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A specific and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of the anticoagulant rodenticide diphacinone (DPN) in mouse and rat liver. Tissue samples were extracted with a mixture of water and acetonitrile containing ammonium hydroxide. The extracted sample was cleaned up with a combination of liquid-liquid partitioning and dispersive solid phase extraction. Chromatographic separation was achieved using a Waters X-Bridge BEH C-18 LC column (50 mm, 2.1 mm ID, 2.5 μm particle size) with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The monitored transition for DPN was m/z 339.0 → 167.0 for quantitation and 339.0 → 172.0 and 339.0 → 116.0 for confirmation. The linear range was 0.5 to 375 ng/mL. The average precision of DPN, represented by the relative standard deviation of the observed concentrations, was 7.2% (range = 0.97% - 20.4%) and the average accuracy, represented by the relative error, was 5.8% (range = 1.06% - 14.7%). The recovery of DPN fortified at 3 different levels averaged 106% in rat liver and 101% in mouse liver. The established method was successfully used to determine DPN residue levels in Polynesian rats (Rattus exulans) and mice (Mus musculus) fed two different formulated baits containing DPN. The observed residue levels were consistent with values observed in other rodent studies. However, the amount of bait consumed was lower for the novel baits evaluated in this study. [ABSTRACT FROM AUTHOR]