In vivo biotransformation and metabolic profiling of dimyricetin-yl-diselenide (DMS), a structurally defined organoselenium flavonoid, in rats after oral and subcutaneous administration by UHPLC-HRMS.

Dimyricetin-yl-diselenide (DMS) is a structurally defined organoselenium flavonoid composed of two myricetin-derived units linked via a diselenide (Se-Se) bond. Despite its potential as a drug candidate or functional supplement, its in vivo biotransformation and route-dependent metabolic profiles remain unclear. Here, we characterized the metabolic fate of DMS in rats after subcutaneous (40 mg/kg body weight, BW) or oral (1000 mg/kg BW) administration using UHPLC coupled with high-resolution mass spectrometry (UHPLC-HRMS). Pooled plasma (time points) and pooled urine, feces, and bile (collection intervals) were analyzed to comprehensively capture DMS-related components. Overall, the in vivo metabolic network converged on two hub intermediates: M318 (myricetin) and putative myricetin-SeH. The latter was not detected in any matrix but was extensively represented by downstream conjugates. After subcutaneous administration, 57 DMS-related metabolites were detected; 46 were annotated with proposed molecular formulas and biotransformation pathways, supporting 14 postulated pathways. Phase II metabolites derived from both myricetin and myricetin-SeH were observed, including methylation, glucuronidation, and sulfation, alone or in combination, yielding di-, tri-, and multi-conjugated derivatives. In plasma, DMS and M764e (a methylated di-glucuronidated myricetin-SeH) predominated (31.7% and 39.4% of total MS peak area, respectively). In feces, M318, M332e (methylated myricetin), and M412 (methylated myricetin-SeH) were major components (38.6%, 15.8%, and 30.5%, respectively). Bile and urine exhibited the broadest metabolite coverage; in bile, M602a (di-methylated and glucuronidated myricetin-SeH) and M670e (di-glucuronidated myricetin) accounted for 15.3% and 18.0%, respectively, while in urine, M602b and M588c (methylated and glucuronidated myricetin-SeH) each exceeded 10%. Notably, 11 unknown metabolites containing a myricetin-SeH moiety were detected exclusively in urine. In contrast, oral administration yielded a simplified profile with 17 metabolites, largely centered on M318 and its phase II metabolites; only one myricetin-SeH-related metabolite (M412') was detected at trace level in feces. Six hydroxylated positional isomers of M318 (M334a-f) were observed exclusively in feces after oral dosing. Feces were dominated by M318 (≥65% across all intervals) together with the parent compound DMS. In orally dosed rats, M318 was the only DMS-related component detected in plasma, di-glucuronidated myricetin (M670'a-c) was the only class detected in bile, and urine contained M318 and five methylated myricetin isomers (M332'a-e), with DMS present only at trace levels. In vitro co-incubation experiments in phosphate buffer and rat plasma further supported M318 as a key intermediate that can be readily and abundantly formed from DMS, and suggested that M318 may undergo further transformation into hydroxylated products under physiological-like conditions. In phosphate buffer, a trace methylated myricetin-SeH derivative was also detected at a very low level. Although it was considered more likely to be an extraction-related artifact than a true biotransformation product, its detection may still provide limited support for the possible occurrence of myricetin-SeH-derived metabolites. Overall, this work provides the first multi-matrix, route-dependent metabolic map of DMS in rats, informing future pharmacokinetic, efficacy, and safety evaluations.