Separation and measurement of plant alkaloid enantiomers by RP‐HPLC analysis of their Fmoc‐Alanine analogs

Abstract

Ammodendrine (1), anabasine (2) and coniine (3) can cause congenital malformations in livestock. They appear naturally in both enantiomeric forms, and can cause variable physiological responses. A method to measure the enantiomeric ratio of these natural toxins is needed. To develop a simple and economical method in order to determine the enantiomeric ratios of piperidine and pyrrolidine alkaloids in small samples of plant material. Mixtures of isolated or purified plant alkaloids were converted to their Fmoc-L-Ala-alkaloid analogues forming diastereomeric mixtures, which were then analysed by high pressure liquid chromatography (HPLC) with mass spectrometry (MS) and ultraviolet (UV) detection to determine enantiomeric ratios. The diastereomeric analogs for ammodendrine, anabasine and nornicotine could be separated and the enantiomeric ratios determined. The Fmoc-L-Ala-coniine analogue was not resolved under the HPLC conditions studied. The enantiomeric ratios of the selected plant alkaloids were measured and found to differ between both location within a species and location between species. A low-cost HPLC method to analyse the enantiomeric ratio of plant alkaloids containing primary or secondary amine nitrogens via conversion to their respective diastereomeric analogues has been developed.