularly in the bursal membrane, for the polymeric POx:PTX formulations. Optimization of the ion optics parameter S-lens level increased ion transmission through the atmospheric pressure interface, thereby increasing analyte ion abundance. Normalization to structural analogue showed marked improvement in the form of reduced variability and linearity of calibration curve for tissue-matched quantitative MSI analyses.
References & Acknowledgements:
(1) Bokhart M, Rosen E, Thompson C, Sykes C, Kashuba AM, Muddiman D. Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization. Analytical and Bioanalytical Chemistry 2015, 407: 2073-2084.
(2) Thompson CG, Bokhart MT, Sykes C, Adamson L, Fedoriw Y, Luciw PA, Muddiman DC, Kashuba ADM, Rosen EP. Mass Spectrometry Imaging Reveals Heterogeneous Efavirenz Distribution within Putative HIV Reservoirs. Antimicrobial Agents and Chemotherapy 2015, 59: 2944-2948.
The authors would like to gratefully acknowledge the financial support received from the National Institutes of Health (R01GM087964), W. M. Keck Foundation, and North Carolina State University.
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