Comprehensive Evaluation of an HPLC–MS-MS Method for Quantitation of Seven Anti-Coagulant Rodenticides and Dicoumarol in Animal Serum

Anti-coagulant rodenticides (ARs) are commonly utilized for controlling rodent populations; however, non-target companion and wildlife animals are also exposed. A method was developed for quantitation of seven ARs (chlorophacinone, coumachlor, bromadiolone, brodifacoum, difethialone, diphacinone and warfarin) and dicoumarol (a naturally occurring anti-coagulant) in animal serum. Analytes were extracted with 10% (v/v) acetone in methanol and analyzed by reverse phase high-performance liquid chromatography–tandem mass spectrometry using electrospray ionization (negative mode) combined with multiple reaction monitoring. In-house method validation in the originating laboratory using non-blinded samples revealed method limits of quantitation at 2.5 ng/mL for all analytes. The inter-assay accuracy ranged from 99% to 104%, and the relative standard deviation ranged from 3.5% to 20.5%. Method performance was then verified in the originating laboratory during an exercise organized by an independent party using blinded samples. The method was successfully transferred to two naïve laboratories and further evaluated for reproducibility among three laboratories by means of Horwitz ratio (HorRat_(R)) values. Such extensive validation provides a high degree of confidence that the method is rugged, robust, and will perform as expected if used by others in the future.