A multi-dimensional multi-mode multi-column preparative chromatography strategy for systematic purification of...

Natural products stand as a critical source in drug discovery, with alkaloids representing the most promising drug leads. This study introduces a multi-dimensional multi-mode multi-column (3 M) preparative chromatography strategy for the effective purification of alkaloids. Crude alkaloids derived from Menispermum dauricum DC (M. dauricum) were chosen over reference standards for method development. By integrating different separation modes, a variety of stationary phases, and diverse mobile phase conditions, excellent orthogonality (r² < 0.1) was achieved. Accordingly, the 3 M prep-chromatography was designed as follows: the first dimension employed a modified ion-exchange column for alkaloid enrichment; the second and third dimension used two reversed-phase columns under different pH conditions for orthogonal separation; the fourth-dimension addressed highly similar components utilizing mix-mode and unique stationary phases; and the final dimension enabled isolation of high-purity compounds by enhancing peak capacity while reducing sample loadability. Using the 3 M prep-chromatography strategy, 81 alkaloids were successfully purified from 13.7 kg of M. dauricum, including 37 novel compounds with 7 previously unreported scaffolds. Based on these results, it is predicted that 445 compounds, each weighing more than 2 mg, could be purified from 100 kg of M. dauricum, demonstrating the significant resolving power of the method. Further investigations of the 3 M prep-chromatography, based on 14 molecular descriptors ranging from charge properties to hydrophobicity, confirmed its rationality and effectiveness in the efficient purification of alkaloids.