Identification of etamiphylline and metabolites in equine plasma and urine by accurate mass and liquid chromatography/tandem mass spectrometry

Etamiphylline camsylate (Millophylline V) was administered intravenously to two horses at a dose of 2.8 mg/kg. Urine and blood samples were taken up to 32 h post administration. Unhydrolyzed plasma and urine was extracted using solid phase extraction (SPE). The identity of the parent drug and metabolites was confirmed using a linear ion trap mass spectrometer and accurate mass analysis on an orbitrap mass spectrometer. Desethyletamiphylline (molecular weight 251) was the main metabolite observed in the urine and plasma samples and resulted from the N-deethylation of etamiphylline. The second metabolite detected in urine and plasma resulted from the demethylation of etamiphylline (molecular weight 265). The third minor metabolite detected in urine was proposed to have resulted from a simultaneous N-deethylation and demethylation of etamiphylline (molecular weight 238).