Distinctive gas-phase fragmentation pathway of the mitorubramines, novel secondary metabolites from Hypoxylon fragiforme Ljubica Svilar 1,2 , Vesna Stankov-Jovanovic 1 *, Marc Stadler 3 , Hristo Nedev 2 and Jean-Claude Tabet 2 * 1 University of Nis, Faculty of Science and Mathematics, Department of Chemistry, Visegradska33, 18000 Nis, Serbia 2 University Pierre and Marie Curie, Paris Institut of Molecular Chemistry, UMR 7201-FR2769, Case Courrier 45, Batiment F, 716, 4, place Jussieu, 75252 Paris cedex 05, France 3 Department Microbial Drugs, Helmholtz-Centre for Infection Research, Inhoffenstrasse 8, 38124 Braunschweig, Germany 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 mitoru- bramines, 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. Mitorubrin azaphilones from Hypoxylon fragiforme (Scheme 1), containing a pyrone-quinone group, have been reported, and elucidation of their structures was performed by a range of analytical methods, [1,2] such as liquid chromatography (LC) with ultraviolet/diode-array detection (UV/DAD), nuclear magnetic resonance (NMR), infrared (IR) and mass spectrometry (MS), using pure, isolated compounds. [3–5] Because of their versatile biological activities (anti-fungal activity, inhibition of dihydrofolate reductase and geranylgeranyl transferase), [6] these compounds represent a group of highly interesting secondary metabolites. Azaphilones generally react with nitrogen from amines to form vinylogous 4-pyridone derivatives. However, very few azaphilone vinylogous 4-pyridone derivatives have been found to be present as natural products in biological samples, [7–9] although some syntheses of these derivatives have been reported. [10,11] To the best of our knowledge, there have been no reports on the presence of vinylogous 4-pyridone derivatives in Hypoxylon fragiforme or other species of the Xylariaceae. Mitorubramines (Scheme 1) have been detected in the current study in the methanolic stromatal extract of Hypoxylon fragiforme, although in low concentration. LC was coupled to high-resolution mass spectrometry (HRMS) and collision-induced dissociation (CID) to provide unambiguous information about the structures, molecular masses and some chemical properties of this novel group of secondary metabolites. Mass spectra obtained by soft ionization techniques often do not provide sufficient information about the molecular structure of interest, and consequently further tandem mass spectrometric (MS/MS) fragmentation must be undertaken to obtain more detailed data. CID MS/MS is widely utilized in forming reference libraries for the * Correspondence to: V. Stankov-Jovanovic, University of Nis, Faculty of Science and Mathematics, Department of Chemis- try, Visegradska33, 18000 Nis, Serbia. E-mail: sjvesna@pmf.ni.ac.rs ** Correspondence to: J.-C. Tabet, University Pierre and Marie Curie, Paris Institut of Molecular Chemistry, UMR 7201- FR2769, Case Courrier 45, Batiment F, 716, 4, place Jussieu, 75252 Paris cedex 05, France. E-mail: jean-claude.tabet@courriel.upmc.fr Copyright © 2012 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2012, 26, 2612–2618 Research Article Received: 14 July 2012 Revised: 22 August 2012 Accepted: 24 August 2012 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2012, 26, 2612–2618 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6382 2612