Quantitative multi-residue analysis of tetracyclines and their 4-epimers in pig tissues by high-performance liquid chromatography combined with positive-ion electrospray ionization mass spectrometry

Abstract

Tetracycline antibiotics, including tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC) and doxycycline (DOX), are commonly used in veterinary medicine because of their broad-spectrum acitivity and cost effectiveness. The purpose of this study was to develop and validate a multi-residue method to determine tetracycline residues in edible tissues of pig. Extraction of the tetracyclines (TCs) and their 4-epimers (4-epiTCs), in the presence of the internal standard demethylchlortetracycline (DMCTC), was performed using a liquid extraction with sodium succinate solution (pH 4.0), followed by protein removal with trichloroacetic acid and paper filtration. Further solid-phase clean-up on an HLB polymeric reversed phase column was performed to obtain an extract suitable for LC–MS/MS analysis. Chromatographic separation of the TCs, the 4-epiTCs and DMCTC was achieved on a PLRP-S polymeric reversed phase column, using a mixture of 0.001 M of oxalic acid, 0.5% (v/v) of formic acid and 3% (v/v) of tetrahydrofuran (THF) in water (A) and tetrahydrofuran (B) as the mobile phase, and at a column temperature of 60 °C. All TCs and their 4-epimers could be identified using the MS/MS detection technique, and subsequently quantified. The method has been validated according to the requirements of the EC at the MRL (100 ng/g for muscle, 300 ng/g for skin+fat and liver, and 600 ng/g for kidney), half the MRL and double the MRL levels, as well for the TCs as for the 4-epiTCs. Calibration curves were prepared for all tissues and good linearity was achieved over the concentration ranges tested ( r>0.99 and goodness-of-fit <10%). Limits of quantification of half the MRLs were obtained for the analysis of the TCs and the 4-epiTCs in muscle, skin+fat, liver and kidney tissues of pig. Limits of detection ranged between 0.5 and 4.5 ng/g. The results for within-day precision (R.S.D.%) and for accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of doxycycline in tissue samples of pigs medicated with doxycycline in the feed and of oxytetracycline in tissue samples of calves medicated with oxytetracycline by intramuscular injection. It is also routinely used in our laboratory for the confirmation of tetracyclines in chicken and pig muscle samples which had positive screening results.