Development and validation of an LC-MS/MS/MS method for the sensitive quantification of trace β-agonists in complex...

Conventional multiple reaction monitoring (MRM) quantification of β-agonists in complex matrices was hindered by insufficient sensitivity due to co-extracted matrix interferents. 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, and fenoterol) in high-fat milk and pork. The 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 the 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^-1 and 0.03 to 0.15 µg kg^-1, 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^-1) and LOQs (0.3-0.45 µg kg^-1) 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.