In Situ Derivatization Combined with DESI-MRM-MS/MS for Spatial Mapping Intratissue Amine-Containing Metabolites, Trimethylamine as a Case

Mass spectrometry imaging (MSI) can directly detect surface analytes, enabling spatial mapping of metabolite synthesis and migration within tissues. However, MSI often lacks the sensitivity for routine analysis of low exposure, low molecular weight endogenous metabolites. Amine-containing metabolites, such as methylamine, dimethylamine, trimethylamine, lysine, dopamine, and 5-hydroxytryptamine, are widely used as medical biomarkers due to their potential biological activity or toxicity. Herein, we innovatively developed a universal in situ derivatization workflow combined with desorption electrospray ionization MS/MS (DESI-MS/MS) for mapping endogenous amine-containing metabolites, with trimethylamine (TMA) selected as a case study. For the first time, tert-butyl bromoacetate (TBBA) was used as an in situ derivatization reagent, which improved sensitivity and reproducibility by overcoming the strong matrix effect and signal instability of low molecular weight compounds in MS analysis. Our data suggested that the derivatization efficiency was affected by the alkalinity of the tissue surface, while spray/extraction solvent significantly affected the sensitivity for TMA-TBBA. Notably, DESI-MS/MS exhibited good linearity, reproducibility, and stability in the analysis of amine-containing metabolites derived from TBBA. The optimized workflow was utilized for the visual analysis of endogenous TMA distribution in the brain and serum of mice, revealing the enrichment characteristics of TMA in the cortex and hippocampus, as well as the severe accumulation of TMA in aged animals. Therefore, this targeted DESI-MS/MS-MSI approach, combined with in situ derivatization, offers novel avenues for the routine quantitation of intratissue amine-containing metabolites. In principle, this assay can be extended to a wide variety of metabolites in different biological samples.