High-performance countercurrent chromatography fractionation of epimeric pairs intermedine/lycopsamine and amabiline/supinine by an off-line electrospray mass spectrometry injection profiling of the roots of Lappula squarrosa

Abstract The toxicity of pyrrolizidine alkaloids (PAs) and how they endanger the health of livestock and humans have been known for decades. However, quantitative measurements of these toxic PAs in food sources and herbal products are still hindered by poor availability of some authenticated reference standards. In this study, a PA fortified extract from a lyophilized Lappula squarrosa root sample (564 g) was separated using high-performance countercurrent chromatography (HPCCC) with the solvent system n-hexane − n-butanol − 1% aqueous ammonia (1:1:2, v/v/v). The HPCCC fractions were initially monitored by an off-line electrospray ionization mass-spectrometry (ESI-MS), and the targeted alkaloid fractions were subsequently elucidated by GC-EI-MS, ESI-MS/MS injection profiling, coupled with 1D/2D-NMR analysis. This approach led to accurate fractionation of some L. squarrosa PAs, while high recovery rates were reached since adsorptive losses of sample is neglected as countercurrent chromatography is an all-liquid chromatographic technique.