Stereoselective HPLC separation and configurational stability study of the N-nitrosamine impurity of indapamide

2-Methyl-1-nitroso-2,3-dihydro-1H-indole, known as impurity A in the European Pharmacopoeia, is a chiral nitrosamine impurity potentially related to the antihypertensive drug indapamide. Its chirality arises from a stereogenic center on the indoline ring. Additionally, the asymmetric substitution at the nitrogen atom and the partial double-bond character of the N-N bond give rise to E/Z stereoisomerism. This study aimed to achieve simultaneous HPLC separation of all four possible stereoisomers using polysaccharide-based chiral stationary phases under both normal and reversed-phase conditions. Optimal enantio‑ and diastereoselective separation was achieved using a chiral stationary phase consisting of amylose tris[(S)-α-methylbenzylcarbamate] immobilized onto 3 μm silica particles, operated at 5 °C with a 50:50 (v/v) acetonitrile-water mobile phase. The chromatographic profiles indicated on-column E/Z interconversion, as evidenced by dynamic chromatogram shapes. The energy barrier for interconversion was estimated, and the full stereochemical characterization of all isomers was accomplished by integrating theoretical calculations, HPLC data, and chiroptical analysis.