Preparative Chiral Separation of the Pyrrolizidine Alkaloids Intermedine and Lycopsamine on Immobilized Amylose and Cellulose Based Stationary Phases

Pyrrolizidine alkaloids (PA) of Symphytum species (comfrey) have been of interest for many years due to their hepatotoxicity [1]. Many of the PAs in comfrey occur as isomeric mixtures, hence, their separation presents a challenge. In recent years, HPLC separation of many chiral compounds has been successful due to the development of new chiral stationary phases, especially carbohydrate based material. Chiral stationary phases of immobilized amylose and cellulose provide the advantages of using a wide range of solvents and modifiers in both reverse and normal phase conditions, thus serving as universal chiral stationary phases [2].

In this study, 1.0kg Symphytum uplandicum roots were extracted with methanol and PAs were reduced with zinc dust to convert their N-oxides to free bases. The extract was enriched on Chem elut cartridges (Varian Inc.) and fractionated on a silica gel column to obtain a racemic mixture of the PAs intermedine and lycopsamine. Separation of this mixture was attempted on semi-preparative chiralpak IA and IC columns (Chiral Technologies, Inc,) with PDA detection. The performance of each column was evaluated by comparing the capacity factor (K₁), enantioselectivity factor (α), and resolution (R_S), on each column under different chromatographic conditions. In the present case, Chiralpak IA provided the best separation. The two peaks were collected and their structures were determined by mass and NMR spectroscopy. References: [1] Prakash AS, et al. (1999) Mutation Research 443: 53–67. [2] Zhang T, et al. (2008) Journal of Chromatography A 1191: 214–222.