Abstract: During the development and analysis of low-concentration epinephrine injections, two unreported trace impurities (impurity H and impurity M) have been newly identified. Their analysis and identification were particularly difficult due to their low content, poor stability, and difficult separation in some cases. For impurity H with short retentiontime, poor separation, low content and poor stability, a two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) with column switching technique was employed. This enabled on-line separation, desalination and structure characterization. In contrast, impurity M which exhibits better separability and stability, was isolated and prepared through enrichment and concentration processes. Its structure was then identified using techniques such as liquid chromatography-high resolution mass spectrometry (LC-HRMS) and NMR, enabling the acquisition of more comprehensive structural information. Experimental results showed that impurity H achieved effective separation in the first-dimensional chromatography and successful on-line desalination and fraction cutting in the second-dimensional chromatography. Its potential chemical structure was deduced through multi-stage mass spectrometry data analysis. For impurity M, efficient enrichment and concentration were accomplished; its structure was further confirmed by combining molecular weight and fragment information from LC-HRMS with spectral data from NMR. This study compared the performance of 2DLC-MS/MS with column switching technology with the traditional impurity concentration and identification technology. The experimental results not only provide a valuable strategy for analyzing and identifying trace impurities in complex matrix samples, but also offer an effective analytical tool for the quality control of trace impurities in low concentration epinephrine injections.