A gradient method was found necessary to optimize the separation of major degradation products formed under various stress conditions. The best resolution was achieved with initial run of
acetonitrile: phosphate buffer (pH 5.0) in the ratio of 15:85 (v/v) for 1 min, which was then changed to 30:70 (v/v) for next 14 min and finally equilibrated back to the same ratio of 15:85 (v/v) from 15 to 20 min. The method worked well with the mixture of degradation solutions and was even applicable to degraded formulations. Fig. 1a–c shows the chromatographic resolution
of the blank, mixture of stressed samples, and a degraded formulation, respectively.