Simultaneous multiresidue determination of metronidazole and spiramycin in fish muscle using high performance liquid chromatography with UV detection

An efficient multiresidue method for the simultaneous determination of metronidazole (MET) and spiramycin (SPY) in tilapia fish muscle, based on high performance liquid chromatography with UV detection (HPLC-UV), has been developed. The drugs were extracted with 0.2% orthophosphoric acid–methanol (6:4), and the extracts were cleaned up on a solid phase extraction cartridge, C18 Sep-Pak® light column. The LC separation was performed on a RP stainless-steel C-18 analytical column (150 mm × 4.6 mm, 5 μm) with a gradient elution system of 0.05 M phosphate buffer adjusted to pH 2.4–acetonitrile as the mobile phase at the flow rate of 1.0 ml min−1. A wavelength programming was applied for the UV detection of the analytes. The method not only enabled the determination of the parent drugs, MET and SPY, but also permitted the determination of their metabolites, hydroxymetronidazole (HMET) and neospiramycin (NSPY). The calibration graphs for each drug were rectilinear in the range of 0.005–1.000 μg g−1 for MET and HMET and 0.025–1.000 μg g−1 for SPY and NSPY. With this method, the cited drugs with their metabolites were determined in fortified fish muscle tissues at levels of 0.025, 0.1 and 1.0 μg g−1 with good accuracy and precision. LOD and LOQ obtained for each drug were as follows: 0.002 and 0.005 μg g−1 for MET and HMET and 0.005 and 0.025 μg g−1 for SPY and NSPY. Utilization of the method to successfully analyze tilapia fish muscle samples incurred with MET and SPY was described.