Diagnostic Fragmentations of Animal and Fungal Sterols/Stanols Obtained by APCI–Tandem Mass Spectrometry: A Route Towards Unknown Free Sterol Identification

<b>Background/Objectives</b>: Animal and fungal sterols and stanols exhibit remarkable structural diversity, driven by variations in the number and position of C=C bonds within the steroidal tetracyclic core and side chain, along with diverse branching patterns of the latter. Similarly to phytosterols, these metabolites produce highly complex tandem mass spectra, whose interpretation has so far been limited. To address this gap, the fragmentation behavior of selected animal/fungal sterols and stanols was studied in this paper. <b>Methods</b>: Higher-Collisional-energy Dissociation-High-resolution tandem mass spectrometry (HCD-HRMS/MS) of protonated/dehydrated species generated via atmospheric pressure chemical ionization (APCI) was performed on structurally diverse compounds, including lathosterol, desmosterol, zymosterol, lanosterol, ergosterol, chalinasterol, and the stanols coprostanol and cholestanol. <b>Results</b>: Structurally diagnostic product ions originating from the side chains were unveiled, shedding light on the intramolecular migration of positive charge from the initial ionization site at C3 to alternative stable sites across the molecular structure, which is a typical mechanism also noted in cholesterol and phytosterols. In addition, characteristic fragmentation patterns related to the steroidal backbone were found and discussed for Δ⁷, Δ^5,7 and Δ⁸-sterols, and a novel elucidation of the fragmentation behavior of 4,4-dimethyl-Δ⁸-sterols, based on lanosterol as a model compound, was achieved. The relative intensities of diagnostic product ions allowed a correlation with specific structural motifs, and "cholesterol-like" and "stigmasterol-like" fragmentations pathways were recognized. These findings were integrated with prior data on cholesterol and plant sterol fragmentation acquired under identical analytical conditions. Moreover, as a proof of its relevance for novel sterol identification, MS/MS-related information was successfully applied to the identification of a positional isomer (Δ⁷) of zymosterol in baker's yeast extract, along with typical fungal major sterols. <b>Conclusions</b>: The comprehensive archive of sterol/stanol fragmentations obtained by APCI-HCD-MS/MS might prove very useful for the future characterization of novel sterol/stanol species in complex matrices.