Integrated chiral separation, recognition mechanism and enantioselective pharmacokinetic study of tetrahydrojatrorrhizine enantiomers by HPLC-MS/MS method

A robust HPLC method employing a polysaccharide-based chiral stationary phase was developed and optimized for the enantiomeric separation of tetrahydrojatrorrhizine. Investigations into the effects of chromatographic conditions on enantiomeric separation revealed that a specific mobile phase, comprising methanol and ammonium acetate solution, significantly enhanced enantioselectivity with a resolution of 1.89 at a lower column temperature. Thermodynamic analysis based on van’t Hoff plots was carried out, which demonstrated a clear enthalpy-driven potential for chiral recognition, suggesting binding interactions played the dominant role in enantiomeric separation. To elucidate molecular mechanisms of chiral recognition, the complementary docking simulation was also performed, identifying that the observed enantioselectivity originated from differences in the key interactions, including hydrogen bond, π-π stacking, and steric hindrance. Additionally, a chiral HPLC-MS/MS method was fully validated for the determination of tetrahydrojatrorrhizine enantiomers in rat plasma, and applied to a preliminary enantioselective pharmacokinetic study. The results showed significantly different pharmacokinetic profiles between the two enantiomers, highlighting that the area under the curve of S -(−)-tetrahydrojatrorrhizine was statistically higher than that of R -(+)-tetrahydrojatrorrhizine. The integrated work presented herein provided a reliable analytical method and mechanistic insights into chiral recognition of tetrahydrojatrorrhizine enantiomers, supporting the precise evaluation of enantiomeric behaviors in vivo .