An online packing strategy for high volume injection in sensitivity-enhanced UPLC-MS/MS analysis of 30 veterinary drugs in beef.

In this study, an online packing strategy was proposed for vortex mixing-enhanced high volume injection in ultrasensitive UPLC-MS/MS analysis of 30 veterinary drugs in beef. Spherical glass beads were densely packed into a custom-fabricated hollow stainless steel pre-column to achieve efficient vortex-induced mixing with the initial weak mobile phase, thus enabling online sample stacking at the analytical column inlet. Sample elution strength was effectively attenuated via online vortex-enhanced mixing within the packing column assembly, and the inherent solvent imbalance in high volume injection was effectively mitigated. Key parameters including particle size, inner diameter, column length, flow direction, and flow rate demonstrated significant influence for targeted drugs. Method validation demonstrated excellent linearity across 0.125-100 μg/kg, with coefficients of determination (R²)>0.998. Matrix effects were quantified as -17.7% to 17.9%. Spiked recovery assays at 2, 10, and 20 μg/kg yielded acceptable accuracy: 70.9%-111.8%, 60.5%-100.1%, and 63.0%-104.8%, respectively. Intra- and inter-day precision displayed favorable relative standard deviations (RSDs) ≤17.3%. Analytical sensitivity was characterized by limits of detection (LODs, 0.03-1.7 μg/kg) and quantification (LOQs, 0.1-4.2 μg/kg). The utilized strategy achieved 10.9 ± 3.7-fold peak area enhancement for target veterinary drugs via 20 μL injection (vs. conventional 2 μL), resolving the persistent "solvent effect" imbalance in high loading injection and exhibiting excellent compatibility with conventional LC-MS systems without hardware/software upgrades. The packed column exhibits intrinsic practical advantages, encompassing streamlined structural design, straightforward installation, and economical packing materials, thereby underscoring its versatility as a valuable analytical tool for widespread application in trace-level substance monitoring.