Isolation and characterization of thermal degradation impurity in brimonidine tartrate by HPLC, LC–MS/MS, and 2DNMR

Abstract

One potential unknown impurity was detected during the analysis of stability batches of brimonidine tartrate (BMT) in the level ranging from 0.03 % to 0.06 % by high-performance liquid chromatography (HPLC). Based on the liquid chromatography-mass spectrophotometry (LC–MS) analysis, the unknown impurity structure was presumed as 3,6,11,13,16-pentaazatetracyclo [8.6.0.0²,⁷.0¹²,¹⁶] hexadeca-1,3,5,7,9,12-hexaene. The proposed structure was elucidated, after its isolation using preparative liquid chromatography from the impurity enriched reaction crude sample, using analytical applications such as 1D NMR (1H, 13C and DEPT-135), 2D NMR (HMBC and COSY), high-resolution mass spectrometry (HRMS) and infrared spectroscopy (IR). The unknown impurity was prepared from brimonidine by following Ullman coupling reaction in the presence of CuBr2 in gram scale with optimum purity to use further in analytical developments. The identification, structural elucidation and synthesis of unknown degradation impurity such as BMT-cyclized impurity, and HPLC method validation were reported for the first time in this paper.