RESUMEN
Analytical solutions of clofazimine drug substance stored in glass HPLC vials were found to undergo degradation at room temperature occasionally. At the time of each sample preparation, it was unpredictable if a particular solution would undergo such solution degradation. Once the degradation peak was observed in a particular vial, typically within 24h, it would keep growing until reaching a total yield of approximately 2%. By using a strategy that combines LC-PDA/UV-MSn with mechanism-based stress studies, followed by preparative HPLC separation and subsequent structure characterization by 1D and 2D NMR, the unknown peak was identified as a clofazimine nitrite ester. It apparently results from nucleophilic substitution of clofazimine by residual nitrite leaching out of the inner surface of the glass HPLC vials used in the sample preparation. Overall, the percentage of the sample solutions that underwent solution degradation is approximately â¼10% to 15%, when the sample solutions were stored in glass HPLC vials at room temperature. Over the period of the analytical method development, it was found that the occurrence of the degradation can be suppressed when the solutions were stored under refrigerated condition (2 - 8°C) or when the samples were prepared in less acidic diluents.