MALDI Imaging Through Laser-Induced Post-Ionization Pushes the Boundaries of Pharmaceutical Development Studies Florian Barré (Presenter) M4I, Maastricht University Presenter Bio: I obtained a bachelor specialized in cell biology and physiology that I got at Lille 1 University in France. I graduated with honors in 2015 with a Proteomics master that I also got at Lille 1 University with Isabelle Fournier and Michel Salzet as directors of the master. During my master, I went to Copenhagen at BRIC institute for 6 months for my first year internship where my project was to create luminescent and fluorescent cancer cell lines in Lund Anders’s laboratory. For my second year of master I went to Uppsala University, at Per Andrén’s laboratory for a project concerning the quantitation of a drug in the brain by MSI coupled to LC-MS for 6 months as well. Afterwards, Ron Heeren and Berta Cillero-Pastor offered me the opportunity to work on cartilage regeneration within a Marie Curie consortium. During PhD, I tried to be involved in the MSI community. For this reason, I was p

Authors: Florian P.Y. Barré(1), Martin R.L. Paine(1), Bryn Flinders(1), Rima Ait-Belkacem(2), João P. Garcia(4), Laura B. Creemers(4), Jonathan Stauber(2), Rob Vreeken(1,3), Berta Cillero-Pastor(1), Shane R. Ellis(1), Ron M.A. Heeren(1)
(1)The Maastricht Multimodal Molecular Imaging Institute (M4I), Division of Imaging Mass Spectrometry, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands (2)ImaBiotech, MS Imaging Dept., Loos, France (3)Discovery Sciences, Janssen Research & Development, Beerse, Belgium (4)University Medical Centre (UMC) Utrecht, Orthopedics Department, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands

Drug imaging is one of the major mass spectrometry imaging (MSI) applications. However, drug development through MSI has some limitations. Indeed, some compounds are not easily detectable due to poor ionization efficiency and ion suppression effects. In this work, we used post-ionization matrix assisted laser desorption/ionization (MALDI-2) to overcome these problems and we are showing that 80% of the drugs we tested directly benefit from ionization enhancement. We are now able to detect pharmaceuticals that are non-detectable by conventional MALDI pushing the boundaries of MSI for drug studies.

Introduction

Drug imaging is one of the key applications of mass spectrometry imaging (MSI) and enables monitoring local drug metabolism and pharmacokinetics (DMPK), mapping, and quantification of pharmaceuticals and their metabolites. However, some compounds are not easily ionized with matrix assisted laser desorption/ionization (MALDI), limiting the capabilities of MALDI-MSI for pharmacological applications. Using a wavelength-tunable laser, we have optimized the recently described method of MALDI-2 MSI to initiate secondary gas-phase MALDI ionization processes through laser-induced post-ionization. In this work we demonstrate for the first time the utility of laser-induced post-ionization to dramatically improve the sensitivity of MALDI-based MSI for a suite of drugs and metabolites from a variety of different tissues.

Methods

20 different drugs have been mixed with 2,5-dihydroxybenzoic acid (20 mg/mL in 70%MeOH) and sprayed on indium tin oxide (ITO) slide using the TM-Sprayer (HTX-technologies). An orbitrap Elite (Thermo Fisher Scientific) with an intermediate pressure source1 has been combined with optical parametric oscillator (OPO) laser system (NT-230, Ekspla) to generate post-ionization. For each compound, laser energy, post-ionization delay, and the OPO wavelength have been optimized. Using triamcinolone acetonide (TAA), we compared our derivatization method combined with regular MALDI and MALDI-2 to test the sensitivity limits for each combination.2 Mice livers and lungs; guinea pig intestines and human cartilage respectively treated with two drugs in development phase, tofacitinib, and TAA, were measured using MALDI-2 (between 20 and 45 µm spatial resolution).

Results

Initially, TAA was used to investigate the benefits of MALDI-2 over conventional MALDI and explore the parameter space, i.e., OPO wavelength, laser power, and delay time. Using MALDI-2, it was possible to detect the protonated drug at m/z 435.217, whereas detection using regular MALDI was only possible following on-tissue derivatization with Girard’s-reagent-T at m/z 548.312 [M+H]+. Following these results, we tested the limits of detection (LOD) for different combinations of derivatized and non-derivatized TAA with MALDI and MALDI-2 using a dilution series (0.005 ng/µL – 5 ng/µL). From this study, we observed that derivatization with MALDI and MALDI-2 allowed the detection of TAA at a LOD of 0.005 ng/µL while MALDI-2 by itself had a LOD of 0.025 ng/µL. This shows that long sample preparation and optimization (derivatization) could be reduced or avoided using MALDI-2. Following these results, 20 compounds were tested and an improved ionization efficiency with MALDI-2 for 16 of these drugs was observed. Among these, a 300 fold gain was recorded for paclitaxel, and doxorubicin – a notoriously difficult compound to detect – was readily observed. The experiments on drug standards were followed by a study on dosed tissues/animals. TAA dosed in human cartilage (up to 43.5 ng/µL) was easily detected demonstrating that MALDI-2 is applicable for direct on-tissue drug imaging. Subsequently, two drugs in the pre-clinical phase were tested and both drugs and their metabolites were detected for the first time in mice lungs and liver (2h and 8h post-administration). Interestingly, when penetration of tofacitinib in guinea pig intestines was investigated with MALDI, it was exclusively present in the intestinal lumen. From this experiment, the conclusion could have been that the drug is not able to penetrate the tissue. However, using the higher sensitivity of MALDI-2, drug penetration into both mucosa and the submucosa was observed. Moreover, MALDI 2 experiments allowed us to correlate the drug presence with a change in the lipid profile. The derivatization procedures involve solvents that cause suppression and/or delocalization of lipid signals that hamper sensitive drug detection in tissue.

Conclusions & Discussion

With this innovative approach, long sample preparation procedures and optimization (derivatization) for not easily ionized compounds can be reduced or simply skipped. Moreover, MALDI-2 has the potential to be applied as an universal tool to study pharmaceuticals and their effects on tissue. In fact, we were also able to detect and map amino acid in treated tumor sections such as Proline, Arginine or their metabolites such as Kynurenine which are downregulated by chemotherapeutic treatments and were detectable so far only by using on-tissue chemical reactions.

MALDI-2 allows in situ detection of non-detectable drugs by conventional MALDI expanding impact of MSI for drug development studies.

(1) Belov, M. E.; Ellis, S. R.; Dilillo, M.; Paine, M. R. L.; Danielson, W. F.; Anderson, G. A.; de Graaf, E. L.; Eijkel, G. B.; Heeren, R. M. A.; McDonnell, L. A. Anal Chem 2017, 89, 7493-7501.

(2) Barre, F. P.; Flinders, B.; Garcia, J. P.; Jansen, I.; Huizing, L. R.; Porta, T.; Creemers, L. B.; Heeren, R. M.; Cillero-Pastor, B. Anal Chem 2016, 88, 12051-12059.

This research has been made possible with the support of the Dutch Province of Limburg. F.Barré has received funding from the European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 642414.