Full-spectrum HPLC fingerprint with binary evaluation system and chemometrics for herbal medicine quality-efficacy...

Traditional chromatographic fingerprints for multi-herb preparations often collapse a diode-array detector (DAD) λ-t cube to one or two wavelengths, masking weak- or non-UV components and limiting their value for stringent quality control. Seeking a more comprehensive yet routine friendly approach, we developed a full-spectrum time-domain integral accumulation (FTIA) strategy that numerically integrates each voxel of the DAD cube and fuses it with evaporative light-scattering detector traces to generate a single, information-rich fingerprint. 32 batches of Zhenqi Fuzheng preparation (ZQFZP) sourced from three manufacturers were profiled, and a binary evaluation system (BES) was designed to separate pattern similarity (Sₘ) from quantitative agreement (Pₘ), overcoming the equal-weight bias of cosine metrics. The method showed excellent analytical performance and, when coupled with principal component analysis, resolved clear inter-manufacturer clusters that the conventional single-wavelength fingerprint failed to distinguish. BES quantified these differences: one supplier produced batches with ten-fold deficits in several phenylethanoid glycosides (Pₘ 3-21 %), whereas another yielded systematic enrichment (Pₘ>120 %). Partial least squares regression attributed 98 % of Pₘ variance to four orthogonal markers (salidroside, specnuezhenide, ononin and formononetin). A marker-rich batch restored spleen and thymus indices and normalized serum IgG, interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) to 85-95 % of baseline in a cyclophosphamide-induced immunosuppressed mouse model, supporting a direct chemistry to efficacy link. These findings demonstrated that FTIA coupled with BES could deliver a regulator friendly, high resolution quality assessment strategy that unites exhaustive chemical coverage with biologically relevant metrics, offering a transferable method for the consistent production of complex natural medicines.