Simultaneous Determination of Seven β₂-Agonists in Livestock Products Using an LC-MS/MS System

Abstract

A simultaneous determination method for seven β₂-agonists (clenbuterol, ractopamine, zilpaterol, cimaterol, isoxsuprine, salbutamol and terbutaline) was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) to evaluate their residual status in livestock products commercially available in Japan. The livestock sample was homogenized with ethyl acetate under alkaline conditions and degreased with n-hexane. The target β₂-agonists were then purified by dispersive solid-phase extraction with C18 particles. For the LC separation, a semi-micro scale column packed with an octadecylsilica (ODS) stationary phase was employed, and gradient elution was performed for 10 min using aqueous acetonitrile containing ammonium acetate as the mobile phase solvent, where the target compounds were eluted within 5-8 min. The developed method was validated using pig muscle, a processed pork food, cattle muscle and a processed beef food, and the obtained trueness values were 60.1-103.5%. The obtained relative standard deviation (RSD) values were 1.26-8.94% (repeatability) and 1.93-10.55% (intermediate precision). In some of the imported livestock products, small but clear peaks of ractopamine were detected by the developed LC-MS/MS method, and the presence of ractopamine was further confirmed by an LC-MS/MS/MS analysis with higher selectivity.