Isomer Differentiation by UHPLC‐Q‐Exactive‐Orbitrap MS led to Enhanced Identification of Daphnane Diterpenoids in Daphne tangutica

ABSTRACT Introduction Liquid chromatography‐mass spectrometry (LC–MS) has enhanced the rapid, accurate analysis of complex plant extracts, eliminating the need for extensive isolation. Tandem mass spectrometry (MS/MS) further enhances this process by providing detailed structural information. However, differentiating structural isomers remains a challenge due to their minor spectral and structural differences. Objective This study aimed to extend the applicability of LC–MS/MS for the structural identification of daphnane diterpenoids, with a particular focus on distinguishing functional isomers. Methods LC–MS analyses were performed using an UHPLC‐Q‐Exactive‐Orbitrap MS. The MS conditions for distinguishing isomers were optimized using in‐source CID and HCD modes with reference compounds. A qualitative analysis was then conducted on the extract of Daphne tangutica . The chemical structures of the detected daphnane diterpenoids were estimated by analyzing the fragmentation patterns in both the mass spectra and product ion spectra. These identifications were further validated by isolation and comparison with an in‐house daphnane diterpenoid library. Results By optimizing MS conditions, especially in the negative ion mode, it was possible to accurately distinguish structural isomers such as yuanhuajine and gniditrin. Qualitative analysis of D. tangutica identified a total of 28 daphnanes, including seven previously unreported compounds. Furthermore, a novel geometric isomer of gniditrin was isolated by conducting isolation on the crude diterpenoid fraction. Conclusion This study demonstrated that LC–MS/MS analysis can effectively distinguish functional isomers of daphnane diterpenoids, thereby enhancing the identification of daphnanes in plant extracts and highlighting its potential as a powerful tool for phytochemical analysis.