Characterization of saponins in peas (Pisum sativum L.) by HPTLC coupled to mass spectrometry and a hemolysis assay

• A HPTLC method for the reliable estimation of pea saponins has been developed.
• Hyphenation of HPTLC with MS is applicable for saponin identification.
• Saponin concentration varies depending on the cultivar.
• Evidence for the temperature dependent decrease of DDMP saponin was given.
• Pea saponins have no hemolytic activity.

Peas (Pisum sativum L.) contain two kinds of monodesmosidic, triterpene saponins, structurally comparable to those from soybeans — saponin B (soyasaponin I) and DDMP saponin (soyasaponin βg).A high-performance thin-layer chromatography (HPTLC) method for the fast and reliable estimation of saponins in selected pea cultivars has been developed. Identification of the substances was achieved by coupling HPTLC directly to electrospray ionization mass spectrometry (HPTLC-ESI-MS). Quantitative assessment of saponin B and DDMP saponin was carried out densitometrically after post-chromatographic derivatization with p-anisaldehyde sulphuric acid. The thermal stability of DDMP saponin was investigated at 50 °C and 60 °C with a time period of 24 h. Additionally, particular attention was on the hemolytic activity of the saponins analyzed directly on blood-gelatin covered TLC plates.The presence of DDMP saponin and saponin B in the pea samples was proven by HPTLC-ESI-MS and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Their concentration varied depending on the cultivar, with saponin B being always the more abundant compound, predominantly located in the hulls. The temperature dependent decrease of DDMP saponin concentration was more pronounced at 60 °C than at 50 °C. When treating peas for 24 h, nearly all of the DDMP conjugate was converted to saponin B whereas the total amount of saponins was more or less constant. Neither pea saponins nor those from soybeans possess hemolytic activity. In sum, the results are consistent with previous reports and gave evidence that HPTLC is an adequate methodology facilitating a fast and simultaneous analysis of numerous legume and further samples.