HPLC-HRMS methods in horse doping control and forensic veterinary toxicology : a case report about the analysis of anticoagulants

A routine high pressure liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) screening method used for horse doping control in blood and urine was used to identify the likely poisoning of a horse in a 1 ml of gastric, hemorrhagic blood. The veterinarian diagnosis hypothesized the ingestion of a substance belonging to the class of anticoagulants, widely used in agricultural and urban rodent control. Three methodological protocols for the detection of anticoagulant rodenticides in biological tissues were used. As a first step, the screening method of the laboratory, consisting in solid phase extraction (SPE) extraction and HPLC-HRMS analysis, was used: the unknown poison was thus identified as Coumachlor (anticoagulant of the first generation). On the basis of this result, we performed a simple and very fast liquid-liquid extraction followed by ultra high pressure liquid chromatography (UHPLC) HRMS (6 min) analisys for the screening of 8 rodenticides plus embutramide, an active principle of Tanax, a medicine used for euthanasia in veterinary practice. All these substances are of interest in forensic veterinary toxicology. The HRMS analysis was coupled to activated Collision Induced Dissociation (CID) to obtain a further confirmation. The instrument was operated in electrospray ionization, with positive-negative polarity switching mode (1 sec). The resolution was set at 25,000. The final analyses were performed both with a Triple Quadrupole and a Linear Ion Trap mass spectrometer and confirmed the results obtained by HRMS operating in CID mode, so demonstrating the fitness of the method also for confirmation analyses. The limit of detection (LOD) and limit of quantification (LOQ) were calculated for all the analytes. Operating in CID-HRMS, classic doping substances like Methylprednisolone, Bumethanide, was detected and confirmed. The UHPLC-HRMS analisys was also tested on blood from an horse in treatment, for navicular syndrome, with warfarin .