Summary:
This thesis describes the development and validation of methods for the
determination of sulfadiazine (SDZ), trimethoprim (TMP), and
acetyl-sulfadiazine (AcSDZ), the main metabolite of SDZ, with dapsone as
internal standard, in tissues of cultured fish, like muscle plus skin in
natural proportion, liver and serum. In Greece, both SDZ and TMP are approved
drugs for use in aquaculture. They are usually used in a ratio SDZ:TMP of 5:1,
for the promotion of growth and for prophylactic and therapeutic purposes
mainly against bacteria diseases.
The wide use of antibiotics in aquaculture results in the presence of drug
residues in fish tissues, that can have undesirable effects on consumer health.
Liquid Chromatography-Mass Spectrometry with high sensitivity and selectivity
was used for the detection and quantitation of residues in concentrations below
the Maximum Residue Limits (MRLs) which have been set by the European Union.
The levels below MRLs are considered to be safe on consumer health. The MRL for
the sum of sulfonamides has been set to 100 μg kg-1 and for TMP 50 μg kg-1 in
muscle plus skin.
Analysis was performed by using High Performance-Liquid Chromatographic system
and the analytical column XTerra MS C18 (100,02,1 mm, 3,5 μm) kept at 40 oC.
The mobile phase was consisted of water and acetonitrile, both solvents
containing formic acid 0,05 % (v/v), and was pumped at a programme of gradient
elution and a constant flow rate of 0,2 mL min-1. The detection of analytes and
internal standard was performed by using the Mass Spectrometric system model ZQ
2000, monitoring the positive (protonated) ions of the drugs.
Extraction of drugs from muscle plus skin and liver samples was carried out by
using water in Accelerated Solvent Extraction. Abselut Nexus 60 mg /3 mL Solid
Phase Extraction cartridges were used for sample cleanup. Liquid-liquid
extraction with ethyl acetate and cleanup with n–hexane were used for serum
sample preparation.
The methods were validated according to the 2002/657/EC European Decision and
were used for the determination of SDZ, TMP and AcSDZ in tissues of gilthead
seabream after a two diet trial. Withdrawal times (Wts) of the drugs were
calculated by WT1.4 software.
Keywords:
Aquaculture, liquid chromatography-mass spectrometry, Method development and validation, Withdrawal time