Analytical strategy to document horse fatalities related to Oenanthe crocata poisoning

Abstract

To report the analytical procedure that was developed and applied to identify oenanthotoxin (OT) and its metabolite 2,3-dihydro-oenanthotoxin (2,3DHOT) in biological and vegetal samples using both LC-DAD and LC-HRMS methods in two cases of equine fatal intoxication by Oenanthe crocata. : (case#1) a 7-year-old Arabian horse was found dead within one hour after the ingestion of «wild carrots», and was autopsied the following day. The gross and microscopic examination was unremarkable, except the presence of several root pieces in the animal's stomach. Sections of the same roots found on the site showed hexagonal subdivisions and a yellowish latex. These findings were consistent with Hemlock water-dropwort (Oenanthe crocata) and confirmed by an experienced botanist. (case#2) an 8-year-old pony was unexpectedly found dead and autopsied two days later. The body conservation was poor; the only significant finding was the presence of several root pieces of undetermined origin in the stomach. Autopsy samples (root pieces found in the stomach, stomach content, liver, kidney, and spleen of case#1; stomach content of case#2) were stored at -20 °C before analysis, while the on-site roots (case#1) were stored at room temperature. After grinding organ samples, 1 to 10 g were extracted using methanol (5 mL), vortex mixed for 30 seconds, and kept at 4 °C for 2 hours. These methanolic suspensions were centrifuged firstly at 2500 g for 10 min, then the supernatant was secondly centrifugated at 8000 g for 14 min. In order to enhance detection sensitivity, one mL of each supernatant from the liver, kidney, and spleen samples was dried under nitrogen and then each extract was resuspended with 100 μL of methanol. Extracts were analysed using two analytical methods (LC-DAD and LC-HRMS) both using the same ACQUITY HSS C18 column (150 × 2 mm, 20 μm) for chromatographic separation. The acquired data were analysed using Empower 3 (LC-DAD) and Unify and Progenesis QI softwares (LC-HRMS) for investigation of specific fragmentation spectra of identified substances. Thanks to combined analytical data (i.e. characteristic UV spectra [Kite. J Chrom B. 2006;838:63-70.] together with specific fragmentation HRMS spectra), oenanthotoxin (OT) and its hydrogenated metabolite, 2,3-dihydro-oenanthotoxin (2,3DHOT*), were identified (case#1) in roots found on the siteand in the stomach content, in the stomach content, kidney, and liver (*also in spleen), and (case#2) in the stomach content. In France, three species of the Apiaceae family can cause life threatening poisoning: the Hemlock (Cicuta virosa), the big Hemlock (Conium maculatum) and the Hemlock water-dropwort (Oenanthe crocata). In the latter one, OT and 2,3DHOT act as non-competitive antagonists of GABA receptors. Clinical signs of poisoning in horses (non-vomiting animals) are dominated by hypersalivation, mydriasis, muscle tremors, respiratory distress, and convulsions, but sometimes sudden death is observed. In humans, the clinical signs are completed/supplemented by ataxia, bruxism, hallucinations, nausea, and vomiting seizures; they occur between 15 min and 10 hours after ingestion [Schep. Clin Tox.2009;47:270–278.]. Analytical detection of Oenanthe crocata toxins in biological samples is a challenge due to their instability (e.g. OT and 2,3DHOT stabilities in post-mortem samples is unknown), the lack of commercially available analytical standards, and the exceptionally low response of these toxins using mass spectrometry detection. Nevertheless, identification of OT and 2,3DHOT can be performed using a combination of LC-DAD and LC-HMRS (for enhanced detection in organs, e.g. liver) methods, in order to support poisoning diagnosis. In this context, we consider that the reported analytical data can be helpful to diagnose oenanthotoxin poisonings.