Development and validation of a reversed phase liquid chromatographic method for analysis of oxytetracycline and related impurities

A simple, robust and fast high-performance liquid chromatographic method is described for the analysis of oxytetracycline and its related impurities. The principal peak and impurities are all baseline separated in 20 min using an Inertsil C8 (150 mm × 4.6 mm, 5 μm) column kept at 50 °C. The mobile phase consists of a gradient mixture of mobile phases A (0.05% trifluoroacetic acid in water) and B (acetonitrile–methanol–tetrahydrofuran, 80:15:5, v/v/v) pumped at a flow rate of 1.3 ml/min. UV detection was performed at 254 nm. The developed method was validated for its robustness, sensitivity, precision and linearity in the range from limit of quantification (LOQ) to 120%. The limits of detection (LOD) and LOQ were found to be 0.08 μg/ml and 0.32 μg/ml, respectively. This method allows the separation of oxytetracycline from all known and 5 unknown impurities, which is better than previously reported in the literature. Moreover, the simple mobile phase composition devoid of non-volatile buffers made the method suitable to interface with mass spectrometry for further characterization of unknown impurities. The developed method has been applied for determination of related substances in oxytetracycline bulk samples available from four manufacturers. The validation results demonstrate that the method is reliable for quantification of oxytetracycline and its impurities.

Typical chromatogram obtained by analyzing a bulk sample of oxytetracycline dihydrate using the final method. Chromatographic conditions: Inertsil C8 (150 mm × 4.6 mm, 5 μm) column, mobile phase A (Water 0.05% TFA) and mobile phase B (80:15:5, v/v/v, ACN, MeOH and THF), flow rate 1.3 ml/min, column temperature 50 °C, injection volume 10 μl and UV detection at 254 nm. The gradient program [time (min)/%B] was set as 0/10, 5/10 to 20/35 to 25/10. Unk = unknown.Figure optionsDownload as PowerPoint slideHighlights
► A new reversed phase high-performance liquid chromatographic method for analysis of oxytetracycline and its related impurities is described.
► The proposed method avoids the use of non-volatile buffers in the mobile phase, suitable to interface with mass spectrometry (MS).
► Satisfactory separation of oxytetracycline and its related impurities was achieved.
► MS was utilized for identification and characterization of unknown impurities.
► The developed method has been successfully applied for purity control of oxytetracycline bulk samples.