Objective: To explore the impurities of berberine hydrochloride extracted from plant and its tablets, identify the structure of unknown impurity, establish the anal. procedure of related substances and provide reference for the revision and addition of the related substances of berberine hydrochloride and berberine hydrochloride tablets in the Chinese Pharmacopoeia 2020.Methods: The major unknown impurities in berberine hydrochloride were identified by LC-MS, and the anal. procedure of related substances of berberine hydrochloride in the Chinese Pharmacopoeia 2020 was optimized and verified.The related substances of multiple batches of active pharmaceutical ingredients and its tablets were determinedResults: In addition to the known impurities of jatrorrhizine hydrochloride and palmatine hydrochloride listed in the related substances of berberine hydrochloride in the Chinese Pharmacopoeia 2020, three unknown impurities of demethyleneberberine, berberrubine and thalifendine beyond the identification limit(0.1%) were identified for the first time.The HPLC method was established for simultaneous determination of those impurities, and the methodol. validation was made.The system suitability, specificity, linearity, limit of detection, limit of quantitation, repeatability, intermediate precision, accuracy and robustness met the requirements.The multiple batches of samples were determinedConclusion: On the basis of the current anal. method and limit requirements of related substances of berberine hydrochloride in the Chinese Pharmacopoeia 2020, the chromatog. condition is optimized and the control limit of known impurities is set to better control the quality of berberine hydrochloride and berberine hydrochloride tablets.