Summary:
DEVELOPMENT AND VALIDATION OF A METHOD FOR THE DETERMINATION OF TETRACYCLINE
RESIDUES IN FISH TISSUES BY LIQUID CHROMATOGRAPHY - UV DETECTION / TANDEM MASS
SPECTROMETRY
Tetracycline antibiotics (TCAs) are broad-spectrum medicinal drug compounds
active against a number of gram-positive and gram-negative bacteria. TCAs have
been successfully used worldwide in both veterinary and human medicine. These
antibiotics are widely used as veterinary drugs for food-producing animals
because of their broad-spectrum activity and cost effectiveness. The commonly
used TCAs in veterinary medicine are tetracycline (TC), oxytetracycline (OTC),
chlortetracycline (CTC) and doxycycline (DC). TCAs are susceptible to
conformational degradation to their 4-epimers. This degradation can occur in
aqueous solutions, but also in biological matrices during sample preparation.
In order to protect the consumer for exposure to residues of TCAs in edible
tissues of animal origin, maximum residue limit (MRL) values were laid down by
the EU. The tolerable MRL values for TC, OTC, CTC and DC and their epimers in
fish tissues has been established at 0.1 μg/g (100 ng/g).
In the present study, a LC – UV-MS/MS method for the determination of four
tetracycline residues and three epimers in fish tissues has been developed.
Initially, different analytical columns were tested and parameters of the LC
system such as composition, flow rate and pH of mobile phase and the timetable
for gradient elution were carefully selected to achieve satisfactory resolution
of the analytes and their epimers. Subsequently, the extraction of the
tetracyclines and the three epimers was studied, using two different extraction
solutions, Mcllvaine/EDTA (pH 4) and CH3COONH4-CH3COOH/EDTA (pH 4). Moreover,
the effect of different clean-up procedures of the extract, such as solid phase
extraction (C18 or polymeric SPE) and dispersive solid phase extraction (C18
and PSA dSPE) were investigated, choosing the latter as the faster and more
robust and the method was transferred to HPLC – MS/MS system.
The final method was validated and the estimated parameters were linearity,
trueness (recoveries were > 66,9%), precision (RSDR <15%) and instrumental and
method limits of detection (LOD: 0.03 – 1.24 ng/g and LOQ: 0.09 – 3.72 ng/g).
Quantification was performed by the standard addition method to blank tissue
samples and especially for DC, due to low recoveries (45,0%) the quantification
was performed coupled with an internal standard method.
Keywords:
Tetracyclines, Epimeres, Spe, Dspe, Ic ms ms
