Identification, separation and structural characterization of process impurity present in Doxepin hydrochloride and quantification by using high performance liquid chromatography

This study aimed to investigate a novel process-related impurity generated during the production of doxepin hydrochloride. A new high-performance liquid chromatography (HPLC) method with a gradient program was developed, revealing that the impurity eluted at approximately 1.5 RRT of the drug substance. Liquid chromatography-mass spectrometry (LC-MS) analysis identified the impurity with a molecular mass of 474.2. Further, the impurity generated during doxepin production was isolated using column chromatography, and its structure was confirmed as a dimer of doxepin through nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy, and high-resolution mass spectrometry (HRMS). The impurity was effectively separated using an HPLC system equipped with a Zorbax Eclipse Plus C18 column ((250 × 4.6) mm, 5 μm) at a column temperature of 40°C. The mobile phase, consisting of 10 mM ammonium acetate and methanol, was delivered in gradient mode, with detection at 254 nm using a diode array detector (DAD). The HPLC method was validated for specificity, linearity, precision, accuracy, and detection threshold, yielding satisfactory results.