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Deuterated ginkgolides were obtained by H/D exchange method.Fragmentation pathways of ginkgolides were elucidated by comparing the MS/MS data of deuterated and non-deuterated molecules.A systematic research strategy for qualitative analysis of ginkgolides was developed by HPLC-Q-TOF-MS.H/D exchange is a potential method for analysis research of complex active constituents in TCMs.Ginkgolides, the main active constituents of Ginkgo biloba, possess significant selectively inhibition on platelet-activating factor and pancreatic lipase and attract wide attention in pharmacological research area. In our study, an effective hydrogen/deuterium (H/D) exchange method was developed by exchanging the α-Hs of lactone groups in ginkgolides with Ds, which was very useful for the elucidation of the fragmentation patterns of ginkgolides in Quadrupole Time-of-flight Mass Spectrometry (Q-TOF-MS), especially in accurately distinguishing the type and position of substituent in framework of ginkgolides. Then, a systematic research strategy for qualitative and quantitative analysis of ginkgolides, based on H/D exchange, tandem solid-phase extraction and LC-Q-TOF-MS, was developed, which was successfully applied in each medicinal part of G. biloba, which indicated that ginkgolide B was the most abundant ginkgolide in the seeds of G. biloba (60.6 μg/g). This research was the successful application of H/D exchange in natural products, and proved that H/D exchange is a potential method for analysis research of complex TCMs active constituents.