Enhancing Hydroxyl Metabolite Analysis through In Situ Derivatization-Enabled Desorption Electrospray Ionization-Mass Spectrometry

Unlocking the full potential of metabolomics hinges on significantly improving the detection of metabolites, particularly those containing hydroxyl groups, which often remain challenging to ionize. Our previous work established 2-(4-Boronobenzyl) isoquinolin-2-ium bromide (BBII) as a highly effective derivatization reagent for enhancing hydroxyl metabolite sensitivity in liquid chromatography-mass spectrometry. Building upon this, we herein introduce a novel and robust extension: BBII derivatization integrated with desorption electrospray ionization (DESI) for <i>in situ</i> hydroxyl metabolite detection. This innovative approach involves incorporating BBII directly into the DESI spray solvent, leading to significant sensitivity enhancement through instantaneous derivatization reaction. We demonstrate substantial signal increases ranging from 1.8- to 17.2-fold for hydroxyl metabolite reference standards, making previously undetectable compounds such as glucose, hexadecanol, and estradiol readily observable. When applied to mouse brain tissue sections for mass spectrometry imaging (MSI), BBII-DESI successfully revealed the distinct spatial distributions of representative hydroxyl metabolites, including glucose and cholesterol that are typically invisible to conventional methods. A key advantage of this methodology is the characteristic boron isotopic pattern of BBII-derivatized features, facilitating rapid and precise screening and identification. This BBII-assisted DESI strategy effectively unveils the "dark metabolome" of hydroxyl compounds, providing access to previously inaccessible metabolic information. Our method broadens the utility of ambient ionization techniques by enabling direct analysis of biological samples with minimal preparation, which is crucial for high-throughput applications. This advance offers substantial potential for accelerating biomarker discovery and disease diagnostics through direct visualization of metabolic alterations within native tissue environments, thereby marking a significant leap forward in spatial metabolomics applications for health and disease research.