Systematic assessment of different wash solvents for the analysis of small molecule metabolites using mass...

Wash protocols are a simple, commonly used approach to enhance the detectability of low-abundant or poorly ionisable compounds in mass spectrometry imaging (MSI). The washing procedures aim to enhance analyte ionisation by removing interfering metabolites that affect the ionisation efficiency and detection of the target metabolites. However, despite the widespread use of wash protocols in MSI, their impact on small molecule metabolites (SMM) has not been systematically evaluated. In this study, 12 different aqueous and organic wash solvents were investigated to assess their impact on the signal intensities of SMMs in tumour tissue using desorption electrospray ionisation mass spectrometry imaging (DESI-MSI). The added wash steps proved to be a promising tool for increasing detection sensitivity for targeted metabolites, with >90% of analytes investigated here showing increased sensitivity following the optimum wash solvent step. While chloroform was found most efficient in removing lipids overall, the most versatile solvent to significantly enhance the detection of polar and semi-polar metabolites, including amino acids, nucleic acid compounds, sugars, and organic acids, was found to be ethyl acetate. In contrast, water-based washes enhanced fatty acids and lipids while removing hydrophilic metabolites. This study emphasises the importance of adjusting pretreatment protocols to the molecular class of interest and provides a targeted guide for increasing ion detection sensitivity across a broad range of metabolites.