DEVELOPMENT AND VALIDATION OF A LIQUID CHROMATOGRAPHY - MASS SPECTROMETRIC METHOD FOR THE DETERMINATION OF SULFADIAZINE IN FISH MUSCLE

摘要

Sulfadiazine (SDZ) belongs to antibacterial agents used in fish-farm. Several methods have been developed for the determination of SDZ in fish tissues because of the increasing concern for consumers protection from drug residues. In this study a method by Liquid Chromatography - Mass Spectrometry for the determination of SDZ in muscle tissue of the cultured fish gilthead sea bream (Sparus aurata) and gilthead sea bass (Dicentrarchus labrax) is developed and validated, using dapsone (DPS) as internal standard. Sulfadiazine is extracted by Accelerated Solvent Extractor (ASE) using water as extraction solvent. Optimized extraction conditions are: temperature 100 °C, pressure 1500 psi, static time 15 min, cell size 11 mL, 1 extraction cycle, flush volume 60 %. The ASE extract is diluted to 20 mL, then is cooled and centrifuged. A portion of 4 mL is loaded onto a Nexus (60mg/3mL) SPE cartridge, which has been conditioned with 2 mL methanol and 2 mL water. The cartridge is washed with 4 mL of mixture methanol/water 5/95 (v/v). The cartridge is dried and sulfadiazine is eluted with 2 mL methanol. The mixture is dried and reconstituted with 0.2 mL of water/acetonitrile mixture 90/10 (v/v). The chromatographic analysis was performed using an HPLC system model Alliance 2695 (Waters, USA). An XTerra MS C18 2.1x100 mm, 3.5 nm analytical column was used (Waters, USA). Separation was performed at a column temperature of 40 °C with a mobile phase flow rate of 0.2 mLmin~(-1). Mobile phase was consisted of solvent A (water containing formic acid 0.05 %) and solvent B (acetonitrile containing formic acid 0.05 %) and a gradient elution programme was applied. Initially 90 % of solvent A was reduced to 2 % in the first ten minutes. In the next half of minute is increased to 98 % and maintained for the next six minutes. Injection volume was 5 μL. A ZQ-2000 Micromass mass spectrometer with the MassLynx software (Waters, USA), was operated in positive ESI mode measuring 251 m/z ion for SDZ and 249 m/z ion for DPS. Optimized conditions for MS were: capillary voltage 3.0 kV, cone voltage 20 V, source temperature 120 °C. The method was validated for major analytical characteristics. A calibration curve was found linear in the concentration range 9-150 ngmL~(-1). The Limit of Detection was found 3 μgkg~(-1) and the Limit of Quantification was 9 μgkg~(-1). Method mean Recovery achieved was 90.0±3.6 (mean ± SD) for blank fortified samples (n=6) range at 50,100 and 150 μgkg~(-1). The method is suitable for fast and accurate determination of SDZ residues in cultured sea bream and sea bass.