Acid-Base speciation and stability of ceftiofur: overcoming alkaline degradation via point-by-point...

Ceftiofur is a third-generation cephalosporin extensively used in veterinary medicine. This study examines its chemical stability, acid-base equilibria, and quantitative determination by UV-Vis spectrophotometry. We demonstrate that conventional spectrophotometric titration procedures lead to inaccurate acidity constants due to significant degradation of the molecule under alkaline conditions, which compromises equilibrium measurements. To overcome these kinetic and pH-induced instabilities, a point-by-point analysis methodology was implemented, allowing independent evaluation at each pH value and minimizing exposure to degrading conditions. This strategy effectively suppressed decomposition effects and enabled the reliable determination of three acidity constants: pK_a1 = 1.75 ± 0.11, pK_a2 = 2.99 ± 0.03, and pK_a3 = 10.81 ± 0.02. Based on these experimentally determined constants, a deprotonation mechanism is proposed that is consistent with both spectroscopic evidence and computational predictions. Additionally, a robust and sensitive analytical method was developed for ceftiofur quantification, achieving limits of detection (0.130 ± 0.001) μM and quantification (0.45 ± 0.003) μM, suitable for determination in pharmaceutical formulations and animal-derived samples, in compliance with international regulatory standards.