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.

在分析稳定性批次的酒石酸溴莫尼定 (BMT) 期间，通过高效液相色谱 (HPLC) 检测到一种潜在的未知杂质，其含量范围为 0.03% 至 0.06%。根据液相色谱-质谱法 (LC-MS) 分析，未知杂质结构被推测为 3,6,11,13,16-五氮杂四环 [8.6.0.0²,⁷.0¹²,¹⁶] 十六进制-1,3 ,5,7,9,12-己烯。在使用制备型液相色谱法从富含杂质的反应粗样品中分离后，使用分析应用，如 1D NMR ( ¹ H, ¹³C 和 DEPT-135)、2D NMR（HMBC 和 COSY）、高分辨率质谱 (HRMS) 和红外光谱 (IR)。未知杂质是通过在 CuBr ₂存在下以克级的Ullman 偶联反应从溴莫尼定制备的，具有最佳纯度以进一步用于分析开发。本文首次报道了BMT环化杂质等未知降解杂质的鉴定、结构解析和合成，以及HPLC方法验证。