Simultaneous determination of azaperone and azaperol in animal tissues by HPLC with confirmation by electrospray ionization mass spectrometry

A simple method is described for the determination of azaperone and its metabolite, azaperol, in animal tissues by high-performance liquid chromatography with ultraviolet detection (HPLC/UV). Chromatography was performed using an ODS column, an acetonitrile–0.025% aqueous diethylamine mixture (2:3, v/v) as a mobile phase and UV detection at 250 nm. Peak heights were found linearly related to the concentrations injected from 0.05 to 2 μg/mL (r > 0.999). Azaperone and azaperol spiked into several animal tissues were solubilized in 1 mol/L NaOH, extracted with hexane, transferred to 0.1 mol/L H2SO4 and re-extracted with hexane in a mild basic condition. Recoveries of both compounds from 12 types of samples (swine muscle, swine adipose tissue, swine liver, bovine muscle, bovine adipose tissue, bovine liver, poultry muscle, poultry adipose tissue, poultry liver, bovine milk, poultry egg, and salmon muscle) were more than 72%. The lower limit of quantification of was 0.025 μg/g. Azaperone and azaperol at 0.1 μg/g were confirmed by LC/MS. In conclusion, we found this method is both simple and useful for the determination of azaperone and azaperol in a variety of animal tissues for food safety and veterinary applications.