Tandem mass spectrometric characterization of acetylated polyhydroxy hellebosaponins, the principal steroid saponins in Helleborus niger L. roots(#).

Abstract

Isolation and extensive nuclear magnetic resonance (NMR) analyses revealed polyhydroxy steroid saponins to be characteristic constituents in Helleborus niger L. roots. A comprehensive study including various multi-stage mass spectrometry (MS(n) ) experiments provided first solid chromatographic and mass spectrometric information facilitating future analysis and structural assessment of polyhydroxy saponins by LC/MS(n) techniques without isolation and NMR analyses. The polyhydroxy saponins were analyzed by direct syringe injection or chromatographically separated on a capillary high-performance liquid chromatography (HPLC) system coupled to an electrospray ionization (ESI) source. MS(n) spectra were recorded on an ion trap mass spectrometer including up to four fragmentation stages (LC/ESI-MS/MS). Additionally, high-resolution mass spectra were recorded on an Orbitrap Fourier transform (FT) mass spectrometer equipped with a nanospray-ESI interface. The polyhydroxy hellebosaponins A and D were discovered to be significant constituents from H. niger roots. Extensive study of their MS(n) data revealed that they readily fragmented in the positive ion mode providing diagnostic fragments for elucidation of the steroidal character and number of OH groups. The negative ion mode yielded valuable information on the [M-H](-) ion, number and location of acetyl groups and sugar units. Additionally, fragmentation pathways for positive and negative ion modes were proposed. These results not only extend the knowledge about H. niger saponins, but also provide a facilitated approach to the analysis of polyhydroxy saponins by LC/MS(n) without prior isolation and extensive NMR identification. Additionally, proposed fragmentation pathways for positive and negative ionization modes provide a solid complementary database for further, more detailed MS(n) studies.