Establishment of an integrated profiling-fingerprinting-chemometrics strategy for quality control and mechanistic investigation of Fuhuo Capsule.

Fuhuo Capsule is widely prescribed for the treatment of osteonecrosis of the femoral head; however, its chemical composition and reliable quality markers have not been comprehensively elucidated. In this work, comprehensive chemical profiling of Fuhuo Capsule was conducted using ultra-high-performance liquid chromatography in combination with quadrupole Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap-MS), enabling detailed identification of its principal constituents. On this basis, an integrated quality evaluation strategy incorporating high-performance liquid chromatography (HPLC) fingerprinting, chemometric analysis, and multi-component quantification was established. In total, 183 constituents were provisionally characterized based on the analytical data obtained, and 38 common chromatographic peaks were consistently detected across 11 batches. Ten characteristic constituents were further confirmed as representative quality markers. Multivariate statistical approaches such as hierarchical clustering (HCA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to analyze the acquired data, effectively discriminated batch-to-batch variability and collectively identified 11 differential chemical markers. An HPLC-based quantitation assay was set up afterwards for the simultaneous quantitation of ten selected marker components, which exhibited satisfactory linearity, precision, repeatability, and stability. Furthermore, network pharmacology analysis indicated that these key constituents exert their therapeutic effects by interacting with critical targets such as protein kinase B (AKT1), signal transducer and activator of transcription 3 (STAT3), and interleukin-1 beta (IL-1β), and by modulating pathways including the advanced glycation end product- receptor for advanced glycation end product (AGE-RAGE) and the mitogen-activated protein kinase (MAPK) signaling pathways. Overall, this integrated strategy provides a systematic framework linking chemical profiling, fingerprinting, chemometrics, and quantification with mechanistic insights through network pharmacology and molecular modeling, thereby enhancing both the quality control and mechanistic understanding of Fuhuo Capsule.