Abstract: AimTo establish a simple method for simultaneous determination of ergosterol, nucleosides and their bases in Cordyceps by HPLC coupled with pressurized solvent extraction (PSE). MethodsThe extraction was performed by using PSE and the PSE condition was optimized by using orthogonal test, the contents of ergosterol, nucleosides and their bases in Cordyceps were determined by HPLC. ResultsThe optimized condition of PSE extraction for ergosterol, nucleosides and their bases in Cordyceps was obtained as follow: solvent, methanol; temperature, 160 ℃; static extraction time, 5 min; pressure, 10 MPa and one extraction cycle and one time. A ZORBAX NH₂ column (250 mm×4.6 mm ID, 5 μm) and a ZORBAX NH₂ guard column (12.5 mm×4.6 mm ID, 5 μm) were used for simultaneous determination of ergosterol, nucleosides and their bases. Solvents that constituted the mobile phase were A (acetonitrile) and B (10 mmol·L^-1 ammonium acetate in water). The elution conditions applied were: 0-5.0 min, linear gradient 0→15% B; 5.0-25.0 min, linear gradient 15%→20% B; 25.0-35.0 min, linear gradient 20%→40% B; 35.0-45.0 min, linear gradient 40%→80% B; 45.0-50.0 min, 80% B isocratic. The flow-rate was 0.6 mL·min^-1 and the injection volume was 20 μL. The system operated at 25 ℃. Ergosterol was monitored and quantified at 275 nm, nucleosides and their bases at 254 nm.ConclusionHigh performance liquid chromatography coupled with pressurized solvent extraction is a rapid and simple method for simultaneous determination of ergosterol, nucleosides and their bases in Cordyceps.