Reversed-Phase Ion-Pair High-Performance Liquid Chromatographic Quantitation of Difethialone Residues in Whole-Body Rodents with Solid-Phase Extraction Cleanup

A reversed-phase ion-pair high-performance liquid chromatographic method was developed for the determination of difethialone residues in laboratory rats (Rattus norvegicus). Difethialone was extracted from rat tissue with chloroform/acetone/formic acid. The extracts were cleaned up by a solid-phase extraction (SPE) procedure using both silica and aminopropyl SPE sorbents, concentrated, and analyzed by reversed-phase ion-pair high-performance liquid chromatography. Difethialone was quantitated via ultraviolet absorbance at 262 nm. A surrogate compound, brodifacoum, was used to correct for method performance. The mean surrogate-corrected recoveries for whole ground rodent fortified at 0.2, 1.0, and 20 µg/g difethialone were 92.3 +/- 7.7, 84.8 +/- 6.6, and 90.2 +/- 3.1%, respectively. The method limit of detection was 0.054 µg/g.