Analytical and semipreparative separation of 25 (R/S)‐spirostanol saponin diastereomers using supercritical fluid chromatography

Spirostanol saponins are a class of steroidal saponins with many pharmacological activities. The structural complexity of the spirostanol saponins presents a daunting challenge in separating their 25 R/S diastereomers. Using two CHIRALPAK IC columns coupled in series, six 25 (R/S)‐spirostanol saponin diastereomers from the Trigonella foenum‐graecum L. seed were successfully separated using supercritical fluid chromatography (SFC) for the first time. In addition, three 25 (R/S)‐spirostanol saponin diastereomers were isolated into their respective individual isomers. The structures of the isolated isomers were unambiguously confirmed by NMR analysis. The SFC method development strategy and its associated underlying principles presented in this paper are generally applicable. SFC is a viable addition to the natural product research toolbox, especially for stereoselective analysis and purification.