Widespread occurrence of in-source fragmentation in the analysis of natural compounds by LC-ESI-MS.

Abstract

The In-Source Fragmentation (ISF) of analyte or co-eluting substances produces unintentional fragment ions, which hampers the identification and quantification by liquid chromatography-mass spectrometry (LC-MS). Natural compounds derived from plants also contain fragile moieties that may undergo ISF. However, the characteristics of ISF of natural compounds in LC-MS are still unclear. The ISF behavior of 214 natural compounds was assayed in liquid chromatography-Q/orbitrap mass spectrometry in electrospray ionization mode (ESI) and the extent of ISF was evaluated. Up to 82% of tested compounds undergo ISF and half of the tested natural compounds that contain more than one fragile moiety underwent successive and severe ISF to generate serial structurally-related ISF products. The major ISF-altering moieties for natural compounds were hydroxyl, lactone, glycosyl, and ether, resulting in neutral loss of H2 O or CO, deglycosylation, or cleavage of ether bond, respectively. Some compounds such as terpenoids undergo severe ISF and less than 1% parent form can be observed. For natural compounds, the ISF product with similar structures more likely to cause interference in analysis because the ISF products may share identical mass-to-charge ratio and similar MS2 fragmentation patterns with precursor ions of the homologs in plants. Furthermore, the severe ISF may cause a false negative in the identification of the parent form. In summary, ISF was a highly frequent phenomenon for analysis of natural compounds by LC-ESI-MS, extensive and successive ISF of natural products may cause misannotation and misidentification with the homologs in plants. It should raise awareness of ISF interference during the analysis of natural compounds.