Abstract
The Forensic Chemistry Laboratory of Johannesburg (FCL JHB) is tasked with the chemical analysis of a variety of samples to assist in determining the cause of death where unnatural cause is suspected. Some of the samples submitted to the laboratory have a herbal or muti connotation, but a large portion of these cases turn out to have no herbal components present as only pharmaceutical or agricultural products are detected in these samples. This study combined, for the first time, forensic investigation, chemistry and botany to create a unique platform needed for the identification of poisonous plants and their components in forensic exhibits and viscera. The research was focussed on the poisonous plants previously detected at the laboratory, as well as the requests received for the analysis of muti/toxic plant components. The selection of plants included Nicotiana glauca, Datura stramonium / Datura ferox, Callilepis laureola, Boophone disticha / Ammocharis coranica, Abrus precatorius, Ricinus communis, Nerium oleander / Thevetia peruviana and Bowiea volubilis. All these species are known to have caused fatalities, hence their choice.
Nicotiana glauca has been implicated in the deaths of at least 15 people since 2001. It was previously detected by GC-MS (EI) in plant exhibits, but could not be detected in a viscera matrix. A selective extraction method for alkaloids was used to extract botanical and viscera samples. Anabasine was successfully detected on the HPLC-MS (EI) system but this detection technique was not considered sensitive enough. A very sensitive HPLC-MS method was developed on the ZMD detector by using electrospray technology. This method outperformed both electron impact detectors (GC and HPLC) and could detect 1ng/ml anabasine with relative ease in full scan mode.
Datura stramonium and D. ferox have not been previously positively linked to any human poisoning or death due to exposure to botanically derived products at the FCL JHB. Atropine and scopolamine were successfully ionised in ESI positive mode and could be detected at 10 pg/ml and 100 pg/ml level respectively. The identities of the compounds were confirmed by characteristic ISCID fragmentation patterns. The developed method was successfully applied to a suspected heart attack case. The results proved conclusively that the deceased was given D. ferox seeds as part of his meal and an overdose of atropine and scopolamine contributed to his death.
Callilepis laureola is reputed to be one of the more commonly used medicinal plants in South Africa, and although its use has been indicated by the specific mention of a possible nephrotoxin and/or hepatotoxin as causative agent, it has not been detected in any of the forensic chemistry laboratories in South Africa. This was mainly due to the absence of a reliable method for the analysis of the main toxic component of C. laureola, atractyloside, by mass spectrometry. A sensitive and very selective HPLC-ESI-MS method was developed that could detect atractyloside, carboxyatractyloside and their monodesulfated analogues in botanical and viscera matrices. The method was successfully applied to a variety of forensic samples and proved that C. laureola may play an important role in herbal poisonings. In a selection of suspected herbal poisonings where the cause of poisoning was unknown, 30% of the samples tested positive for the presence of atractyloside, carboxyatractyloside or their monodesulfated analogues.
The bulbs of Boophone disticha are rich in isoquinoline alkaloids and some of the alkaloids were detected by GC-EI-MS and LC-EI-MS, but the detection of these alkaloids in viscera samples was not successful. A routine method used for the screening for drugs of abuse in forensic samples, were successfully used for the analysis of the bulb extracts of B. disticha and the bulb scales of A. coranica. The chromatographic profile of these two plants appeared very similar at a first glance, but a closer evaluation of the mass spectra highlighted significant differences between the two plants. Six alkaloids from B. disticha were isolated and characterised by LC-MS and NMR and these compounds were detected in suspected herbal poisoning cases. It has been shown that B. disticha is one of the commonly used plants to “clean the system” but frequently results in the death of the patient.
Abrus precatorius contains one of the most toxic compounds known to mankind, namely abrin that collectively refers to a group of glycoproteins. The seeds of A. precatorius also contain two indole alkaloids, abrine and hypaphorine. The two alkaloids were fractionated and characterised by LC-MS and NMR. Due to the fact that the instrumentation of the FCL JHB is not suited to the detection of proteins, an LC-ESI-MS method was developed for the detection of the two alkaloids in plant and viscera matrix as markers for A. precatorius. The presence of these two alkaloids was indicated on the TMD system (EI spectra) in a suspected herbal poisoning case. The LC-ESI-MS method was applied to the analysis of the samples and the absence of abrine and hypaphorine were proven in the samples.
Ricinus communis is similar to A. precatorius in that it also contains a group of extremely poisonous glycoproteins, collectively refered to as ricin. The analysis of R. communis seeds encountered the same problems as the analysis of A. precatorius seeds, and the analysis was again focused on the detection of the minor piperidine alkaloid ricinine. The LC-ESI-MS method developed for abrine was modified to detect ricinine and functioned well in botanical and viscera matrices. This method will enable the forensic analyst to detect ricinine in very low levels when the presence of ricinoleic acid in samples indicates the use of a R. communis-based product.
Nerium oleander is a common decorative garden plant that is used medicinally. The plant is rich in cardenolides with oleandrin the main compound. A reversed-phase chromatographic method with ESI mass spectral detection was developed to separate and detect 11 cardiac glycosides. The compounds were adequately separated to allow unambiguous identification, and displayed very stable cationisation with sodium. An extraction method was developed to extract the cardiac glycosides from the leaves of N. oleander and Thevetia peruviana and was also evaluated in a viscera matrix. The extraction method functioned well and extracted a variety of compounds that produced unique chromatographic fingerprints, allowing for the easy differentiation between the two plants. The method is ideally suited for the detection of oleandrin in high concentrations (full scan mode), low concentrations (selected masses) or trace levels (SIM analysis of ion clusters). The method is able to distinguish between extracts derived from N. oleander and T. peruviana and was able to detect and confirm neriifolin, odoroside and neritaloside in N. oleander leaf extracts. Analysis of forensic case exhibits were also successfully done with this method and performed well with liquid and solid matrices. With the new method oleandrin could be detected at trace levels in viscera samples that did not produce positive results in the past.
Bowiea volubilis is widely used as a medicinal plant, but is also an extremely toxic plant. It is freely available at traditional healer markets, and is one of the most highly traded plants on the Durban market. Despite the high usage of the plant, it has not been detected by any of the forensic laboratories in South Africa. Bovoside A, a bufadienolide, is reported to be the main cardiac glycoside in the bulb of B. volubilis. The cardiac glycoside method was successfully applied to the analysis of the bulb extract of B. volubilis and bovoside A was identified as the main bufadienolide present in the bulb. Bovoside A was fractionated and characterised by LC-MS. Four extracts of botanical origin could be successfully distinguished from each other by monitoring the main masses of bovoside A, oleandrin and thevetin A and thevetin B. These marker compounds were well separated from each other and made the identification of the botanical extracts quite easy, and the identity of each extract was confirmed by the mass spectrum of each peak.
Prof. F.R van Heerden