A Dual‐Platform UHPLC‐MS Workflow for Bone Metabolomics Reveals BCAT2‐Associated Branched‐Chain Amino Acid Remodeling in Postmenopausal Osteoporosis

ABSTRACT Postmenopausal osteoporosis (PMOP) is characterized by progressive bone loss and microarchitectural deterioration. However, the metabolomic characterization of localized metabolic remodeling in bone tissue remains limited by its structural complexity and chemical heterogeneity. In the study, a bone‐oriented dual‐platform UHPLC‐MS analytical workflow was established, integrating untargeted metabolomics, targeted quantification, and network pharmacology‐assisted pathway prioritization to characterize metabolic remodeling in PMOP and its modulation by Gushudan (GSD). Using biphasic extraction in combination with complementary HILIC (ACQUITY UPLC Amide) and RP (BEH C18) UHPLC‐Q‐Orbitrap HRMS platforms, expanded coverage of polar and non‐polar metabolites in rat bone tissue was achieved. In ovariectomized rats, pronounced metabolic perturbations in bone were revealed by this workflow, together with a partial shift toward metabolic normalization following GSD treatment. A total of 33 differential metabolites were putatively annotated, with pathway analysis highlighting branched‐chain amino acid (BCAA) metabolism as a major perturbed pathway. Integration with network pharmacology prioritized branched‐chain amino acid transaminase 2 (BCAT2) as a candidate regulatory target. To validate this pathway, a targeted UHPLC‐MS/MS analysis for BCAAs and branched‐chain keto acids in bone tissue was developed and fully validated, demonstrating satisfactory selectivity, linearity, precision, accuracy, recovery, matrix effect, and stability. Quantitative analysis confirmed BCAA remodeling in osteoporotic bone and its partial normalization after GSD treatment. Western blotting further supported BCAT2 dysregulation in PMOP bone tissue and its reversal by GSD, while molecular docking and enzyme inhibition assays suggested that salvianolic acid D and naringenin may contribute to BCAT2‐related modulation. This study establishes an integrated analytical strategy for resolving localized metabolic alterations in bone tissue and identifies BCAT2‐associated BCAA remodeling as a candidate target modulated by GSD in PMOP.