Chemical Composition Analysis of Shengmaiyin (Red Ginseng Formula) Based on UHPLC-Q-Orbitrap MS/MS and Molecular Networking

Traditional Chinese medicine (TCM), with its long history in Asia, has become a focal point in the life sciences in recent years. Among the classic TCM formulas, Shengmaiyin has been widely used in the treatment of coronary heart disease and diabetes. However, the complex chemical composition, low abundance of various constituents, and a large number of isomeric compounds, together with interactions among diverse bioactive ingredients, render the effective separation and identification of components in TCM herbs highly challenging via traditional analytical methods. As a result, the active chemical constituents responsible for the therapeutic effects of Shengmaiyin remain unclear. In this study, ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-MS/MS) combined with GNPS molecular networking was employed to rapidly analyze the chemical components of Shengmaiyin (Red Ginseng Formula). A Supelco C18 column (3.0 mm×50 mm, 2.7 μm) was used with 0.1% formic acid aqueous solution and acetonitrile as the mobile phase for gradient elution. Data acquisition was then performed in full MS scan/data-dependent MS/MS scan (Full MS/dd-MS²) mode. After data collection, chromatographic peak alignment and extraction were conducted using Xcalibur software. Mass spectrometry data were acquired in both positive and negative ion modes. A molecular network was constructed based on the similarity of MS/MS fragmentation patterns, and component identification and species attribution were performed by combining information including retention time, accurate molecular weight, mass spectrometry fragmentation patterns, and characteristic fragment ions of reference standards. Molecular networks of ginsenosides, ophiopogonins, flavonoids, and lignans were established. A total of 91 chemical components were identified from Shengmaiyin (Red Ginseng formula) oral liquid, including 65 saponins, 4 flavonoids, 19 lignans, and 3 nortriterpenoids. Through comparison with published literature on the individual herbs, 41 compounds were attributed to red ginseng, 27 compounds to Ophiopogon japonicus, and 23 compounds to Schisandra chinensis. This method provides a basis for clarifying the pharmacodynamic material basis of Shengmaiyin (Red Ginseng formula), and thus furnishes a scientific foundation for further exploration of its pharmacological effects and mechanisms of action. However, this method is not yet able to identify and attribute unknown compounds, and it is essential to recognize the inherent limitations and applicable scopes of various analytical methods and instruments. Therefore, to achieve efficient and accurate analysis of active components in TCMs, it is advisable to further optimize separation and identification strategies by combining diverse techniques such as nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, thin-layer chromatography, and capillary electrophoresis-mass spectrometry.