A high performance liquid chromatography method for the detection of brodifacoum in serum

A procedure for the detection of brodifacoum (BDF) in serum was developed. Extraction of BDF was achieved by acidification of 2 ml of serum with 1 ml of 1.5% acetic acid followed by dual extractions with 10 ml diethyl ether and ether: acetonitrile [1:1]. In spiking experiments, 68 +/- 3, 61 +/- 4 and 65 +/- 5% of added BDF was recovered from serum containing 1000, 100 and 25 ng BDF/ml, respectively. Two high performance liquid chromatography solvent systems were used for chromatographic separation (A: 1.5% acetic acid, pH 4.5: acetonitrile [1:2] with 1% dibutylamine; and B:O.2 M tris(hydroxymethyl)aminomethane, pH 7.5:acetonitrile [1:3]). Detection limits were 75 and 3 ng BDF/ml of serum using ultraviolet absorption (254 nm) and fluorescence measurement (313 nm excitation, 375 nm emission), respectively. This method has been used successfully to monitor serum concentrations of BDF in experimental and field cases of exposure.