Library-assisted UHPLC-Q-TOF-MS/MS bioanalytical profiling of Qiling Hushen granules: In-vivo metabolic mapping and...

Qiling Hushen Granules (QLHSG), a multi-herb prescription widely used for diabetic kidney disease (DKD), lacks system-level evidence linking chemical features with in-vivo disposition. A library-assisted UHPLC-Q-TOF-MS/MS workflow was developed to integrate in-vitro phytochemical profiling with in-vivo prototype/metabolite elucidation and organ-level biodistribution. An in-house spectral library was constructed using reference standards, open databases and diagnostic-ion rules, enabling high-confidence annotation based on retention time, accurate mass and MS/MS fragmentation. In total, 166 in-vitro constituents were cataloged. In vivo, 78 prototype constituents were characterized. Metabolites were assigned by matrix (bile 46, feces 44, plasma 27, urine 156), delineating phase I/II biotransformation and elimination routes. Tissue mapping demonstrated kidney-biased accumulation of multiple prototypes and metabolites, indicating that renal targeting is consistent with the therapeutic context. Dominant exposure classes included iridoids and flavonoids. Representative bioactives were astragaloside IV, calycosin, paeoniflorin, secologanoside and sweroside. Network analysis combined with molecular docking connected absorbed constituents to DKD-relevant targets (e.g., TP53, EGFR, AKT1, STAT3, HIF1α), providing plausible mechanistic insights. Overall, this workflow establishes a reproducible pathway, from chemical profiling to systemic exposure and organ targeting in complex herbal formulations. For the first time, it provides system-level chemical evidence supporting the renoprotective effects of QLHSG.