Multi-residues determination of antimicrobials in fish tissues by HPLC–ESI-MS/MS method

• A simple LC–MS/MS method was developed for the determination of four commonly used antimicrobials in fish tissues.
• The studied drugs belong to different chemical classes.
• Liquid extraction followed by a post-extraction clean-up step with n-hexane was employed.
• The developed method was applied for detection and quantification of the studied antimicrobials in real samples.

A rapid, simple, sensitive and specific LC–MS/MS method was developed and validated for the simultaneous quantification of four antimicrobials commonly used in aquaculture, namely ciprofloxacin (CPX), trimethoprim (TMP), sulphadimethoxine (SDM) and florphenicol (FLOR) in fish tissues. The LC–MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Sample preparation involves simple liquid extraction step followed by post-extraction clean-up step with n-hexane. The purified extracts were chromatographed on Agilent Poroshell 120 EC, C18 (50 mm × 3 mm, 2.7 μm) column by pumping an isocratic mobile phase consisting of 0.1% formic acid in water:0.1% formic acid in methanol (20:80, by volume) at a flow rate of 0.4 mL/min. A detailed validation of the method was performed as per FDA guidelines and the standard curves were found to be linear in the range of 1–100 ng/g for both CPX and TMP, 0.5–100 ng/g for SDM and 1–50 ng/g for FLOR. The intra-day and inter-day precision and accuracy of the results were within the acceptable limits. A run time of 1.5 min for each sample made it possible to analyze multiple fish tissue samples per day. The developed assay method was successfully applied for the detection of antimicrobials in real fish tissue samples obtained from different fish farms.