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 <i>Hormonema dematioides</i>

ABSTRACT 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.