In-depth characterization of quinolizidine alkaloids via highly efficient enrichment and multi-stage mass spectrometry

The identification of new natural products is a critical step in the discovery of lead compounds, and is a prerequisite for the development of novel pharmaceuticals. Traditional Chinese medicines (TCMs) are renowned for their diverse pharmacological activities, and represent a valuable source of novel chemicals. The quinolizidine alkaloids are abundant in plants of the Fabaceae family and exhibit significant bioactivities, including anticancer and anti-inflammatory effects. However, the structural diversity of quinolizidine alkaloids and the chemical complexity associated with TCMs pose great challenges in the discovery of novel quinolizidine alkaloids. In the present study, Sophora flavescens Ait. was used as a model to establish a comprehensive strategy for characterizing quinolizidine alkaloids. A simple and selective method was developed using an FC8HL column for the efficient enrichment of polar quinolizidine alkaloids. Through the integration of high-resolution multi-stage mass spectrometry and feature-based molecular networking, the enriched alkaloids were thoroughly characterized, including matrine-type, cystine-type, aloperine-type, anagyrine-type, lupinine-type, and dimeric species. A total of 186 quinolizidine alkaloids were identified, including 131 newly discovered compounds. A series of novel substituents was also identified for the first time. The findings of this study not only deepen our understanding of the structural diversity of quinolizidine alkaloids, but they also offer a novel research strategy for the comprehensive characterization of quinolizidine alkaloids in other plants, potentially facilitating the discovery of new drug candidates.