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

Abstract

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. The methods were validated (linearity, precision, trueness, limit of quantification, limit of detection, specificity) according to the requirements defined by the European Community. Good linearity was achieved over the concentration ranges tested (0–0.5 μg ml −1 and 0–4.0 μg ml −1, r>0.99, goodness-of-fit <10%). Limits of quantification of 0.025 μg ml −1 and 0.1 μg ml −1 were achieved for the LC–MS/MS and LC-UV methods, respectively. The limits of detection were 0.009 and 0.077 μg ml −1, respectively. The results for precision (within-day and between-day) fell within the ranges specified. The methods have been successfully used for the determination of levamisole in plasma samples from two pigs medicated via drinking water. A good correlation was observed between the results of both methods ( r 2=0.9831, slope=1.13, intercept=0.005 μg ml −1), proving their usefulness for the application in the field of pharmacokinetic and residue analysis.