Detection of tetracycline antibiotic residues in pork,pig viscera, chicken meat and chicken viscera

Residues of oxytetracycline (OTC), tetracycline (TC), doxycycline (DC), and chlortetracycline (CTC) in livestock products were analyzed by paper disk diffusion method, microbial receptor assay (Charm II), enzyme-linked immunosorbent assay (ELSIA), and high performance liquid chromatography (HPLC). The positive samples with paper disk diffusion method, Charm II, ELISA, and HPLC were further confirmed with liquid chromatography tandem mass spectrometry (LC/MS/MS).
LC/MS/MS method was carried out by Oasis® HLB column (2.1 × 20 mm, 15 μm) with mobile phases of 0.3 % formic acid and 0.3 % formic acid in acetonitrile using a linear gradient elution at a flow-rate of 0.3 ml/min. The ionization mode of mass spectrometry was set up with electrospray in positive ion mode, and parent ions of OTC, TC, DC, and CTC were m/z 461, 445, 445, and 479, respectively. The daughter ions of m/z 426 and 443 for OTC, m/z 410, 427, and 428 for TC, m/z 428 for DC, and m/z 444 and 462 for CTC were chosen for the confirmation by product ion spectrum. They were both qualified and quantified by multiple reaction monitoring (MRM) simultaneously. The retention times of OTC, TC, DC, and CTC were 4.96, 4.99, 5.10, and 5.05 min, respectively. Standard curves were plotted by daughter ions against areas from 1 to 100 ppb, and the correlation coefficients (R2) were 0.97 to 0.99.
Compare with each analytical method, the analysis time of Charm II was the shortest, paper disk diffusion method cost minimum and was easily operated, HPLC, ELISA, and LC/MS/MS methods can qualitate and quantitate residual antibiotics concentrations correctly. Method detection limits (MDLs) for OTC and TC with paper disk diffusion method were both 50 ppb, and 10 ppb for DC and CTC. The MDL for total tetracycline antibiotics (TCs) with Charm II was 50 ppb. The MDLs with HPLC were 5 ppb for OTC and TC, and 10 ppb for DC and CTC. The maximum residues limits (MRLs) set up by Department of Health (DOH) of total TCs residues in muscle, liver, and kidney were 0.2, 0.6, and 1.2 ppm, respectively. The analytical methods used in this study including paper disk diffusion method and Charm II for screening, and HPLC and LC/MS/MS for confirmation were suitable for analysis of tetracycline antibiotics in these products.
Residues of TCs were detected from 209 livestock samples including 55 pork, 50 pig viscera, 58 chicken meat, and 46 chicken viscera collected from the supermarkets, sale stores, and traditional markets by the public health bureau from March, 2003 to April, 2005. The results showed that two black-bone-chicken meat were both found DC.