Separation and characterization of bufadienolides in toad skin using two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography coupled with mass spectrometry

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• 作者：Yun Zhang^a ; ¹ ; Hongli Jin^a ; ¹ ; Xiaolong Li^a ; Jianqiang Zhao^b ; Xiujie Guo^a ; Jixia Wang^a ; Zhimou Guo^a ; Xiuli Zhang^a ; Yanduo Tao^b ; Yanfang Liu^a ; ^{liuyanfang@dicp.ac.cn" class="auth_mail" title="E-mail the corresponding author} ; Deliang Chen^c ; ^{deliangchen@sohu.com" class="auth_mail" title="E-mail the corresponding author} ; Xinmiao Liang^a
• 关键词：Bufadienolides ; Normal-phase liquid chromatography ; Two-dimensional liquid chromatography ; MS/MS ; Toad skin
• 刊名：Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2016
• 出版时间：15 July 2016
• 年：2016
• 卷：1026
• 期：Complete
• 页码：67-74
• 全文大小：1407 K

文摘

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.