Quantitative determination of chlortetracycline content in animal plasma at controlled keto-enol tautomerism 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 plasma – necessary to study its pharmacokinetics – 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–ESI-MS/MS method to analyze the CTC content in animal plasma, with the originality that it apports a solution to overcome the possible interference of the keto-enol tautomerism equilibrium, which is influenced by several factors. Therefore, the plasma samples were incubated at 56 °C, forcing the equilibrium completely in favour of the CTC keto-form. The CTC keto-tautomer was stable after deproteinization of the samples with concentrated trifluoroacetic acid, and was quantified by LC–ESI-MS/MS, as a measure of the total CTC content. The method could be validated for pig and chicken plasma by a series of parameters – linearity, trueness, precision, limit of quantification, specificity, stability – in accordance with the European guidelines, and was used for pharmacokinetic studies on pigs and chickens medicated with CTC via the feed.