We aimed to develop a rapid, simple and reproducible method based on LC–tandem mass spectrometry (LC–MS/MS) to analyze β‐agonist residues (clenbuterol, zilpaterol, ractopamine and isoxsuprine) in bovine tissues. The method was validated in accordance with the European Council Decision 2002/657/EC. The samples were homogenized, and then 10 mL of an acetate buffer was added to a 5‐g sample. The sample was then centrifuged at 12,000 rpm and filtered. Sodium hydroxide (2 m) was added to adjust pH of the sample that was centrifuged again. The extract was filtered through a solid‐phase extraction column. The residue was re‐dissolved in 250 μL acetonitrile and then subjected to LC–MS/MS. The separation was done on a C18 column. The mobile phase consisted of 0.1% formic acid in deionized water and 0.1% formic acid in methanol. The mean recoveries of β‐agonists were in the range of 84.3%–119.1% with relative standard deviations (%RSDs) of 0.683%–4.05%. Decision limits and detection capabilities of the analytes ranged from 0.0960 to 4.9349 μg/kg and from 0.0983 to 5.0715, respectively. This method was used to detect four β‐agonists in 100 bovine muscle, 100 liver and 100 kidney tissues from a slaughterhouse. No residue was found above the maximum residue limit level.

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