Abstract: Objective: To establish a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of hyperoside in Tibetan Acanthopanax leaves from 5 different species. Methods: Hyperoside was identified by liquid chromatography-mass spectrometry (LC-MS). Hyperoside in samples were extracted by refluxing with 60% ethanol for 105 min. The chromatographic separation was achieved on a Kromasil C18 column at 30 ℃ through gradient elution using methanol as mobile phase A and 0.5% phosphoric acid at a flow rate of 1 mL/min. The detection wavelength was set at 355 nm. Results: A calibration curve equation describing the relationship between hyperoside concentration (Y) and peak area (X) was established as follows: Y＝2 × 106X ＋ 1.94883 (r＝0.990, n＝6). The calibration curve was linear over the concentration range of 0.17－2.55. The average spike recovery rate of hyperoside was 100.31% with a RSD of 0.95% (n＝9). Conclusion: A simple, accurate and repeatable method to determine hyperoside in Tibetan Acanthopanax leaves has been developed. Tender leaves of Tibetan Acanthopanax from the same species grown in Tibetan and Qiang Autonomous Prefecture and Ganzi Tibetan Autonomous Prefecture, Sichuan Province were found to be much richer in hyperoside than old ones. Low-temperature dried tender leaves of A. leucorrhizus (Oliv.) Harms var. fulvescens Harms & Rehd and A. setchuenensis Harms ex Diels had approximately equal hyperoside levels followed by tender leaves of A. giraldii Harms; tender leaves of A. sessiliforus Rupr had the lowest hyperoside level. Different drying methods were ranked in descending order from the highest to the lowest hyperoside content in dried A. leucorrhizus (Oliv.) Harms var. fulvescens Harms & Rehd as follows: shade drying ＞ processing into tea ＞ oven drying ＞ blanching ≈ microwave drying ≈ sun drying >> salting ＞ 1-month frozen storage ≈ 1-month frozen storage after blanching.

Key words: Tibetan Acanthopanax, hyperoside, RP-HPLC, LC-MS, content