title: Separation and characterization of bufadienolides in toad skin using two-dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography coupled with mass spectrometry.
abstract: Bufadienolides possess various bioactivities especially antitumor. Due to the high structural diversity, the separation of bufadienolides often suffers from coelution problem on conventional RP columns. In this work, an off-line two-dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography (2D-NPLC×RPLC) method was developed to separate and characterize bufadienolides in toad skin. Several RP and NP columns were evaluated with five reference bufadienlides. The XUnion C18 and XAmide columns exhibited superior chromatographic performances for bufadienlide separation, and were selected in RPLC and NPLC, respectively. RPLC was used in the second-dimension for the good compatibility with MS, while NPLC was adopted in the first-dimension. The orthogonality of the 2D-NPLC×RPLC system was investigated by the geometric approach using fifteen bufadienolide mixtures. The result was 49.6%, demonstrating reasonable orthogonality of this 2D-LC system. By combining the 2D-LC system with MS, 64 bufadienlides including 33 minor ones and 11 pairs of isomers in toad skin were identified. This off-line 2D-NPLC×RPLC allowed to solve the coelution problem of bufadienlides in one-dimension RPLC, and thus facilitated the identification significantly.
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Hongli
authors.initials: H
authors.lastname: Jin
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, PR China.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Xiujie
authors.initials: X
authors.lastname: Guo
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Jixia
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Zhimou
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Xiuli
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, PR China.
authors.forename: Yanduo
authors.initials: Y
authors.lastname: Tao
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: liuyanfang@dicp.ac.cn.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Wuhan Univ., Zhongnan Hosp., Dept. Internal Med., Div. Cardiol., Wuhan 430071, Hubei Province, PR China. Electronic address: deliangchen@sohu.com.
authors.forename: Deliang
authors.initials: D
authors.lastname: Chen
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Xinmiao
authors.initials: X
authors.lastname: Liang
mesh_terms: D000818:Animals; D002018:Bufanolides / Q000737:chemistry / Q000302:isolation & purification*; D002025:Bufonidae; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D002018:Bufanolides
keywords: Bufadienolides; MS/MS; Normal-phase liquid chromatography; Toad skin; Two-dimensional liquid chromatography