Detection of Anticoagulant Residues by a New HPLC Method in Specimens of Poisoned Animals and a Poison Control Case Study

Abstract

Poisonings of domestic and wildlife animals are frequently caused by anticoagulants. Between 1996 and 2003, 70% of all suspected animal poisoning cases were confirmed in this laboratory following analysis of a variety of specimens. Thirty-nine percent of all animal poisonings detected in this laboratory were caused by anticoagulants. Suspicious veterinarians and pet owners involved with animal poisonings ask for toxicological analyses. With respect to these demands, a new high-performance liquid chromatography (HPLC) method (system-I, reversed-phase C-18 column, methanol/ammonium acetate) combined with specific extraction procedures was developed for the sensitive detection of nine substances with anticoagulative properties. This method yields high recoveries of the analytes, which can simultaneously be detected. In addition, another HPLC (system-II, acetonitrile/potassium phosphate) was used to confirm and separate chromatographic peaks, which could not be resolved by HPLC system-I. The reliable method has good reproducibility and is most suitable for forensic analyses. Using these analytical procedures, concentrations of eight organic anticoagulants were determined in more than 100 aliquots of animal liver, blood, bait, gastric content, bloody intestinal content, and water. Over the last eight years, dogs and wild birds were the most frequently poisoned species. Interestingly, lethally poisoned birds had lower anticoagulant residues in their organs than mammals.

Postmortem findings of secondary brodifacoum poisoning in a kestrel (Falco tinnunculus)

“…F I G U R E  High-performance liquid chromatography (HPLC) chromatograms of the liver sample alone (a) and spiked with internal standards (b). Peaks from the left: sulfaquinoxaline (1), racumin (2), warfarin (3), bromadiolone (4) difenacoum (5), flocoumafen (6) and brodifacoum (7). Note that the sample and standard peak of brodifacoum overlap.…”

Analytical methods for determination of anticoagulant rodenticides in biological samples

“…Various organic solvents and their mixtures such as acetonitrileethylether [8], chloroform-acetone [10], acetone-diethyletherchloroform [11], acetonitrile-acetone [12], dichloromethaneacetone [14], 1-chlorobutane [18], ethyl acetate [19][20][21]28,32], acetone [22,23], acetonitrile [24][25][26]29] and methanol [30,33] were used to extract anticoagulant rodenticides from biological samples. Among all organic solvents used in LLE, acetonitrile-ethyl ether (9:1) [8] produced the highest recovery (92-109%) in plasma and liver tissues.…”

“…Similar approaches, with combined LLE and SPE, have successfully been developed [11,12,14,25,26,31].…”

“…However these methods have low detection sensitivity mostly ranging from 10 to 100 ng/ mL. Similarly HPLC-FLD has also been employed for rodenticides detection [6][7][8][9][10][11][12][13][14]30]. Although fluorescence detection offers enhanced sensitivity for hydroxycoumarin based rodenticides, but it still requires an extensive sample clean-up.…”

The Usefulness of MS3 to Confirm Poisoning on the Example of Dog Poisoning with Strychnine

“…Intentional or accidental poisonings with strychnine occur mainly in small animals, especially dogs and occasionally cats, and rarely in farm animals. From animals, for the purposes of toxicological examinations, the most frequently used matrices are stomach contents, blood, and liver, whereas from environmental samples they are baits and feeds [4,5,6,7,8,9,10,11,12]. Cases of strychnine poisoning are usually acute or hyperacute.…”