High-performance counter-current chromatography purification and off-line mass spectrometry monitoring and identification of pyrrolizidine alkaloid markers of tropical Ghanaian honey.

The nutritional and medicinal properties of honey have been well-documented. However, honey has occasionally been contaminated with hepatotoxic pyrrolizidine alkaloids as a result of bees foraging on the flowers of pyrrolizidine alkaloid plants. This study establishes a simple and rapid method to determine the marker pyrrolizidine alkaloids in honey using high-performance counter-current chromatography and an off-line electrospray ionization-tandem mass spectrometry, in order to identify the botanical sources responsible for the contamination. The honey sample was initially liquid-liquid extracted (sulfuric acid/hexane, 2:3, v/v) to enrich the pyrrolizidine alkaloids and subsequently purified by a semi-preparative high-performance counter-current chromatography using a solvent system, hexane/butanol/1% aqueous ammonia, 1:1:2, v/v, based on partition coefficient measurements of the target alkaloids. The recovered fractions were profiled by injecting them sequentially into an off-line electrospray ionization-tandem mass spectrometry device to monitor the preparative molecular weight based on elution and extrusion modes. The monitored lycopsamine-type pyrrolizidine alkaloids and their N-oxides (m/z 300, 316; lycopsamine, intermedine, rinderine, and echinatine) were used as the phytochemical markers to identify plants like Chromolaena odorata, Ageratum spp., or Heliotropium spp. to be responsible for the pyrrolizidine alkaloid contamination. Identification of these pyrrolizidine alkaloid plants could guide beekeepers in locating their beehives in order to minimize their potential liver damaging effects.