Characterization of Bromethalin and its Degradation Products in Veterinary Toxicology Samples by GC-MS-MS

Bromethalin is a neurotoxicant with unusual instability and chromatographic behavior that make it difficult to analyze by gas chromatography (GC) in forensic examination of non-target animal deaths. Physicochemical breakdown of bromethalin produced multiple unique products with discernible mass spectra. This paper describes an investigation of the GC electron impact-mass spectrometric properties of bromethalin and its capacity to generate up to twenty heat- or light-induced breakdown products. Two principal breakdown products are isomeric with one another and involve release of both fluorine and methyl groups to develop dehydrofluorodesmethylbromethalin products. These compounds have proven to be excellent surrogate markers in screening forensic samples for bromethalin exposure, particularly in veterinary samples in which the active metabolite desmethylbromethalin has not yet accumulated to any appreciable extent, such as baits and animal stomach contents. The compounds as well as their parent bromethalin were easily monitored by GC interfaced with a tandem-quadrupole mass spectrometer using multiple-reaction monitoring (MRM) modes. Unusual gas chromatographic properties of bromethalin included: (i) specific requirements for a maximum oven temperature; (ii) non-linear increases in detector response on increased injection volumes, hypothesized to result from variable diffusion coefficients. We report here the development of GC strategies that facilitate detection of bromethalin and its breakdown products, as well as their MRM analysis by tandem-quadrupole mass spectrometry. The developed approaches are applicable to feed, baits and stomach contents as well as extracted tissue samples such as liver and kidney.