Isolation and structural elucidation of an unknown novel impurity in sulfasalazine by high performance liquid chromatography coupled to mass spectroscopy and toxicology prediction

• One novel SFLE impurity have been spotted in SFLE bulk drug. • Used spectroscopic (NMR) and chromatographic (HPLC and LC-MS) methods. • Covered the possible process associated with this novel SFLE impurity's generation and control. • First to disclose this new SFLE impurity with the structure and toxicology prediction data. Sulfasalazine is authorised for the management of long-term inflammatory conditions, including ulcerative colitis as well as juvenile and adult rheumatoid arthritis. Using high performance liquid chromatography analysis, one unknown impurity with a level greater than 0.2% was found in prototype batches of sulfasalazine during production. The intended impurity under investigation eluted at retention time of around 33 min and had a relative retention time value of 1.79. Preparative high performance liquid chromatogram was used to isolate this unknown impurity from the crude sulfasalazine. A thorough examination of the 1 H-nuclear magnetic resonance spectroscopy ( 1 H-NMR) and mass spectrometry (MS) data revealed the structure of the novel impurity. The management of new impurity creation and the potential molecular pathways leading to its formation were also examined. A new sulfasalazine impurity was characterized and toxicology prediction data was also obtained.