Control of the keto-enol tautomerism of chlortetracycline for its straightforward quantitation in pig tissues by liquid chromatography–electrospray ionization tandem mass spectrometry

Chlortetracycline (CTC) is a member of the group of the tetracycline antibiotics used in human and veterinary medicine. Its reliable measurement in edible tissues is hampered by the fact that CTC is subjected to keto-enol tautomerism resulting in its keto- and enol-form. Also, it undergoes epimerization to form its 4-epimer. In this paper, we present an LC–electrospray ionization (ESI)–MS/MS method to analyze the CTC content in pig tissues, with the originality that it apports a solution to overcome the possible interference on the measurement of the keto-enol tautomerism equilibrium, which is influenced by several factors. Therefore, the minced tissue samples were incubated at 56 °C, forcing the equilibrium in favour of the CTC keto-form. The CTC keto-tautomer, as a measure of the total CTC content, was then quantified by LC–ESI–MS/MS, after further extraction of the tissue samples, consisting of a liquid extraction with sodium succinate solution (pH 4.0), followed by protein removal with trichloroacetic acid and paper filtration, and finally solid-phase clean-up on an hydrophilic-lipophilic balance (HLB) polymeric reversed-phase column. The method could be validated for pig muscle, liver and kidney tissues – for which maximum residue limits (MRLs) have been fixed by the European Union – with respect to linearity, trueness and precision, in accordance with the European guidelines.