A novel dereplication strategy for the identification of two new trace compounds in the extract of Gastrodia elata using UHPLC/Q-TOF-MS/MS

• UHPLC/Q-TOF-MS/MS analysis offered complementary structure information of parishins.
• 64 compounds including 15 new ones were identified from Gastrodia elata.
• LC/MS guided isolation of G. elata yielded two new parishins.
• NMR analysis of two new parishins was consistent with LC/MS analysis.

An ultra performance liquid chromatography (UHPLC) coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS/MS) was used in the structural determination of natural compounds in Gastrodia elata. A total of 64 compounds were identified or tentatively characterized. The strategy used for characterization was comparing their retention time and fragmentation behaviors with those of the reference standards, or investigating their accurate mass measurements and characteristic fragmentation patterns followed by low-energy collision dissociation tandem mass spectrometry (CID-MS/MS). Phenolic conjugates mainly underwent consecutive losses of gastrodin residues and combined losses of H2O and CO2 from their citric acid units under negative MS/MS conditions. According to these rules, we have successfully characterized fifteen potential novel compounds. To confirm the reliability of this strategy, two targeted unknown trace parishins were obtained from G. elata by LC/MS-guided isolation. Based on the analysis of data from NMR spectroscopy and other techniques, the two unknown parishins were identified as 2-[4-O-(β-d-glucopyranosyl)benzyl]-3-methyl-citrate (parishin J) and 1,2-di-[4-O-(β-d-glucopyranosyl)benzyl]-3-methyl-citrate (parishin K), respectively. The fully established structures were consistent with the MS-oriented structural elucidation. This study expanded our knowledge on parishins in Gastrodia species, and the proposed strategy was proven efficient and reliable in the discovery of new minor compounds from herbal extracts.