Comprehensive UHPLC–Orbitrap–MS Profiling of Phloroglucinol α-Pyrones in Helichrysum italicum (Roth) G. Don (Asteraceae)

Helichrysum italicum is a Mediterranean medicinal plant whose phloroglucinol (PG) α-pyrones constitute a structurally distinctive and pharmacologically relevant metabolite class, yet no systematic high-resolution mass spectrometric (HRMS) study has comprehensively characterized them. In this study, the acetone extract of inflorescences from Helichrysum italicum (Roth) G. Don was profiled by UHPLC–Orbitrap–MS in negative ion mode using full-scan and data-dependent MS/MS acquisition; exact-mass elemental composition assignment and HCD fragmentation analysis were combined with unsupervised clustering of fragment-ion and neutral-loss intensity matrices, validated against a manually curated annotation table using the adjusted Rand index (ARI) and cluster purity. From a total of 59 annotated compounds, 23 corresponded to known PG-α-pyrones previously reported from Helichrysum and 36 represented structures not previously described from the genus. Diagnostic fragment ions (FIs) and neutral losses (NLs) were established for identifying specific substituents of the α-pyrone and PG moieties. Semi-quantification gave a total PG-α-pyrone content of 0.019% dry weight, dominated by arzanol. This study establishes a UHPLC–HRMS framework for profiling phloroglucinol α-pyrone derivatives and confirms H. italicum as a rich source of this class.