Separation of bacoside A3 and bacopaside II, major triterpenoid saponins in Bacopa monnieri, by HPTLC and SFC. Application of SFC in implementation of uniform design for herbal drug standardization, with thermodynamic study

Development, optimization, and validation of new analytical methods for standardization of bacoside A3 and bacopaside II, the major triterpenoid saponins present in Bacopa monnieri extract, are needed to improve the quality assurance of derived extracts and phytomedicines. Two chromatographic methods are described for evaluation of the quality of Bacopa monnieri extract and its commercial formulations. The first is reversed-phase high-performance thin-layer chromatography (RP-HPTLC), the second is packed column supercritical-fluid chromatography with photodiode- array detection (PC–SFC–DAD). SFC conditions were optimized by uniform design. The effect of temperature on the separation of the saponins was studied in detail. The Van’t Hoff plots for retention and selectivity were found to be linear. To obtain a better understanding of the different separations, the temperature dependence was studied to determine the thermodynamic data ΔH°, ΔS°, Δ ΔH° and Δ ΔS°. These data revealed that separation of bacoside A3 was enthalpically favoured in the range of temperatures investigated whereas entropy-controlled separation was observed for bacopaside II. Both methods were validated for precision, robustness, recovery, and limits of detection and quantitation. Analysis of variance (ANOVA) and Student’s t-test were used to correlate results from quantitative determination of the markers by RP-HPTLC and PC-SFC–DAD.