An integrated approach for structural characterization of Gui Ling Ji by traveling wave ion mobility mass spectrometry and molecular network

Gui Ling Ji (GLJ), an ancient reputable traditional Chinese medicine (TCM) formula prescription, has been applied for the treatment of oligospermia and asthenospermia in clinical practice. However, its inherent compounds have not yet been systematically elucidated, which hampers developing standards or guidelines for quality evaluation and even the understanding of pharmacological effects. In this study, an integrated approach has been established for comprehensive structural characterization of GLJ. Mass spectrometry datasets of GLJ and each of the single herb medicines in this prescription have been developed by dynamic exclusion fast data-dependent acquisition and high-definition data-independent acquisition modes on ultra-high-performance liquid chromatography coupled with travelling wave ion mobility quadrupole time-of-flight mass spectrometry (UPLC-TWIMS-QTOF-MS). A global natural product social molecular networking (GNPS) platform was then applied for the visualization of chemical space of GLJ and further for the high throughput identification of the targeted or untargeted compounds due to the support of data-transmitting from each single herbal medicine to the formula GLJ. Moreover, drift time, predicted CCS, and diagnostic fragment ions were induced for annotating isomer compounds. Consequently, based on molecular network and library hits, a total of 257 compounds from GLJ, which were classified into 4 structural types, were positively or tentatively characterized. Among them, 20 potential new compounds were detected and 30 pairs of isomers were comprehensively distinguished. The established strategy was effective for attribution, classification, recognition of various constituents, and also was valuable for integrating large amounts of disordered MS/MS data and mining trace compounds in other complex chemical or biochemical systems.