Analysis of Temocillin and Impurities by Reversed Phase Liquid Chromatography: Development and Validation of the Method

A robust, specific, precise and sensitive high-performance liquid chromatographic method has been described for purity control of temocillin. Chromatographic separation was achieved using a Symmetry C₁₈ (150 × 4.6 mm, 5 µm) column kept at 30 °C. The mobile phase consisted of a gradient mixture of mobile phases A (5 g/L solution of Na₂HPO₄·2H₂O, pH 7) and B (ACN-MeOH-H₂O, 50:10:40 v/v/v) pumped at a flow rate of 1.0 mL/min. UV detection was performed at 235 nm. The developed method was validated according to the ICH guidelines for its robustness, selectivity, sensitivity, precision and linearity. An experimental design was applied for the robustness study. Linearity was assessed both at impurity level in the range from LOQ to 10 % and assay level from 25 % to 150 % (0.6 mg/mL = 100 %). It is the first liquid chromatographic method described for the separation of temocillin and its potential impurities. It was possible to identify four degradation products from the forced degradation studies. The degradants do not interfere with the main peak and other known impurities showing that the method is specific and stability-indicating.