An integrated analytical approach based on enhanced fragment ions interrogation and modified Kendrick mass defect filter data mining for in-depth chemical profiling of glucosinolates by ultra-high-pressure liquid chromatography coupled with Orbitrap high resolution mass spectrometry

High resolution mass spectrometry (HRMS)-based analytical technique promotes the discovery and development of new bioactive molecules from natural sources. However, challenges for MS analysis of natural products include their structural diversity, numerous trace components, as well as the interference from complex matrices that limits the rapid detection and identification of all target analytes in the extracts. Herein, we presented an integrated analytical approach to obtain chemical profile of glucosinolates (GLSs) in Eutrema yunnanense, a perennial herb, which is used as a condiment (Wasabi), by ultra-high-pressure liquid chromatography coupled with Orbitrap high resolution mass spectrometry (UHPLC-Orbitrap/HRMS). The intelligent AcquireX deep scan greatly improved the detection efficiency and coverage of data-dependent acquisition (DDA) mode, and enhanced structurally significant product ions interrogation by generating exhaustive MS/MS spectra with more informative fragmentation. Massive HRMS data mining for searching GLSs was then achieved by a modified Kendrick mass defect filter (MKMDF), which enabled the visualization of their homologous characteristics and reduced the complicacy of data post-processing. Ultimately, a total of 175 GLSs were tentatively identified or characterized based on the MS fragmentation patterns, including 52 potentially new compounds among which 37 malonylated GLSs were first discovered. These compounds were then applied to analyse the chemical differentiation between the rhizome and leaf of E. yunnanense. This study provides a feasible approach for screening and confident structure characterization of GLSs and has practical implications for profiling other natural bioactive homologous compounds.