Analysis of anticoagulant rodenticide residues in Microtus arvalis tissues by liquid chromatography with diode array, fluorescence and mass spectrometry detection

We describe here a fast and selective analytical method to determine the levels of four anticoagulant rodenticides (chlorophacinone, bromadiolone, brodifacoum and difenacoum) in animal tissues by liquid chromatography (LC) using different detection methods: fluorescence (FLD), diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS). Rodenticides were extracted from freeze-dried and homogenized tissue samples (liver, intestine and muscle) that had been obtained from the common vole (Microtus arvalis). These samples were diluted in 5 mL of methanol, the solution was shaken and centrifuged, and the supernatant was removed and evaporated to dryness. The residue was reconstituted in 1 mL of methanol (liver samples) or 1 mL of the mobile phase (muscle and intestine samples), and injected onto an LC–DAD–FLD–MS system coupled to electrospray ionization (ESI) in negative mode. After conducting an LC optimization study, we selected a Gemini 5 μm C18 column, a mobile phase composed of a mixture of 30 mM ammonium formate in water and methanol (26:74, v/v), and we used an isocratic elution mode. The method was fully validated and shown to be selective, precise, accurate, and linear in the range from ∼5 μg/kg (ESI-MS) or ∼50 μg/kg (DAD–FLD) to 10,000 μg/kg, depending on the compound analyzed. Recoveries ranged from 82% to 103%, while the limits of detection and quantification ranged from 9–89 μg/kg (FLD–DAD) and 0.6–4.6 μg/kg (ESI-MS). This method was successfully used to simultaneously measure the aforementioned compounds in M. arvalis tissues.