A RP-HPLC method was developed that successfully quantified mixtures of oxytetracycline (OTC), 4-epiOTC, á- and â-apoOTC down to 40, 20, 50 and 140 ng/ml at ambient temperature in less than 35 minutes. A 0.1M ammonium acetate buffer (pH 3.0)-acetonitrile-tetrahydrofuran (72.5:12.5:15, v/v/v) mobile phase was found by means of the statistical simplex method of solvent optimization to give excellent separation of compounds. Quantitative and reproducible isolation of OTC from aqueous and tissue matrices using solid-phase extraction (SPE) C18 cartridges was successful, whereas isolation of the degradation compounds, particularly á- and â-apoOTC from tissue, was not. Using this method, the degradation kinetics of OTC upon exposure to various meat processing parameters (heat, water activity, and the presence of certain additives) in aqueous and tissue matrices were studied. Regression analysis of ln-linear data revealed that pseudo-first order kinetics were observed for all matrices and parameters. Between 60 and 80°C, the rate of OTC degradation (kobs) increased several-fold. Glycerol-adjusted water activities (0.6 to 1.0) had only a minimal effect upon kobs (p > 0.05), although changes in the secondary kinetic parameters (enthalpy and entropy) were evident. Monomeric phosphate increased kobs in aqueous solution in accordance with a base-catalyzed reaction mechanism. Polymeric phosphates significantly decreased (p 0.05), although their secondary effects on the matrix altered the apparent stability of OTC. Binding studies provided evidence that OTC binding to tissue was due in part to complexing protein-bound cations. Experimental evidence indicated that mineral content was the primary factor affecting OTC degradation. Parameter manipulation did not appear to alter the rate-limiting mechanism (E2 elimination) by which OTC breakdown occurred, as was evidenced by the occurrence of enthalpy/entropy compensation. This research has provided a detailed database for the degradation of OTC. Enthalpy/entropy compensation was shown to be a potentially useful tool for studying OTC degradation
