Generation of Pseudo Metabolites as a Side Reaction in On‐Tissue Chemical Derivatization in Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) is a key spatial omics technology for spatially resolved visualization and direct on‐tissue identification of multiple lipid and metabolite classes. Recently, many on‐tissue chemical derivatization (OTCD) reagents targeting defined functional groups have been introduced to improve sensitivity and/or molecular specificity for compound annotation. Some act as reactive matrices that introduce permanent charges and no longer require (de)protonation for ionization. However, OTCD side reactions are understudied, and it is presently unknown what pseudo metabolites can be generated as OTCD‐derived artifacts. These are compounds that, in principle, exist as bona fide metabolites in tissues but that can also be products of OTCD side reactions. Here, it is demonstrated that besides expected reaction products, the reactive matrix 4‐(anthracen‐9‐yl)‐2‐fluoro‐1‐methylpyridin‐1‐ium iodide (FMP)‐10 can also cause unexpected N ‐ and O ‐lactoylation and even apparent poly‐lactoylation of multiple amino acids through Mukaiyama esterification. Side reactions are unrelated to MALDI laser photochemistry, as they happen in solution and can be readily observed by liquid chromatography–MS/MS. Since N ‐lactoylated amino acids have recently received much attention in sports physiology and obesity research, much care needs to be taken to distinguish true endogenous N ‐lactoylated amino acids from those that are formed by Mukaiyama side reactions.