Diagnostic Ion-Guided Isolation and Characterization of Trace Periplocin-Derived Cardenolide Metabolites from Rat Urine

Cardiac glycosides from Periplocae Cortex are bioactive constituents, but isolating and structurally confirming their trace in vivo metabolites remains challenging because authentic standards are often unavailable. We developed a steroid-core fragment-guided strategy based on the diagnostic ion at m/z 355.2270 that integrates metabolite screening, targeted purification, and structural characterization. Periplocin-related analytes were detected in rat urine and plasma after oral periplocin administration using HPLC-QQQ-MS/MS and UPLC-Q-TOF-MS/MS. Guided by the diagnostic ion, 13 L of rat urine was fractionated using macroporous resin, Sephadex LH-20, ODS chromatography, and preparative HPLC. Two trace metabolites were isolated and identified as gomphogenin (M1, 4 mg) and 17α-asclepioside (M2, 8 mg) by HR-MS and NMR spectroscopy. Periplocin, periplogenin, and periplocymarin were confirmed using reference standards. Structural comparison supported a putative transformation pathway involving deglycosylation and steroid-core modification. In a hypoxia/reoxygenation-injured H9c2 cell model, M1 and M2 increased cell viability at 50 μM, supporting further evaluation using complementary cellular endpoints. This diagnostic-ion-guided workflow enables the targeted isolation of trace cardenolide metabolites from biological matrices and provides reference compounds for future pharmacokinetic and pharmacological studies.