Development and validation of an LC-MS/MS/MS method for the sensitive quantification of trace β-agonists in complex animal-derived food matrices

Abstract

Conventional multiple reaction monitoring (MRM) quantification of β-agonists in complex matrices was hindered by insufficient sensitivity due to co-extracted matrix interferences. To address this drawback, we developed a highly selective and specific liquid chromatography triple-stage tandem mass spectrometry (LC-MS/MS/MS, LC-MS3) method via systematic comparison of MRM and MRM3 modes for the determination of nine β-agonists (ractopamine, penbutolol, cimaterol, salbutamol, clenbuterol, tulobuterol, clorprenaline, terbutaline, fenoterol) in high-fat milk and pork. MS3 method overcomes the limitations of conventional MS/MS through the multi-stage fragmentation capability of a hybrid linear ion trap (LIT), enabling deeper structural characterization and superior matrix interference resistance. The results demonstrated that LC-MS3 method not only accurately quantified β-agonists in complex matrices, but also significantly enhanced sensitivity by reducing matrix effects. Extracted ion chromatograms (XICs) revealed nearly interference-free peaks, exhibiting the excellent selectivity and specificity of the LC-MS3 method. The LC-MS3 method exhibited good linearity (R² ≥ 0.9973), and the limits of detection (LOD) and quantification (LOQ) for β-agonists ranged from 0.01 to 0.05 μg/kg and 0.03 to 0.15 μg/kg, respectively. The recoveries were between 85.47% and 105.32%, with intra-day and inter-day relative standard deviations (RSDs) of 1.68%–6.42% and 2.42%–8.68%, respectively. The matrix effect (ME) was within -10.55% to 9.53%. In comparison, the conventional LC-MS/MS method showed higher LODs (0.1–0.15 μg/kg), LOQs (0.3–0.45 μg/kg), and broader matrix effects (-18.12%–19.35%), confirming the superior sensitivity of the LC-MS3 method. This is the first study to employ MRM3 mode for the determination of β-agonists in high-fat milk and pork matrices.