2D-TLC-UV/Vis/FLD-direct bioautography-HRMS/MS-based identification of antibacterial compounds of Myrtus communis L

The search for new antibacterial agents is often hindered by the limited efficiency of traditional isolation-based workflows, which may result in low yields or insufficient detection of bioactive constituents. To address these limitations, an affordable two-dimensional thin-layer chromatography (2D-TLC) combined with direct bioautography was applied for the rapid screening of the hydroalcoholic leaf extract of Myrtus communis. The 2D-TLC-UV/Vis/FLD-Bacillus subtilis assay provided improved separation capacity, including in overloaded conditions, also enabling the visualization of minor antibacterial zones. Bioactivity-guided HRMS/MS analysis performed directly from the inhibition areas allowed the tentative identification of several triterpenoids and fifteen myrtucommulone-type non-prenylated acylphloroglucinol derivatives. Some of these metabolites have not been previously described in the context of antibacterial screening of M. communis. This is the first application of an eight-dimensional workflow integrating 2D-TLC-UV/Vis/FLD-B. subtilis bioassay-HESI-HRMS/MS for the screening and characterization of antibacterials in a complex matrix, exemplified here for M. communis, which provided previously unexplored compounds with potent antibacterial activity.