Development and validation of an HPLC method for the determination of six penicillin and three amphenicol antibiotics in gilthead seabream (Sparus Aurata) tissue according to the European Union Decision 2002/657/EC

Abstract

A confirmatory high performance liquid chromatography method for the determination of six penicillin antibiotics and three amphenicol antibiotics in gilthead seabream (Sparus Aurata) tissue was developed. Ampicillin (AMP), penicillin G (PG), penicillin V (PV), oxacillin (OXA), cloxacillin (CLO), dicloxacillin (DICLO), thiamphenicol (TAP), florfenicol (FFC) and chloramphenicol (CAP) were separated on an Inertsil, C8 (250×4mm, 5μm) column by gradient elution with a mobile phase consisting of ammonium acetate 0.05M and acetonitrile at 25°C. Diode array detection with monitoring at 225nm (for the determination of AMP, PG, PV, TAP and FFC), 240nm (for OXA, CLO and DICLO) and 278nm (for CAP) was applied. Examined antibiotics were isolated from gilthead seabream tissue by liquid–liquid extraction and further clean-up was performed by solid phase extraction using Oasis HLB (200mg/6mL) cartridges. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity according to the European Union Decision 2002/657/EC.