Ultra-Performance Liquid Chromatography–Electrospray Ionization–Ion Trap Tandem Mass Spectrometry for Fragmentation of Cyclic Dipeptides: Methodology and Application to the Chemical Profiling of the Fungus Hormonema dematioides

Cyclic dipeptides (CDPs), distinguished by a 2,5-diketopiperazine core, exhibit a broad spectrum of biological activities. The (un)targeted discovery of CDPs in complex matrices is therefore of considerable interest, and mass spectrometry (MS) offers unrivalled advantages: rapid analysis, high sensitivity, minimal sample consumption, and limited pre-treatment requirements. Yet, confident MS-based identification of CDPs has been hampered by the scarcity of authentic reference standards. To address this deficit, we undertook a systematic investigation of 21 CDPs isolated from the fungus Hormonema dematioides using ultra-performance liquid chromatography–electrospray ionization–linear ion trap multistage MS (UPLC–ESI–LIT-MS n ) in positive ion mode. Hierarchical MS 2 and MS 3 experiments afforded reproducible fragmentation pathways that unambiguously delineated the CDP nucleus and the constituent amino acid residues. For the first time, we herein proposed [M + H-17] + and [M + H-56] + as co-diagnostic ions for the 2,5-diketopiperazine scaffold, together with the previously reported [M + H-28] + , [M + H-45] + , and [M + H-73] + ions. Additionally, in contrast to previous reports, the [M + H-28] + ion consistently appeared as the base peak for the majority of CDPs examined. Amino-acid-specific fragments were found to be strictly dependent on the nature of the incorporated residues. These fragmentation rules were subsequently applied to the untargeted profiling of H. dematioides , enabling the identification or tentative annotation of 30 CDPs through combined UV and MS n ( n = 3) data, corroborated wherever possible by authentic standards. Notably, 10 of the CDPs are reported here for the first time from this fungal species.