An Integrated Strategy for Comprehensive Characterization of Traditional Chinese Medicine (TCM) Formulas: A Case Study of Gegen‐Qinlian Decoction

ABSTRACT Objective An integrated strategy was utilized in this study to comprehensively characterize the chemical composition of Gegen‐Qinlian Decoction (GQD). Methods The major components of GQD (total flavonoids, total saponins, total alkaloids, carbohydrates, and proteins) were determined firstly using colorimetric methods, while free amino acids and trace elements were analyzed by automatic amino acid analyzer and inductively coupled plasma‐mass spectrometry (ICP‐MS), respectively. After that, the small‐molecule components of GQD were characterized using ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (UHPLC‐QTOF‐MS/MS). Furthermore, a quantitative method was established for 26 representative compounds determination simultaneously using ultra‐high performance liquid chromatography coupled with triple‐quadrupole linear ion‐trap tandem mass spectrometry (UHPLC‐QTRAP‐MS/MS) in multiple reaction monitoring (MRM) mode. Results The accurate content and proportion of the major constituents in GQD were determined for the first time. A total of 230 compounds were characterized from GQD, of which four undescribed alkaloids were identified initially by their tandem mass data analyses based on mass spectral fragmentation pathways of isoquinoline alkaloids. A quantitative method was established to determine 26 representative compounds, and validated through linearity, precision, repeatability, stability, and recovery. The total content of the 26 compounds accounted for 27.81%, of which baicalin being the most abundant was up to 107.26 ± 1 mg/g. Conclusions The present study achieved a comprehensive characterization of constituents in GQD, laying the foundation for elucidating the active components, mechanism, and the quality control of GQD, offering a valuable analytical framework for research on other traditional Chinese medicine (TCM) formulas.