Development and validation of an LC–MS/MS method for analysing doxycycline in medicated fish-feed and evaluation of top-coating procedures

Antimicrobials are widely used to control diseases in fish farming. Irregular use of these substances can reduce treatment efficacy and contaminate aquatic environments, promoting the emergence and spread of antimicrobial-resistant bacteria. Doxycycline, a broad-spectrum antimicrobial active against both Gram-positive and Gram-negative bacteria, is authorised for use in aquaculture in several countries and represents a promising therapeutic option. However, to date, no studies have investigated its incorporation into fish feed or its potential to leach into the environment. This study aimed to develop, optimise, and validate an LC-MS/MS method to detect doxycycline in medicated feed and to evaluate top-coating procedures to ensure treatment efficacy while minimising leaching into the environment. The LC-MS/MS method was optimised using a Plackett-Burman design to determine the optimal sample extraction conditions and, subsequently, validated, demonstrating selectivity, precision (CV < 3.5%), accuracy (recovery of 98.4-102%), and linearity (<i>R</i>² > 0.99, 1.6-8.3 g kg^-1). Two top-coating procedures using ethyl cellulose and gelatine polymers were evaluated; both achieved high incorporation efficiency (70%) and homogeneous drug distribution within feed pellets (CV < 2%). Leaching-tests showed that the top-coatings effectively retained doxycycline, with leaching rates below 5% after 15 min of immersion. Overall, these approaches may enhance treatment efficacy and mitigate the environmental impact, promoting the responsible use of antimicrobials in fish farming in accordance with the One Health concept.