In-depth research into the precise evaluation of enzymatic digestion efficiency and the selection of a suitable deuterium-labelled internal standard remains a gap in the accurate determination of β-agonists in animal-derived food by isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC-MS/MS). In this study, the enzymatic digestion conditions were optimized by monitoring the presence of β-agonist conjugates in positive samples, which proved to be reliable for ensuring complete enzymatic digestion. Comparative analysis of deuterium-labelled internal standards for salbutamol (SAL), ractopamine (RAC), and clenbuterol (CLB) revealed that CLB-D and SAL-D were less effective in compensating for matrix effects due to hydrogen‑deuterium exchange during MS fragment formation. Consequently, SAL-D, RAC-D and CLB-D were chosen for the implementation of ID-LC-MS/MS. The developed method demonstrates high accuracy and precision, with the average recoveries ranging from 93.8% to 107.3% with RSD <6.1%, which can provide higher-order measurement results for β-agonists in pork.
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