Solid Ionic Matrices applied via Low-Temperature Evaporation enable High-Resolution and Sensitive MALDI Imaging of Metabolites

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) has become a key technology for spatially resolved molecular analysis. However, imaging of low molecular weight compounds remains challenging due to spectral interferences in the low m/z range caused by conventional organic matrices. Ionic matrices (IMs), in contrast, reduce matrix-related background signals, improve spectral reproducibility, and enhance sensitivity for detecting metabolites and lipids, but their use in MALDI-MSI has so far been limited, primarily focusing on lipid imaging above m/z 500. Here, we present a novel approach combining solid ionic matrices (ISMs) with low-temperature thermal evaporation (LTE) to enhance MALDI-MSI performance for small metabolites. ISMs based on -cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) provided superior sensitivity, with CHCA + N,N-diethylaniline enabling the detection and annotation of 73 small metabolites (m/z <500) in mouse brain at 20 {micro}m spatial resolution. When applied to the pancreas, this matrix enabled the first specific localization of melatonin and 3-iodotyrosine within the islets of Langerhans, opening new avenues for spatial metabolomics in endocrine research.