Cardiac glycosides (CGs), classified as cardenolides or bufadienolides, are toxins found in a variety of flora and fauna. CGs have historically been used to treat heart failure, but despite their place in medicine, they are still toxic to humans and animals. Currently, liquid chromatography paired with mass spectrometry (LC-MS) is used to screen for CGs. However, this requires using specific, costly, and often unavailable CG standards for reference. This study investigated the potential of identifying a cardenolide-containing (CC) plant using a plant’s overall cardenolide composition, represented by unique ions in the 300 to 400 m/z region in their mass spectra, rather than individual toxins. The chromatographic patterns of different plants at these select ions may provide unique “fingerprints” to serve as a means of identification. Not only would this provide an alternative to the standards, but it could also aid the diagnosis of cardenolide toxicosis in humans and animals, especially in cases where exposure history is unknown. To accomplish this, cardenolide standards in methanol were analyzed using UHPLC-HRMS and ions in the 300 to 400 m/z region exhibiting the “loss of 18 m/z” pattern were noted. These ions were grouped into “high”, “medium”, “low”, based on their masses, and “genin”. Next, 14 CC plants were individually homogenized in methanol and analyzed in replicate. The extracted ion chromatograms (EICs) at each ion of interest were observed for all plants. Blind tests were conducted to assess the ability to, using the EICs at the select ions, distinguish between CC and non-CC plants (Blind Test 1) and identify a CC plant (Blind Test 2). Blind Test 1 had a success probability of 1.0 across all ion groups, while Blind Test 2 had a success probability of 1.0 for the “high” and “medium” groups, and a success probability of 0.990 for the “low” and “genin” groups. Overall, this present study shows a promising start in using select ion chromatographic finger prints as a means of qualitatively identifying CC plants.

An efficient and effective method for the detection of a wide variety of toxicants in liver tissue was developed and validated using gas chromatography-mass spectrometry (GC-MS). The aim of this research was to address the need for non-targeted analysis in postmortem liver samples. We wanted to find a faster, less labor-intensive method for fat removal from liver. A variety of clean-up sorbents and cartridges were assessed for their efficacy in lipid removal from liver tissue as well as analyte recovery. The study focused on the evaluation of QuEChERS solvent extraction with Enhanced Matrix Removal-Lipid (EMR-Lipid™) clean-up to remove lipids from liver tissue samples. Qualitative analysis was performed on 15 representative pesticides and 2 drugs commonly analyzed for in veterinary diagnostics. Increased sensitivity was achieved by using 5 μL injections into the GC-MS running on solvent vent mode. Probability of detection (POD) was used as the statistical model for method evaluation and validation. Three sources of liver (bovine, chicken, and caprine), with varying amounts of fat present, were tested. The QuEChERS extraction with EMR-Lipid clean-up was found to be as effective as gel permeation chromatography (GPC) in removing fats from liver tissue with minimal analyte loss. GPC, the typical clean-up method for fatty tissue samples, was the reference method used for comparison. It was determined by the POD model that the candidate method had better detection than GPC at 5 μg/g for all the analytes investigated and that the difference was statistically significant at 5 μg/g but not below that. In addition, EMR-Lipid uses substantially less solvent and is twice as fast compared to GPC.

Second generation anticoagulant rodenticides (SGARs), developed first to exterminate rodent populations, have now become a means of accidental secondary wildlife intoxication. Utilizing highly potent bait formulations designed to overcome resistance, these SGARs now exhibit ongoing persistence in liver and other tissues. Particularly, SGARs persist antemortem within target consumer tissues allowing for non-target wildlife exposure. The aim of this research was to utilize bromadiolone’s and brodifacoum’s diastereomer configurations of cis and trans isomers to determine whether one isomer had less tissue persistence. To accomplish this, High Performance Liquid Chromatography paired with Mass Spectrometry (HPLC-MS/MS) was applied. Through partnership with the California Department of Fish and Wildlife, 45 non-target wildlife livers including coyote (Canis latrans), gray fox (Urocyon cinereoargenteus), and cougar (Puma concolor) were obtained and analyzed for diastereomer proportions of bromadiolone and brodifacoum. Current bromadiolone and brodifacoum baits were tested and isomer ratios were compared to ratios in primary consumer species and secondarily exposed non-target species. All 45 non-target samples contained brodifacoum and 41 had bromadiolone residues. For bromadiolone, trans-bromadiolone was shown to be more persistent in the food chain with 56% (23/41) having ≥91%. For brodifacoum, 35/45 cougar and coyote samples displayed the cis isomer as more persistent. However, gray foxes displayed the trans isomer of brodifacoum. Thus, brodifacoum’s isomer pattern is variable, perhaps reflecting species differences in brodifacoum metabolism and elimination. Moreover, the benefits of reformulating brodifacoum to a certain isomer were not as clear. However, this research establishes that reformulation of bromadiolone bait to include less trans isomer will likely lower both food chain persistency and non-target wildlife exposure.

Background

Case presentation

Conclusion