Enhanced selectivity in RP-HPLC-UV separation of structurally similar pentacyclic triterpenes in Centella asiatica...

The separation of structurally similar pentacyclic triterpenes in Centella asiatica remains a significant analytical challenge because of the co-elution of isomeric glycosides and closely related aglycones. This study developed and validated a selective reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) method using β-cyclodextrin (β-CD) as a mobile-phase additive to enhance chromatographic selectivity. Method development was conducted in two sequential stages. First, chromatographic parameters, including the gradient profile, β-CD concentration, mobile-phase composition, and stationary phase, were systematically optimized using six reference standards: terminoloside, madecassoside, asiaticoside, terminolic acid, madecassic acid, and asiatic acid. The method was subsequently refined using authentic C. asiatica extracts to minimize matrix interference and improve peak purity. The optimized method employed a YMC-Pack ODS-A column with gradient elution using water/acetonitrile (80:20 and 50:50, v/v), containing 0.01% formic acid and 2 mM β-CD, and achieved baseline separation of all target analytes and adjacent matrix components with resolution (Rs) values ≥ 2. Molecular docking supported the formation of β-CD-analyte inclusion complexes, providing a mechanistic basis for the enhanced chromatographic selectivity. The method demonstrated excellent linearity (R² = 0.9987-0.9998), satisfactory precision (intra- and inter-day %RSD ≤ 1.79%), recoveries of 96.29-108.52%, and adequate sensitivity, meeting the ICH Q2(R2) validation criteria. The validated method was successfully applied to the quantitative determination of pentacyclic triterpenes in C. asiatica leaf samples collected from different geographical regions of Thailand. Overall, the proposed method provides a robust and reliable analytical approach for phytochemical profiling and quality control of C. asiatica.