title: Separation of 25R/S-ergostane triterpenoids in the medicinal mushroom Antrodia camphorata using analytical supercritical-fluid chromatography.
abstract: Ergostanes are major bioactive constituents of the medicinal mushroom Antrodia camphorata. These tetracyclic triterpenoids usually occur as 25R/S epimeric pairs, which renders their chromatographic separation difficult. In this study, we used analytical supercritical-fluid chromatography (SFC) to separate seven pairs of 25R/S-ergostanes from A. camphorata. The (R)- and (S)-forms for each of the seven pairs could be well resolved (Rs>1.3) on a Chiralcel OJ-H column (4.6×250mm, 5μm, chiral), eluted by 10% MeOH in CO2 at 2mL/min with a back pressure of 120bar and a column temperature of 40°C. Particularly, this chiral-SFC method could rapidly and efficiently separate low-polarity epimers like antcin A and antcin B, which were very difficult for RP-HPLC. A 3-min preparative-scale method was established to purify (25S)- and (25R)-antcin A for the first time. However, OJ-H column suffered from peak overlapping of different pairs of ergostanes. We found that Princeton 2-ethylpyridine column (2-EP, 4.6×250mm, 3μm, achiral) could effectively separate different pairs, although the resolutions for 25-R/S forms of each epimeric pair were not as good as OJ-H column. Meanwhile, all the (25S)-forms showed stronger retentions than the corresponding (25R)-epimers on the 2-EP column. These results demonstrated different selectivity of chiral- and achiral-SFC in separating 25R/S-ergostane epimers. Aside from high separation efficiency, SFC also showed advantage over HPLC in short analysis time and low consumption of organic solvents. Finally, both OJ-H and 2-EP columns were used on analytical SFC to separate 25R/S-ergostanes in an extract of A. camphorata.
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Qiao
authors.affiliation: Agilent Technologies, 3 Wangjing North Road, Beijing 100102, China.
authors.forename: Rong
authors.initials: R
authors.lastname: An
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Ji
authors.affiliation: Agilent Technologies, 3 Wangjing North Road, Beijing 100102, China.
authors.forename: Lang
authors.initials: L
authors.lastname: Li
authors.affiliation: Institute of Biochemical Sciences and Technology, Chaoyang University of Technology, Taichung 41349, Taiwan.
authors.forename: Yew-min
authors.initials: YM
authors.lastname: Tzeng
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: De-an
authors.initials: DA
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. Electronic address: yemin@bjmu.edu.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Ye
mesh_terms: D055491:Antrodia / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D025924:Chromatography, Supercritical Fluid / Q000379:methods; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000302:isolation & purification; D048690:Fruiting Bodies, Fungal / Q000737:chemistry*; D013237:Stereoisomerism; D013256:Steroids / Q000302:isolation & purification; D014315:Triterpenes / Q000302:isolation & purification*
chemical_list: D013256:Steroids; D014315:Triterpenes; C561899:antcin A; C015190:ergostane; D004875:Ergosterol
keywords: 2-Ethylpyridine; 25R/S-Ergostane; 4-Methylbenzoate cellulose; Antrodia camphorata; Supercritical-fluid chromatography; Triterpenoid