An integrated LC-HRMS workflow for anti-doping-relevant characterization of lapisteride metabolism in vivo and in...

Metabolic characterization is important for poorly characterized compounds, especially when controlled human administration studies are limited by ethical or safety concerns. Lapisteride is a 4-azasteroid-related compound with 5α-reductase inhibitory activity, but its metabolism and disposition remain unclear. Here, we established an integrated LC-HRMS-based workflow combining human liver microsomal incubation, CYP450 isoform verification, rat administration, multi-matrix sampling, LC-Q-TOF-MS metabolite identification, and urine/bile untargeted metabolomics. Seven lapisteride-related metabolites were tentatively identified. The main biotransformation reactions included mono-oxidation, O-demethylation, di-oxidation, side-chain oxidation to carboxylic acid, and glucuronidation. Diagnostic MS/MS fragments, especially the steroidal core ion at m/z 317 and characteristic C17 aryl side-chain ions, supported the proposed structures and indicated that the C17 aryl-containing side chain was a major metabolic region. CYP450 inhibition results suggested that CYP2C9 and CYP1A2 were the main isoforms involved in lapisteride oxidative metabolism, with CYP2D6 playing a possible secondary role. Multi-matrix concentration-time profiling showed distinct distribution and elimination patterns. Plasma and prostate mainly reflected early exposure, whereas bile and feces were more informative for hepatobiliary excretion and delayed elimination. Untargeted metabolomics identified 79 annotated metabolites in urine and 61 in bile. KEGG analysis showed significant enrichment of unsaturated fatty acid biosynthesis in urine, while primary bile acid biosynthesis showed a non-significant but biologically plausible trend in bile. Overall, this study provides systematic evidence for lapisteride metabolism and offers a practical strategy for metabolic characterization of compounds with limited metabolic information that are difficult to evaluate in controlled human studies.