Chlortetracycline (CTC) is a broad-spectrum antibiotic used in veterinary medicine for pulmonary or digestive infections and having a regulatory maximum residue limit (MRL) necessitating an official analytical control method. The purpose of this study was to clarify the identification of different forms of CTC observed in standard solution, in spiked muscle samples and in naturally incurred muscle samples of pigs analysed by LC-MS/MS and to demonstrate the in vivo formation of 6-iso-chlortetracycline and 4-epi-6-iso-CTC as a metabolite of CTC and 4-epi-CTC in muscle. The six following forms were identified, all being isobaric with a protonated molecule at m/z 479 (precursor ion): the keto-enol forms of CTC and the keto-enol forms of 4-epi-chlortetracycline (4-epi-CTC), 6-iso-chlortetracycline (6-iso-CTC) and 4-epi-6-iso-chlortetracycline (4-epi-6-iso-CTC). The 6-iso-CTC and 4-epi-6-iso-CTC were observed only in incurred pig samples so were identified for the first time as metabolites of CTC and 4-epi-CTC. Identification of the different forms was obtained by comparing incurred muscle samples with standard solutions and with spiked samples. Then the differences between the features of the chromatograms obtained by LC-TQ-MS and the fragmentation study of the different forms of CTC obtained by LC-Q-TOF-MS helped us to support this identification. The extraction steps and the LC-MS/MS conditions developed to analyse muscle tissue samples are described. This clarification concerning the rigorous identification of chromatographic peaks allowed us to evaluate the relevance of our monitoring method with regard to the regulations in place in the European Union and could be of help to laboratories involved in official control of antibiotic residues in food of animal origin. Additional results are also presented highlighting the transformation of the CTC when prepared in a mixture with other antibiotics.
