Journal of Chromatography A, 1036 (2004) 239–243 Short communication Simultaneous determination of ergosterol, nucleosides and their bases from natural and cultured Cordyceps by pressurised liquid extraction and high-performance liquid chromatography S.P. Li a,b,∗ , P. Li a , C.M. Lai a , Y.X. Gong a , Kelvin K.W. Kan a , Tina T.X. Dong c , Karl W.K. Tsim c , Y.T. Wang a a Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau, China b National Standard Laboraory for Traditional Chinese Medicines, Nanjing University of Traditional Chinese Medicines, Nanjing, China c Department of Biology and Biotechnology Research Institute, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China Received 29 December 2003; received in revised form 23 February 2004; accepted 27 February 2004 Abstract A simple method is described for the simultaneous determination of ergosterol, nucleosides and their bases in Cordyceps. The samples were extracted by using pressurised liquid extraction (PLE). The effects of experimental variables, such as solvent, temperature, static extraction time and cycles, on PLE efficiency have been studied. The results showed a strong influence of the solvent and temperature on extraction efficiency of PLE. The determination was achieved by high-performance liquid chromatography (HPLC) using a Zorbax NH 2 analytical column (250 × 4.6 mm i.d., 5 m) with diode-array detector (DAD). The automated preparation of the sample permits a very fast analysis which is an important goal for routine purpose. © 2004 Elsevier B.V. All rights reserved. Keywords: Cordyceps spp.; Pharmaceutical analysis; Ergosterol; Sterols; Nucleosides 1. Introduction The extraction step has often proved to be the bottle- neck of most analytical procedures, as it is one of the least evolved parts of the whole method. During the past few years, one of the most promising and recent sample prepa- ration techniques is pressurised liquid extraction (PLE; Dionex trade name ASE for accelerated solvent extraction), which offers the advantages of reducing solvent consump- tion and allowing for automated sample handling [1]. More recently, the distinct advantages of PLE, such as signif- icantly reduced extraction time and low solvent volume requirement, are being exploited in diverse areas, including biology, pharmaceuticals and foodstuffs [2]. An interesting and important new application area of PLE is in the extrac- tion of chemical constituents from plants or herbal materials [2–10]. ∗ Corresponding author. Tel.: +86-3974692; fax: 86-841358. E-mail address: spli@umac.mo (S.P. Li). Cordyceps, one of the well-known traditional Chinese medicines, is a composite consisting of the stroma of the fungus, Cordyceps sinensis (Berk.) Sacc. (Fam. Hypocre- aceae), parasitized on the larva of some species of insects (Fam. Hepialidae), and the dead caterpillar. Recent stud- ies have demonstrated its multiple pharmacological actions [11,12]. The natural Cordyceps (wild C. sinensis) is rare and expensive on the local market. Several mycelial strains, e.g. Pacilomyces hepiali and Hirsutella hepialid, isolated from natural C. sinensis have been manufactured in large quan- tity by fermentation technology [11]. At present, cultured Cordyceps mycelia are commonly sold as health food prod- ucts in South East Asia. Nucleosides and sterols are believed to be the active components in Cordyceps [11,13]. Several methods have been developed for the separation of nucleosides. The vast majority of these separations have been performed by using either reversed-phase high-performance liquid chro- matography (HPLC) with gradient elution [14–16] or using reversed-phase ion-pair chromatography [17]. Capillary electrophoresis [18] and capillary electrochromatography 0021-9673/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2004.02.080