Targeted HPLC and LC-MS profiling of anthraquinones: Linking to hypericin biosynthesis in Hypericum spp

Liquid chromatography with diode-array and mass spectrometric detection (HPLC-DAD, LC-MS) is essential for anthraquinone (AQ) analysis, particularly for glycosides lacking standards. While hypericins in Hypericum are well studied, AQ diversity and its relationship to hypericin biosynthesis remain insufficiently characterised. We developed an optimised workflow for profiling emodin- and skyrin-related AQs and their glycosides in Hypericum species. A liquid-liquid extraction enabled efficient recovery of aglycones and glycosides. HPLC-DAD was optimised for selective detection, ion-trap LC-MS supported structural elucidation, and acid hydrolysis confirmed glycosylation patterns. Analysis of eleven Hypericum species revealed O-pentosides and O-hexosides of skyrin and oxyskyrin, the latter reported here for the first time in the genus and restricted to hypericin-producing species. Correlation analyses showed coordinated accumulation of emodin, skyrin derivatives, and hypericins, consistent with associative relationships within the naphthodianthrone-related metabolic network. The approach improved analytical resolution and highlighted interspecific variation relevant to phytochemical and biosynthetic studies.