Quality evaluation of Rhododendri Mollis Flos formula granules by chemical and dissolution fingerprinting using...

To overcome the limitation of conventional fingerprint profiling in identifying specific compounds, an integrated analytical strategy was developed for comprehensive quality evaluation of Rhododendri Mollis Flos Formula Granules (RMFFG). This strategy is structured as a stepwise, logically connected chain: from detection to identification to marker screening to functional validation. UPLC chemical fingerprints of 28 batches of RMFFG were established, all showing similarity indices above 0.90. However, to transform these anonymous peaks into chemically meaningful information and resolve peak alignment challenges between UPLC‑DAD and UPLC‑Q‑Exactive Orbitrap MS/MS, a "Dual Standards and Single Mass" method was implemented. This approach uses neohesperidin as an exogenous internal standard to predict retention times of unknown peaks via a calibrated formula, achieving deviations of only 0.04-0.20 min, while authenticated external standards confirm compound identities. Together, they eliminate the need for a full set of expensive or commercially unavailable reference standards. Using this method, 26 common peaks in the chemical fingerprint were unambiguously identified. Based on these identified peaks, chemometric analysis (HCA, PCA, OPLS‑DA) classified the 28 batches into two groups and screened 13 critical quality markers (VIP > 1). As a quantitative validation, fourteen bioactive components were quantified with concentration ranges (e.g., catechin 0.0432-0.0746%, quercitrin 0.1570-0.1942%). Furthermore, because a consistent chemical fingerprint does not guarantee consistent drug release, dissolution fingerprinting in three media (pH 1.2, pH 6.8, and aqueous) was performed, also yielding similarity indices above 0.90. The combination of chemical fingerprinting, Dual Standards and Single Mass identification, and dissolution fingerprinting provides a practical, cost‑effective, and industrially applicable quality control framework that ensures batch‑to‑batch consistency of RMFFG, supporting its future industrial scale‑up and clinical application.