Derivatization of Small Molecules using Deuterated Analog of Reactive Matrix to Facilitate Identification in Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) is a powerful tool for spatial metabolomics and biomarker discovery, but molecular identification remains challenging, particularly for low‐abundance analytes with poor signal‐to‐noise ratios. Herein, a novel approach that utilizes a deuterated analog of the reactive matrix FMP‐10 to enhance molecular identification is introduced. This isotopically labeled matrix enables precise determination of derivatization patterns, allowing the number and sequence of reactive functional groups in small molecules to be deduced. By observing specific mass shifts, the method provides additional structural information beyond high‐resolution MS and MS/MS, addressing key limitations in MSI‐based biomarker discovery. This innovative labeling strategy improves identification confidence for neurotransmitters and metabolites, making it a powerful addition to the MSI toolbox for complex tissue analysis. The findings represent a significant advance for spatial metabolomics, with particular advantages for neurochemical mapping in the study of neurodegenerative diseases.