Comprehensive metabolic profiling of rupestonic acid following intranasal administration in rats: Insights from in vitro and in vivo studies using UHPLC-Q/TOF-MS and UHPLC-MS/MS.

Rupestonic acid, a bioactive sesquiterpenoid from Artemisia rupestris L., has demonstrated notable anti-inflammatory and anti-allergic effects. However, the metabolic fate of rupestonic acid following intranasal administration remains unknown. Therefore, the present study aimed to systematically characterize the in vitro and in vivo metabolism of rupestonic acid after intranasal administration and to evaluate its metabolic stability. An integrated analytical strategy was employed to systematically investigate the in vitro and in vivo metabolism of rupestonic acid. A UHPLC-Q/TOF-MS method was established to identify metabolites in rat liver microsomes, plasma, bile, urine, and feces, and their structures were tentatively characterized based on fragmentation patterns. The results revealed that rupestonic acid undergoes multiple biotransformation pathways, including hydroxylation, oxidation, hydrogenation, and glucuronidation, forming a relatively complex metabolic network involving both phase I and phase II reactions. In addition, a validated UHPLC-MS/MS method was developed for the quantitative determination of rupestonic acid in rat liver microsomal incubation samples, meeting the requirements of the U.S. Food and Drug Administration. Metabolic stability studies demonstrated that rupestonic acid exhibits a low metabolic rate in rat liver microsomes, with a relatively long half-life and low intrinsic clearance, indicating favorable metabolic stability. These findings provide new insights into the disposition characteristics of rupestonic acid after intranasal administration and offer a foundation for the future development and pharmacological evaluation of intranasal formulations.