Withdrawal period estimation of a long-acting intramuscular amoxicillin trihydrate–florfenicol combination in olive flounder

This study aimed to scientifically validate the withdrawal periods (WPs) of a long-acting intramuscular formulation of amoxicillin trihydrate (AMXT) and florfenicol (FFC) in olive flounder (Paralichthys olivaceus). WP estimates from the European Medicines Agency (EMA) and U.S. Food and Drug Administration (FDA) statistical frameworks were compared. Healthy olive flounder were given one AMXT–FFC (20 mg/kg [10 mg/kg AMXT and 10 mg/kg FFC]) injection intramuscularly, and muscle tissue (including the injection site) was collected for up to 28 days post-administration. Amoxicillin (AMX), FFC, and FFC amine (FFA) concentrations were quantified using liquid chromatography–mass spectrometry. Log-linear regression with one-sided tolerance limits based on the EMA and FDA approaches (95/95 and 99/95% tolerance limits, respectively) was used to estimate WPs. Both antimicrobials exhibited time-dependent depletion. Total FFC and AMX residues declined below their respective maximum residue limits by day 14 and day 28, respectively. All analytes fell below the quantification limit by day 28. The EMA’s 95% tolerance limit at 21°C yielded a WP of 40 days (840°C-day) for AMX and 16 days (336°C-day) for total FFC (FFC + FFA). Based on the FDA’s 99% criterion, these estimates extended to 46 days (966°C-day) and 20 days (420°C-day), respectively. Withdrawal determination depended on the slower elimination of AMX. A WP of at least 46 days (966°C-day at 21°C) is recommended for conservative regulatory compliance in olive flounder based on the FDA’s 99% tolerance limit.