Objective To address the challenges associated with complex matrices, diverse product types, and the potential illegal addition of amphenicol antibiotics in aquaculture inputs, a sensitive and reliable analytical method was developed for the simultaneous determination of chloramphenicol (CAP), thiamphenicol (TAP), and florfenicol (FF) residues in aquaculture inputs, providing technical support for quality and safety supervision.

Methods A liquid chromatography–tandem mass spectrometry method was established for the determination of CAP, TAP, and FF. Samples were extracted with ethyl acetate under alkaline conditions, followed by lipid removal using n-hexane liquid–liquid partitioning. The extracts were purified using C₁₈ solid-phase extraction cartridges. Chromatographic separation was achieved on a reversed-phase C₁₈ column, and detection was performed in negative electrospray ionization mode using multiple reaction monitoring (MRM). Quantification was carried out using an internal standard method. Key parameters, including extraction solvent, purification procedure, chromatographic conditions, and mass spectrometric parameters, were systematically optimized. The method was validated in terms of linearity, limits of detection (LOD), limits of quantification (LOQ), recovery, and precision.

Results Good linearity was obtained for all three analytes over the concentration range of 0.50–20.0 ng/mL, with correlation coefficients (r) greater than 0.999. The LODs ranged from 0.043 to 0.088 μg/kg, while the LOQs ranged from 0.14 to 0.29 μg/kg. Recovery experiments conducted at three spiking levels (1.00, 2.50, and 5.00 μg/kg) in different aquaculture input matrices showed satisfactory recoveries ranging from 92.0% to 115%. The intra-day precision, expressed as relative standard deviation (RSD), ranged from 1.15% to 4.13%, and the inter-day precision ranged from 1.88% to 5.02%. Analysis of 24 batches of real aquaculture input samples revealed the presence of florfenicol in several samples, with concentrations ranging from 3.58 to 10.6 μg/kg, while chloramphenicol and thiamphenicol were not detected.

Conclusion The developed liquid chromatography-tandem mass spectrometry method demonstrates high sensitivity, good accuracy, and excellent reproducibility for the determination of amphenicol residues in complex aquaculture input matrices. The method is suitable for large-scale screening and quantitative analysis and can be effectively applied in routine regulatory monitoring and risk assessment of aquaculture inputs.