Development and Validation of QuEChERS Extraction Coupled with Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for the Detection of Nine Macrolides in Fish Products

Veterinary drug residues in aquatic products are often overlooked, yet they pose significant environmental risks and potential threats to human health. In this study, a rapid and sensitive analytical method was developed for the simultaneous determination of nine commonly used macrolide antibiotics in largemouth bass (<i>Micropterus salmoides</i>) muscle using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Sample extraction was performed using 80% acetonitrile in water, followed by purification with Cleanert MAS-Q cartridges. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C₁₈ column (50 mm × 2.1 mm; 1.7 μm), equipped with a Waters VanGuard^TM BEH C₁₈ guard column (1.7 μm), using a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Mass spectrometric detection was conducted in positive electrospray ionization mode (ESI⁺) using multiple reaction monitoring (MRM). The method demonstrated excellent linearity in the concentration range of 0.2-30 ng/mL, with determination coefficients (R²) exceeding 0.9980 for all analytes. Average recoveries ranged from 89.3% to 108.4%, with intraday and interday relative standard deviations (RSDs) of 2.9-11.6% and 4.1-12.5%, respectively. The limits of detection (LOD) and quantification (LOQ) for largemouth bass muscle were determined to be 0.4 μg/kg and 2.0 μg/kg, respectively. The decision limits (CC_α) and detection capabilities (CC_β) ranged from 2.13 to 215.71 μg/kg and 2.22 to 231.42 μg/kg, respectively. The developed method was successfully applied to the quantitative analysis of macrolide residues in 20 largemouth bass samples collected from local markets.