An Optimized UHPLC‐Q Exactive MS/MS Separation and Chemometric Characterization of Polyphenols in the Aerial Parts and Rhizomes of <i>Rhodiola fastigiata</i> Using Ultrasound‐Assisted Deep Eutectic Solvent Extraction

ABSTRACT The Rhodiola genus has been extensively utilized in China to manage respiratory infections owing to its anti‐inflammatory, antioxidant, and immune‐regulating properties. Rhodiola fastigiata is commonly used as an adaptogen for relieving cough and clearing phlegm. Although the rhizomes of Rhodiola rosea and crenulata have been well studied, there are few studies reported on R. fastigiata , despite its frequent use as a substitute for Rhodiola crenulata . Moreover, comparative studies of the morphological parts of R. fastigiata remain scarce. Thus, this study aimed to establish a green extraction strategy, systematically identify polyphenol markers in R. fastigiata , and quantitatively compare differential metabolites between the aerial and rhizome parts. An optimized deep eutectic solvent system of choline chloride: Glycolic acid (1:3) yielded the highest total polyphenol content. Ultrahigh‐performance liquid chromatography coupled with a Q Exactive Orbitrap mass spectrometer operated in data‐dependent acquisition mode identified 50 polyphenolic compounds, predominantly flavonoids and phenolic acid derivatives. Subsequent UHPLC–tandem mass spectrometry integrated with multivariate chemometric analysis identified and quantified 15 differential metabolites that corresponded to bioactive compounds predicted by the network pharmacology. Kaempferol, myricetin, salidroside, quercetin, and their derivatives were identified as potential biomarkers of R. fastigiata . Notably, based on the abundance of the differential metabolites, the rhizomes had a higher abundance of metabolites. However, several characteristic markers, such as quercitrin, afzelin, and β‐glucogallin, were enriched in the aerial parts. This study demonstrates the applicability of deep eutectic solvents in polyphenol extraction and a robust high‐performance mass spectrometry characterization and differentiation of compounds in plant tissues. Thus, this work provides an analytical basis for the utilization of different morphological parts of R. fastigiata in phytochemical studies and pharmacological applications.