Liquid chromatographic determination of levamisole in animal plasma: ultraviolet versus tandem mass spectrometric detection Original Research Article

This work presents the optimization and validation of a liquid chromatography (LC)–ultraviolet (UV) detection method and a tandem mass spectrometry (LC–MS/MS) method with positive electrospray ionization for the determination of levamisole, an anthelmintic for veterinary use, in animal plasma. A liquid–liquid extraction procedure in alkaline medium, using hexane-isoamyl alcohol (95:5, v/v) as extraction solvent, was performed to clean-up the plasma samples prior to LC-UV analysis. The sample preparation prior to LC–MS/MS analysis consisted of a protein precipitation step, using acetonitrile. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher® 60 RP-select B (5 μm) column using a mixture of 0.1 M ammonia acetate in water and acetonitrile as the mobile phase. A gradient (flow rate 1 ml min−1) or isocratic (flow rate 0.2 ml min−1) elution was performed in case of the LC-UV and LC–MS/MS methods, respectively. UV detection was at 235 nm. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode.