Non-lethal microsampling and rapid identification of multi-residue veterinary drugs in aquacultured fish by direct analysis in real time coupled with quadrupole-Orbitrap high-resolution mass spectrometry

An analytical protocol for the identification of 52 multi-class veterinary drug residues in aquacultured fish has been developed based on direct analysis in real time (DART) coupled with quadrupole-Orbitrap high-resolution mass spectrometry (Q-Orbitrap HRMS). A “sample-and-release” strategy was adopted where live fishes are captured for non-lethal blood microsampling and then released back into the original farming water. A simple micro liquid-liquid extraction (micro-LLE) was incorporated for the extraction and clean-up of veterinary drug analytes from fish blood samples. The main parameters affecting micro-LLE, DART ionization, and Q-Orbitrap HRMS analysis were systematically studied. Data acquisition was carried out using a synchronous full-scan MS/data-dependent MS/MS (full-scan MS1/dd-MS2) workflow. Suspect screening was conducted by comparing the experimentally measured exact mass of precursor and fragment ions with the custom accurate-mass database and by matching the acquired mass spectra against the mass spectral library. The analytical performance of the developed methodology was assessed in terms of sensitivity, linearity, repeatability, and recovery. The limits of detection (LODs) and quantitation (LOQs) of the 52 analytes were in the range of 0.5–20 μg/L and 2–50 μg/L, respectively. The matrix effect ratios ranged from –23.5% to 25.9%. The developed approach demonstrated promising potential for rapid, real-time monitoring of veterinary drug residues to ensure food safety and consumer health.