Investigation of the on-column oxidative formation of impurity D during amlodipine impurities profiling by UHPLC.

The determination of related substances in amlodipine salts is a critical aspect of pharmaceutical quality control. This study investigated the unexpected formation of impurity D, a substituted pyridine derivative, observed as an on-column analytical artifact during UHPLC analysis. Although the initial impurity level was low (<0.01%), a pronounced time-dependent increase up to 1.0% was observed during routine analytical sequences. Systematic experiments demonstrated that the increase did not originate from intrinsic sample instability. Instead, it was triggered by a synergistic effect of the mobile phase composition, the pH environment, and stationary phase aging. In particular, the use of MS-compatible ammonium formate buffers or mildly acidic conditions (pH 6.4) accelerated the on-column oxidation process. The results further indicate that the phenomenon is associated with the accumulation of reactive sites or contaminants on column hardware. The effect became evident after approximately 400 injections of samples and reference solutions. A regeneration procedure based on a multicomponent organic solvent mixture was subsequently developed. This treatment effectively restored column performance and reduced impurity D levels to approximately 0.01%. These findings provide guidance for distinguishing true degradation from analytical artifacts in amlodipine impurity profiling and contribute to the reliability of stability-indicating methods.