Determination and characterization of two degradant impurities in bendamustine hydrochloride drug product.

Bendamustine hydrochloride is an alkylating antitumor agent with a good efficacy in the treatment of chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin's lymphoma (B-NHL). Under the stressed conditions, two degradant impurities in bendamustine hydrochloride drug product were detected by high-performance liquid chromatography. These two degradant impurities were isolated from preparative liquid chromatography, and were further characterized using Q-TOF/MS and nuclear magnetic resonance (NMR). Based on the MS and NMR spectral data, they were characterized as 4-[5-(2-chloro-ethylamino)-1-methyl-1H-benzoimidazol-2-yl] butyric acid hydrochloride (impurity-A) and 4-{5-[[2-(4-{5-[bis-(2-chloroethyl) amino]-1-methyl-1H-benzoimidazol-2-yl}-butyryloxy)-ethyl]-(2-chloroethyl)amino]-1-methyl-3a, 7a-dihydro-1H-benzoimidazol-2-yl} butyric acid hydrochloride (impurity-B). Isolation, structural elucidation of these two impurities by spectral data (Q-TOF/MS, (1)H NMR, (13)C NMR, D2O exchange NMR and two-dimensional NMR) and the probable formation mechanism of the impurities were discussed.