Integrating offline 2D UHPLC-QTOF-MS/MS-based automated analytical platform and UHPLC-QTRAP®-MS: The chemical...

The modified Gegen Qinlian Decoction (MGQD) is an upgraded formula of Gegen Qinlian decoction (GQD) with two additional herbs, Euphorbiae Humifusae Herba and Zingiberis Rhizoma Preparatum based on traditional Chinese medicine (TCM) clinical practice. To elucidate the impact of these additions on the chemical composition of the decoction, this study conducted a comprehensive chemical profiling of MGQD. Firstly, the major components in MGQD were quantified, including total saponins, flavonoids, phenolic acids, inorganic elements, carbohydrates, proteins, and free amino acids. These components collectively accounted for 95.06 % of MGQD, with flavonoids being the most abundant category at 30.41 %. Subsequently, an integrated identification strategy was employed by establishing an in-house mass spectral library for MGQD chemical components and incorporating compound clustering analysis through molecular networking of MS² data obtained from offline two-dimensional ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (2D UHPLC-QTOF MS/MS). This approach enabled the identification of 477 structurally diverse compounds in MGQD. Finally, a quantitative analytical method was developed using ultra high performance liquid chromatography coupled with triple quadrupole linear ion trap tandem mass spectrometry (UHPLC-QTRAP®-MS/MS) for the simultaneous determination of 35 selected compounds, which collectively constituted 23.16 % of MGQD. Among these, baicalin was the most abundant (89.4 ± 1.7 μg/mg), followed by puerarin (32.9 ± 0.4 μg/mg) and wogonoside (20.6 ± 0.3 μg/mg). By comparing with the chemical components with that of the original GQD, the potential pharmacodynamic substances of MGQD was inferred, laying the foundation for subsequent research into the pharmacological mechanism. This study provides an effective analytical approach for studying the complex chemical profiles of TCM formulas.