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Rationale: Azaphilones, belonging to the class of mitorubrins usually produced in Hypoxylon fragiforme, react easily with amino groups, giving amine derivatives, mitorubramines. These secondary metabolites exhibit a wide range of biological activities. Finding new secondary metabolites from fungi is important, and electrospray ionization (ESI) high-resolution mass spectrometry (HRMS) coupled with sequential MS(n) experiments has become a method of choice for the chemotaxonomic classification of fungi., Methods: High-performance liquid chromatography of methanol extracts coupled to positive electrospray ionization, high resolving power for accurate mass measurements and resonant excitation for selective ion collision-induced dissociation (CID) have been conducted with the aim of resolving the structures of possible novel compounds., Results: Soft desolvation conditions in the ESI source enabled the detection of intact mitorubramines present in the extract. HRMS provided unambiguous information about the elemental composition of the mitorubramines and their product ions, while sequential MS(3) experiments were essential for the structural discernment of already reported mitorubrins and newly discovered mitorubramines. Indeed, specifically from the latter, a series of consecutive dissociations takes place under CID conditions that are useful for structural elucidation., Conclusions: A distinctive method for two families of secondary metabolites has been developed. Information observed using HRMS and sequential MS(n) experiments gave unambiguous information about the structure of mitorubramines, especially the position of the nitrogen atom, which was strengthened by proposed unusual fragmentation mechanisms, such as the rearrangement yielding the release of CO(2) from the hydroxyl-diketone structures. These experiments demonstrated that the fragmentations are facilitated by the nitrogen electron lone-pair in mitorubramines, which does not occur in mitorubrins., (Copyright © 2012 John Wiley & Sons, Ltd.)