title: Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla.
abstract: Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro-Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of ultra-high performance SFC (ultra-performance convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2methods were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and acetonitrile-0.2% diethylamine in CO2as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China.
authors.forename: Wenzhi
authors.initials: W
authors.lastname: Yang
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China.
authors.forename: Yibei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.
authors.forename: Huiqin
authors.initials: H
authors.lastname: Pan
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.
authors.forename: Changliang
authors.initials: C
authors.lastname: Yao
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China.
authors.forename: Jinjun
authors.initials: J
authors.lastname: Hou
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Yao
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China.
authors.forename: Luying
authors.initials: L
authors.lastname: Cai
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China.
authors.forename: Ruihong
authors.initials: R
authors.lastname: Feng
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China. Electronic address: wanyingwu@simm.ac.cn.
authors.forename: Wanying
authors.initials: W
authors.lastname: Wu
authors.affiliation: Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China.
authors.forename: Dean
authors.initials: D
authors.lastname: Guo
mesh_terms: D000470:Alkaloids / Q000032:analysis* / Q000737:chemistry; D025924:Chromatography, Supercritical Fluid / Q000379:methods*; D007211:Indoles / Q000032:analysis* / Q000737:chemistry; D000078183:Oxindoles; D032070:Uncaria
chemical_list: D000470:Alkaloids; D007211:Indoles; D000078183:Oxindoles; C022960:2-oxindole
keywords: Isomerization; Preparative scale supercritical fluid chromatography; Spiro oxindole alkaloid; Ultra-performance convergence chromatography; Uncaria macrophylla