Abstract: This study aimed to develop a high performance liquid chromatography (HPLC) method for the simultaneous determination of the contents of five nucleoside (uridine, inosine, guanosine, adenosine and cordycepin) in cultured Cordyceps militaris. An ultrasonic-assisted ionic liquid microextraction procedure was proposed using [C4MIM]PF6-20% methanol as extraction solvent for sample pretreatment. The method was performed on an Inert Sustain C18 column (4.6 mm × 150 mm, 5 μm) with methanol-0.02 mol/L KH2PO4 solution as mobile phase at a flow rate of 0.6 mL/min by gradient elution. The column temperature was 30 ℃ and the detection wavelength was set at 254 nm. The highest extraction yields of five nucleosides were obtained by using 0.7 mol/L [C4MIM]PF6-20% methanol solution as extraction solvent at a solid-to-liquid ratio of 1:50 (g/L), pulverizing samples to pass through a 50-mesh sieve, ultrasonic irradiation for 50 min, and centrifugation at 3 000 r/min. A good linearity was observed in the range of 0.568?3.408, 0.284?1.704, 0.264?1.584, 0.232?1.392 and 1.672?10.032 mg/mL for uridine, inosine, guanosine, adenosine and cordycepin, respectively, with correlation coefficients above 0.999 8. The average recoveries were in the range of 97.6%?101.5%, with relative standard deviation (RSDs) ranging between 1.43% and 1.97%. The method was rapid, simple and sensitive, and it could be applied for rapid analysis of five nucleosides in cultured C. militaris.

Key words: cultured Cordyceps militaris, ionic liquid, high performance liquid chromatography (HPLC), nucleoside constituents