Comparison and Validation of QuEChERS Extraction Methods Coupled with UHPLC/Orbitrap HR-MS for the Determination of Antibiotics and Related Compounds in Fish and Fish Feed

The widespread presence of pharmaceutical active compounds (PhACs) in aquatic environments raises significant environmental and public health concerns, particularly through their accumulation in marine biota and potential transfer to humans via seafood. In aquaculture, fish feed is essential for production but may also act as a pathway for contaminants in the marine environment. This study aimed to develop and validate an analytical method for the extraction and quantification of 14 antibiotics and ethoxyquin antioxidant in fish tissue and feed. Two QuEChERS-based extraction protocols were compared: the AOAC 2007.01 method (Method A) using Z-Sep⁺ as clean-up, and the original QuEChERS method (Method B) employing Enhanced Matrix Removal (EMR)-lipid. Ultra-performance liquid chromatography coupled with Orbitrap mass spectrometry using electrospray ionization in positive and negative mode was applied for identification and quantification. Validation included assessment of recovery, linearity, precision, limits of detection and quantification, uncertainty, matrix effects, and process efficiency. Both methods showed good linearity (R² > 0.9899) and precision (<19.7%). Method B achieved superior recoveries for most analytes in both fish tissue (70–110%) and feed (69–119%), with lower uncertainties (<18.4%) compared to Method A. Overall, the original QuEChERS method demonstrated better analytical performance, supporting its application as a green, robust tool for monitoring emerging contaminants in aquaculture products.