Ionic Solid 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) enables spatially resolved molecular analysis of biological tissues. However, imaging low-molecular-weight metabolites remains challenging because conventional organic matrices generate intense background signals in the low m/z region. Ionic matrices can reduce matrix-related interferences, improve spectral reproducibility, and enhance sensitivity. However, their application in MALDI-MSI has remained limited, largely due to the need for additional matrix preparation and the challenging wet deposition of viscous ionic liquid matrices, and has mainly focused on lipid imaging above m/z 500. Here, we present a novel approach combining ionic solid matrices (ISMs) with low-temperature thermal evaporation (LTE) to enhance MALDI-MSI performance for small metabolites. ISMs based on α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) provided superior sensitivity and reduced matrix background. In particular, CHCA + N,N-diethylaniline enabled the detection and annotation of 69 metabolites with m/z < 500 in mouse brain tissue at 20 μm spatial resolution. When applied to mouse pancreas, this ISM allowed the first specific localization of melatonin and 3-iodotyrosine within the islets of Langerhans, opening new avenues for spatial metabolomics in endocrine research.