Hundreds of veterinary drugs are widely used in agricultural activities and continuously enter aquatic environments through various pathways, posing potential risks to ecosystem. Considering that sediments function both as sinks and sources of these contaminants, it is crucial to promptly and accurately acquire veterinary drug residue level in sediments. In this study, a highly effective analytical method for simultaneous determination of 103 veterinary drugs from 16 classes in sediments was developed using high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS). The extraction procedure was performed twice by ultrasound-assisted extraction with an acetonitrile-buffer mixture consisting of Na
2EDTA, Na
3PO
4·12H
2O, and Na
3C
6H
5O
7·2H
2O. The supernatant was cleaned using 500 mg/6 mL Oasis HLB solid-phase cartridges. The elution solutions were concentrated and redissolved in formic acid–methanol–water (0.1/20/79.9,
v/
v, FA-MeOH-H
2O) for detection. Results showed that all 103 target drugs exhibited good linearity with R
2 > 0.990 over a concentration range of 0.010 to 1000 μg·L
−1, and method detection limits (MDLs) ranged from 0.025 to 5 μg·kg
−1. The recoveries at three spiking levels (2, 5, and 10 times of the method quantification limits, MQLs) varied from 33% to 150%, 32% to 140%, and 40% to 140%, respectively, with relative standard deviations (RSDs,
n = 3) of 0.7%~29%, 0.8%~23%, and 0.5%~20%. The matrix effects for all compounds ranged from –85% to 84% with 32 targets negligible, 51 moderate, and 20 significant. An isotope-labeled surrogate method was proposed for quantitation to effectively overcome matrix effects and improve accuracy with better recoveries of 60%~120% for 93 target drugs and RSDs (
n = 3) all below 20%. This method was applied to determine 12 sediment samples collected from the Jiulong River, and 16 target drugs were detected in the concentrations range of 0.1~7.6 μg·kg
−1. The method is accurate, sensitive, and efficient, providing a powerful analytical tool for behavior and effect studies of multi-classed veterinary drug residue in sediment environments.
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