A guide of column selection for two-dimensional liquid chromatography method development of natural alkaloids

Natural products, especially alkaloids, are one of the most valuable, potential drug leads in drug discovery. As an efficient tool for complex samples, two-dimensional liquid chromatography (2D-LC) has become a powerful means of analysis and separation of natural alkaloids. Method development of 2D-LC is of great importance because it helps to enhance the selectivity, resolution and peak capacity of a separation system. However, due to the diversity of the nature and subclasses of natural alkaloids, peak tailing occurs frequently, making alkaloid separation complicated and time-consuming. To conquer such difficulties, we proposed a guide for column se-lection and combination in 2D-LC so as to improve the alkaloid separation. Based on a comprehensive evaluation of applicability and orthogonality of several columns, this guide would provide a simple but clear starting point for column selection of 2D-LC method development. The evaluation included seven columns to involve most separation mechanisms reported in alkaloid separation, and 49 natural alkaloid standards of various subclasses and natures. Detailed studies of peak shapes of every column were carried out as well, providing useful refer-ences to better understand the peak tailing issues of some analytes on specific columns. Subsequently, a 2D-LC method was developed using our guide to isolate an alkaloid sample from U. rhynchophylla, generating sym-metrical peaks and a high orthogonality of 80.3%. Further, this evaluation process would help to have a quick understanding when a new stationary phase is designed.