Efficient purification of active bufadienolides by a class separation method based on hydrophilic solid-phase extraction and reversed-phase high performance liquid chromatography

• Two classes of bufadienolides were separated by HILIC-SPE.
• This prevented their co-elution in RP-HPLC.
• A total of 12 compounds from the two classes were purified by this method.

Traditional Chinese medicines (TCMs) have played a significant role in the process of discovering natural bioactive compounds, especially in anticancer therapeutics. However, the components of TCMs are complex mixtures with wide variation in polarity and content, which leads to inefficiency in the process of active compound discovery from TCMs. In this paper, the popular strategy of utilizing “pre-fractionated natural product libraries” has been improved by a new class separation approach to accelerate the process. As an example, the skin of Bufo bufo gargarizans Cantor, a well-known TCM, mainly contains two distinct bufadienolide classes: amino acid-conjugated bufadienolides (AACBs) and free form bufadienolides (AAUBs). We utilized hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE) to resolve the two types of bufadienolides, which co-eluted on C18 columns. By this strategy, twelve bufadienolides of the two types were purified via prep-HPLC from one active fraction, and eight of them were identified by 1H NMR and 13C NMR. These results indicated that the class separation method not only overcame the limited orthogonality in a 2D-RPLC × RPLC system but also accelerated the process of active compound discovery.

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