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An UHPLC-Q/TOF-MS method was applied to profile major constituents in E. fischeriana.The reasonable fragmentation mechanism of four jolkinolide components was depicted.A novel compound named jolkinomethylide was determined and elucidated based on MS and NMR data.Ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) in positive ion mode was used to profile and identify the major constituents in Euphorbia fischeriana Steud. (E. fischeriana). An Agilent ZORBAX SB-C18 column was used to separate the crude extract of E. fischeriana, and the mobile phases were acetonitrile and 0.1% aqueous formic acid (v/v). A total of 40 peaks were detected, and 37 components, including phloroglucinol derivatives, diterpenoids and nitrogenous compounds, were identified based on their accurate masses and fragmentation characteristics. The chemical structures of 7 of the detected compounds were determined by comparing standard compounds to the compounds that were isolated from E. fischeriana. Reasonable fragmentation mechanisms and key fragment ions of the four jolkinolide components were determined to aid in the identification of the other diterpenoids in E. fischeriana. In addition, the compound corresponding to peak 18, namely, jolkinomethylide, was determined to be new, and its chemical structure was determined and elucidated based on MS and NMR spectroscopic data. The present study provided valuable information on the new components in E. fischeriana and established a practical method for evaluating the quality of E. fischeriana.