MALDI MSI Workflow for Pharmaceutical Tablets: Toward a Reproducible Strategy for High-Resolution Imaging

Abstract Mass spectrometry imaging (MSI) can complement liquid chromatography–mass spectrometry (LC-MS), the gold standard for impurity assessment, by enabling spatial visualization of active pharmaceutical ingredients (APIs), byproducts, and excipients in solid formulations. With new advances in matrix-assisted laser desorption/ionization (MALDI) MSI, critical factors such as ablated spot size, raster speed, and standardized tablet surface preparation still need to be properly addressed. This study presents an innovative sample holder and sample preparation protocol enabling reproducible up to 10-μm step size acquisition for MSI analysis of tablets. The workflow was validated by embedding several commercial tablets containing various APIs in blocks with epoxy resin, then transversally cut them to expose their surfaces. Following this, the tablet surfaces were trimmed using a cryostat and subsequently polished with lapping diamond films to achieve flat and even surfaces. The tablets were then placed in a custom metal holder for MALDI-trapped ion mobility spectrometry (TIMS)-MSI analysis using a timsTOF fleX. The optimized protocol provides high-quality sample preparation and avoids loss of laser focus, enabling high-throughput analysis with high sensitivity for tablet imaging. This robustness was essential for spatial resolution measurements using the Smartbeam 3D, where precise initial focusing is required to preserve data quality across imaging areas. Consistent high signal sensitivity was achieved for single pixels in 10 mg cetirizine or 20 mg famotidine tablets and digoxin fragments were detected in a 0.25 mg/130 mg tablet. This approach provides an important tool for pharmaceutical development by enabling reliable, high-resolution spatial analysis of active ingredients and impurities in solid dosage forms. Collision cross section (CCS) values from reference standards and tablets, as well as MS/MS data were used for confident identification. LC-MS-based workflows remain essential for quantitative analysis of impurity assessment, while infrared spectroscopy (IR) and nuclear magnetic resonance (NMR) can support formulation level characterization. MALDI-TIMS-MSI complements these approaches by adding direct spatial localization of APIs and API-related molecular features within the tablet matrix.