Determination of pyrrolizidine alkaloids in Senecio species by liquid chromatography/thermospray-mass spectrometry and liquid chromatography/nuclear magnetic resonance spectroscopy

A new method using direct on-line coupling between HPLC, MS and H-1-NMR was developed for the detection and identification of pyrrolizidine alkaloids (PAs) in crude extracts of Senecio species. The PAs present in the extracts were separated on a C-18 reversed-phase column with an alkaline acetonitrile-water gradient. Molecular weight information of each peak was obtained by LC/MS and specific fragments were recorded by complementary MS/MS experiments. In order to distinguish isomeric structures of PAs, complementary LC/H-1-NMR analyses were performed in both on-flow and stop-flow modes with alkaline acetonitrile-D2O and methanol-D2O as mobile phases. This approach led to the identification of the known PAs of S. vulgaris; retrorsine (1), seneciphylline (2), and senecionine (3). The method was also applied for the analysis of African Senecio species: S. mariettae and S. venosus. Retrorsine was identified as the main PA in both species. Detection of the PAs in small amounts was achieved by LC/MS. Higher amounts of extracts (mg) had to be injected to obtain good quality on-flow LC/H-1-NMR.