Identification and quality control of isomers in Huo-Xiang-Zheng-Qi Mixture using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry and inductive effects analysis

Huo-Xiang-Zheng-Qi Mixture is a renowned traditional Chinese medicine formula used to treat ailments associated with dampness pathogens. This study employed ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry to perform a comprehensive qualitative and quantitative analysis of the chemical compounds in Huo-Xiang-Zheng-Qi Mixture. A total of 155 compounds were identified, including 61 flavonoids and their glycosides, 36 phenylethanoid glycosides, 23 saponins, 14 coumarins, 9 organic acids, 1 amino acid, 2 nucleosides and purines, and 9 additional compounds. For the first time, a practical method based on inductive effects and hydrogen bonding was developed to determine the elution order of PhGs isomers. The relative quantification of 9 isomers and the absolute quantification of 10 compounds in Huo-Xiang-Zheng-Qi Mixture were determined, primarily derived from tangerine peel, licorice and Magnolia officinalis. Notably, 18 (3 - glycyrrhetinic acid and 9 Phenylethanoid glycosides isomers were quantified for the first time in the Huo-Xiang-Zheng-Qi prescription. These findings were compared with corresponding values in Huo-Xiang-Zheng-Qi oral liquid. The research revealed relatively low levels of 18 (3 - glycyrrhetinic acid in the mixture and significant differences in the content of four key compounds: magnolol, honokiol, glycyrrhizic acid and hesperidin. This study offers valuable insights into the chemical composition of Huo-Xiang-Zheng-Qi Mixture and provides a foundation for optimizing preparation processes, improving therapeutic efficacy, and establishing quality standards.