Determination of bromadiolone and brodifacoum in human blood using LC-ESI/MS/MS and its application in four superwarfarin poisoning cases.

Superwarfarin poisoning is a growing health problem. A sensitive and reproducible LC-ESI/MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) method was developed and validated for the determination of bromadiolone and brodifacoum, the most commonly used superwarfarins, in human blood using warfarin-D5 as an internal standard. Bromadiolone and brodifacoum were extracted from whole blood samples by liquid-liquid extraction with ethyl acetate. Multiple-reaction monitoring (MRM) was used to detect bromadiolone and brodifacoum using precursor→product ion combinations of m/z 525→250 and 521→135, respectively. The calibration curves were linear (r(2)=0.9999) in the concentration range of 0.5-100.0 ng/mL for bromadiolone and brodifacoum, with a lower limit of detection of 0.1 and 0.2 ng/mL, respectively, in whole blood. This method detected trace levels of bromadiolone and brodifacoum in whole blood samples and can be used in the diagnosis of poisoned human beings.