Accidental fatal poisoning by Nicotiana glauca: identification of anabasine by high performance liquid chromatography/photodiode array/mass spectrometry

A method, based on reversed phase high performance liquid chromatography (HPLC) was developed for the detection and quantification of anabasine, the toxic alkaloid of Nicotiana glauca, in forensic applications. A standard solid phase extraction (SPE) method was used for the extraction of anabasine from viscera, but was optimized for the extraction of this alkaloid from plant material. The careful selection of mobile phase components allowed the direct coupling of electron impact (EI) and Z spray mass selective detector (ZMD) of the HPLC. Under these conditions, anabasine was well separated from nicotine and could be detected on the PDA (limit of detection, LOD = 250 ng/ml), TMD (LOD = 10 microg/ml) and ZMD (LOD =1 ng/ml) detectors. Three geographically isolated N. glauca trees were analyzed for alkaloid content and it was found that both the leaves and the flowers contain anabasine. The optimized HPLC method was used to analyze two viscera samples (the stomach and contents of a mother and child who putatively died from food poisoning) and a flower exhibit. Anabasine was detected in both the viscera samples, supporting the finding that these fatalities were due to the ingestion of N. glauca accidentally collected with traditional spinach (marog). The alkaloid profile of the flower exhibit submitted with the viscera samples was similar to those obtained from flowers collected from three different N. glauca trees. The results show that anabasine and/or N. glauca poisoning can easily be confirmed using the forensic methodology described.