Configurational Isomer Differentiation Between 20(S)- and 20(R)-Ginsenosides in Red Ginseng via Applying...

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.

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.

Five pairs of ginsenoside epimers were collected for method development. MS¹ and MS² spectra of every trimeric complex ion formed by complexation of ginsenoside, l-Phe, and Cu²⁺ 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.

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.

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.