DART-HRMS allows the detection of toxic alkaloids in animal autopsy specimens and guides the selection of confirmatory methods in accidental plant poisoning

Abstract

Background: In cases of suspected animal poisonings or intoxications, there is the need for high-throughput, rapid and accurate analytical tools capable of giving rapid answers and, thus, speeding up the early stages of investigations. Conventional analyses are very precise, but do not meet the need for rapid answers capable of orienting the decisions and the choice of appropriate countermeasures. In this context, the use of ambient mass spectrometry (AMS) screening methods in toxicology laboratories could satisfy the requests of forensic toxicology veterinarians in a timely manner.

Results: As a proof of principle, direct analysis in real time high resolution mass spectrometry (DART-HRMS) was applied to a veterinary forensic case in which 12 of a group of 27 sheep and goats died with an acute neurological onset. Because of evidence in the rumen contents, the veterinarians hypothesized an accidental intoxication after ingestion of vegetable materials. The DART-HRMS results showed abundant signals of the alkaloids calycanthine, folicanthidine and calycanthidine, both in the rumen content and at the liver level. The DART-HRMS phytochemical fingerprinting of detached Chimonanthus praecox seeds was also compared with those acquired from the autopsy specimens. Liver, rumen content and seed extracts were then subjected to LC-HRMS/MS analysis to gather additional insights and confirm the putative assignment of calycanthine anticipated by DART-HRMS. HPLC-HRMS/MS confirmed the presence of calycanthine in both rumen contents and liver specimens and allowed its quantification, ranging from 21.3 to 46.9 mg kg^-1 in the latter. This is the first report detailing the quantification of calycanthine in liver after a deadly intoxication event.

Significance and novelty: Our study illustrates the potential of DART-HRMS to offer a rapid and complementary alternative to guide the selection of confirmatory chromatography-MSⁿ strategies in the analysis of autopsy specimens from animals with suspected alkaloid intoxication. This method offers the consequent saving of time and resources over those needed for other methods.

Keywords: Alkaloids; Ambient mass spectrometry; Calycanthine; DART-HRMS; Poisoning; Toxins; Veterinary forensic toxicology.