Validated HPLC Method for Simultaneous Quantification of Six Triterpenes with Isomer Separation in Commercial...

Centella asiatica contains pharmacologically important pentacyclic triterpenes that are commonly used as chemical markers for quality evaluation of herbal preparations. However, accurate quantification remains challenging because some analytes are structurally related and chromatographically difficult to resolve. In this study, a validated high-performance liquid chromatography (HPLC) method was developed for the simultaneous separation and quantification of six major triterpenes in C. asiatica products, namely, asiaticoside B (ASB), madecassoside (MS), asiaticoside (AS), terminolic acid (TA), madecassic acid (MA), and asiatic acid (AA). Because β-cyclodextrin (β-CD) is inexpensive, it was thus selected as a mobile-phase modifier to improve discrimination of closely related triterpene isomers. Baseline separation of the two critical isomeric pairs, ASB/MS and TA/MA, was achieved within 15 min with a total chromatographic run time of 25 min on an ACE Excel C18 column (5 μm, 4.6 × 250 mm) using gradient elution with acetonitrile and 0.2% (v/v) phosphoric acid containing β-CD. Unlike previous CD-assisted HPLC methods that focused on the separation of five triterpenes or selected isomeric pairs, the present method enables, for the first time, simultaneous identification and quantification of six triterpenes, including both ASB/MS and TA/MA isomeric pairs. The method was fully validated according to ICH, Eurachem, and AOAC guidelines. The method showed good linearity, acceptable limits of detection and quantification, satisfactory repeatability and intermediate precision, suitable recovery, and good selectivity. The validated method was successfully applied to commercial C. asiatica products, demonstrating its suitability for routine quality control and phytochemical assessment of herbal raw materials and preparations. Analytical greenness assessment using the Analytical GREEnness (AGREE) metric gave a score of 0.50, indicating a moderate greenness profile, while holistic assessment using the Mosaic-White Analytical Chemistry (WAC) tool gave a Whiteness Index of 68.6, suggesting satisfactory overall method quality based on analytical reliability, environmental impact, and practical applicability. This method provides a practical and reliable approach for the simultaneous determination of six triterpenes in C. asiatica matrices.