**2. Results and Discussion**

We reported the isolation and purification of several saponins from the viscera of sea cucumber species, *H. lessoni*, using ethanolic extraction, followed by solvent partition, then HPCPC. The extraction and purification procedures and the mass spectrometry analyses were described in detail in our previous publications [3,12].

The appropriate HPCPC fractions were pooled, based on their similar Rf values when run on thin-layer chromatography (TLC), and concentrated to dryness. Sodium ions were introduced to the samples before conducting the MS analysis, ensuring all saponins observed in the positive ion mode were predominantly singly charged sodium adducts [M + Na]+; triterpene glycosides have a high affinity to alkali cations. The prominence of [M + Na]<sup>+</sup> also facilitated the analysis of saponins in mixtures or fractions. The saponin profile of each HPCPC fraction was then revealed by MALDI MS and ESI-MS [3,12]. MS2 analyses identified key diagnostic ions produced by cleavage of the glycosidic bond including oligosaccharide and monosaccharide fragments [3,12,26]. Other visible peaks and fragments detected corresponded to the loss of other neutral moieties such as CO2, H2O or CO2 coupled with H2O.
