Abstract: A method for the simultaneous determination of 7 nucleosides in Asterias rollestoni was developed using reversed-phase high performance liquid chromatography (RP-HPLC). Analytes were extracted by ultrasonic-assisted extraction and separated on two different C18 columns, which were connected in series, under the gradient elution with the mobile phases of methanol and 0.2% (v/v) acetic acid/water at room temperature. The chromatographic conditions were as follows: flow rate, 0.8 mL/min; detection wavelength, 260 nm; injection volume, 20 μL. Under the optimized conditions, good linear relationships between the values of mass concentrations and the peak areas of hypoxanthine, uridine, xanthine, thymine, inosine, guanosine and thymidine were observed in the ranges of 0.65-40, 0.80-40, 0.80-40, 1.15-40, 0.80-40, 0.50-40, and 0.65-40 mg/L, respectively. The relative standard deviations were around 0.72%-3.23% and the recoveries were around 90.00%-105.00%. The results showed that the developed method is sensitive, accurate and reproducible. It is suitable for the analysis of nucleosides in Asterias rollestoni with high recoveries and it is expected to be used for the quality control and evaluation of Asterias rollestoni.

Key words: Asterias rollestoni, nucleosides, reversed-phase high performance liquid chromatography （RP-HPLC）