Configurational Isomer Differentiation Between 20( <i>S</i> )‐ and 20( <i>R</i> )‐Ginsenosides in Red Ginseng via Applying [Cu ^II ( <scp>l</scp> ‐Phe) ₂ −H] ⁺ to Post‐Column Infusion LC–MS/MS

ABSTRACT Introduction 20( R )‐ginsenosides are formed by Walden inversion during ginseng processing and usually exhibit almost identical MS/MS behaviors, yet display distinct pharmacological activities compared with 20( S )‐configurational isomers. It is thereby crucial for isomer differentiation to facilitate quality control. Objective To pursue a program enabling 20( R )‐ and 20( S )‐ginsenosides discrimination in complicated matrices using MS/MS, red ginseng, the processed form of ginseng, was deployed as a representative. Methods Five pairs of ginsenoside epimers were collected for method development. MS 1 and MS 2 spectra of every trimeric complex ion formed by complexation of ginsenoside, l ‐Phe, and Cu 2+ were recorded using direct infusion (DI)–MS/MS, and the abundance ratio between the two dimeric fragment ions was termed R . Moreover, energy‐resolved MS (ER‐MS) was deployed to monitor fragmentation trajectories of precursor and concerned fragment ions and to determine the R feature at optimal collision energy, denoted R max . To validate the applicability for differentiating epimers in complicated matrices, e.g., red ginseng extract, post‐column infusion (PCI) was introduced to configure PCI‐LC–ER‐MS. Results 20( R )‐ vs. 20( S )‐Rg3 as representatives, [Cuᴵᴵ( l ‐Phe) 2 (20( R )‐/20( S ))‐Rg3)−H] + complex ions were observed and R values of the dimeric fragment ions such as [Cuᴵᴵ( l ‐Phe)(20( R )‐/20( S ))‐Rg3)−H] + and [Cuᴵᴵ( l ‐Phe) 2 −H] + , exhibited differences. R max features obtained by DI–ER‐MS showed significant differences when comparing other 20( R )‐ and 20( S )‐configurational isomers, leading to a reliable way for epimer discrimination. The reproducibility of recognition capability on PCI‐LC–ER‐MS platform was justified and applied to epimer differentiation in red ginseng extract. Successes were reached for the differentiation of two pairs of epimers, 20( R )‐ and 20( S )‐Rg3, and 20( R )‐ and 20( S )‐Rh1. Conclusion In summary, the proposed strategy combined the complexation driven by [Cu II ( l ‐Phe) 2 −H] + with PCI‐LC–ER‐MS enables the differentiation of 20( R )‐ and 20( S )‐configurational ginsenoside isomers in complicated matrices.