Use of Digitoxin and Digoxin as Internal Standards in HPLC Analysis of Triterpene Saponin-Containing Extracts

Introduction - Saponins are widely distributed complex plant glycosides possessing a variety of structure-dependent bioactivities. Quantitation of individual saponins is difficult due to lack of available standards, mainly as a consequence of purification difficulties. Determination of total saponin content can be problematic, often relying on non-specific methods based on butanol solubility, haemolytic activity or formation of coloured derivatives. Objective - To develop a general quantitative method based on the use of the readily available cardenolides, digitoxin (1) and digoxin (2), as internal standards in an HPLC-PAD-based analysis. Methodology - The cardenolides were run at a variety of concentrations to establish linearity and reproducibility of detector response and then evaluated as internal standards for quantitation of triterpene saponins in several plant-derived extracts by HPLC-PAD. Mixtures of saponins, largely freed from other extractables, were obtained by fractionation of total extracts on solid phase extraction columns (SPE) employing a water-methanol gradient and used for construction of calibration curves. Saponin identification and structural information was obtained via a single quadrupole mass detector using ellectrospray ionisation in negative ion mode (ESI-). Results - Saponin contents in six samples from five species were determined and compared with literature results and a gravimetric method based on butanol-water partitioning. Results were generally consistent with literature reports and superior to gravimetric butanol-water partitioning. Conclusion - Digitoxin and digoxin are useful as internal standards in HPLC estimation of saponin content. Saponins from different species having similar structures and molecular weights afford similar calibration curves. Copyright (C) 2008 John Wiley & Sons, Ltd.