Technical Approach for Structural Analysis of an Unknown Compound in Huoxiang Zhengqi Oral Liquid based on Linear Ion Trap Mass Spectrometry

Chinese herbal compounds (CHCs) play an irreplaceable role in Chinese medicine, and identifying their complex constituents has been a significant research focus in recent years. Huoxiang Zhengqi oral liquid is a classic Chinese patent medicine whose chemical composition requires further investigation at the molecular level. Traditional mass spectrometers, such as time-of-flight and Orbitrap, typically only provide secondary fragmentation information. Based on a linear ion trap mass spectrometer, compounds could be broken down more thoroughly, thereby obtaining deeper fragment information. This paper develops a structural exploration technique for unknown compounds in CHCs, encompassing sample pretreatment, ultra-performance liquid chromatography preparation, mass spectrometry preparation, full-spectrum testing, secondary mass spectrometry testing, multi-level mass spectrometry testing, and result analysis. Representative results demonstrate the derivation process of the compound structure. We discuss factors influencing the experimental technique, such as isomers, polyhydroxy compounds, and instrument resolution. Based on the derivation of the microscopic molecular structure of unknown compounds through multistage mass spectrometry fragmentation, the established experimental method is versatile and applicable for structural characterization of bioactive small molecules in traditional Chinese medicine and their linkage to pharmacological mechanisms.