A High-Accuracy Measurement Procedure for Salbutamol, Ractopamine, and Clenbuterol in Pork by Isotope Dilution-Liquid Chromatography/Tandem Mass Spectrometry

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 β2-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 β2-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-D6 and SAL-D9 were less effective in compensating for matrix effects due to hydrogen‑deuterium exchange during MS fragment formation. Consequently, SAL-D3, RAC-D3 and CLB-D9 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 β2-agonists in pork.