Abstract: A method is established for the rapid determination of five alpha-agonists (apraclonidine, brimonidine, tizanidine, clonidine and xylazine) in animal derived food by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) after clean-up by the enhanced matrix removal of lipids (EMR-Lipid). After the pretreatment and UPLC-MS/MS conditions are optimized, the samples are dispersed with 2.0% ammonium hydroxide, then the analytes are extracted with acetonitrile and dried with the extraction salt packet which contains 4.0 g of anhydrous magnesium sulfate and 1.0 g of sodium chloride. Afterward, the acetonitrile layer is purified with EMR-Lipid, and salted out with EMR-Lipid Bond Elut Polish (EMR-Polish). After the solvent is evaporated under nitrogen and reconstituted, the separation is performed on an Eclipse Plus C₁₈ (50 mm×2.1 mm, 1.8 μm) column with 0.10% formic acid aqueous solution and methanol as the mobile phase by gradient elution. The electrospray source is in the positive ion mode and monitored in the multiple reactions monitoring mode. The five alpha-agonists show great linear relationships in the range of 0.20～20 μg/L with correlation coefficients (r²) greater than 0.999. The limits of detection (LOD) and limits of quantitation (LOQ) are in the ranges of 0.20～0.70 μg/kg and 0.60～2.0 μg/kg, respectively. The average recoveries of five targets vary from 76.1% to 103%, and the relative standard deviations are between 1.3% and 12% at the spiked levels of 2.0, 4.0 and 20 μg/kg. The results of matrix effect study show that the matrix has weak effects on all of the five alpha-agonists. The experimental results indicate that the proposed method is simple, quick, accurate and sensitive, and is suitable for the determination of five alpha-agonists in animal derived food.

Key words: Ultra-performance liquid chromatography-tandem mass spectrometry, Enhanced matrix removal of lipids, Alpha-agonists, Animal derived food