Dissolved organic matter (DOM) is a highly complex mixture in composition and structure. DOM from diverse sources contains a large number of identical molecular formulae and similar structural features of isomers. The isomers cannot be distinguished by mass spectrometry alone and the molecular formulas are average results. Therefore, the diversity of DOM has not been fully revealed.

In this study, the composition and structure of marine DOM were analyzed by UHPLC-Orbitrap MS/MS to compare the detailed structure. With the powerful separation capability of UHPLC, the ultra-high mass accuracy and resolution of Orbitrap MS and the rich fragment ion information of MS/MS, the composition characteristics of marine DOM were revealed from three levels: retention time of chromatogram, accurate molecular formula and fragment ion distribution.

The peak pattern of the marine DOM mass spectra conformed to the normal distribution. The CHO-containing compounds have the dominated relative abundance, with the highest abundance of O₈ classes. Surface and deep marine DOM were separated into 3 fractions by UHPLC. The type and abundance of compounds in each fraction differed significantly. The highest abundance of CHO class species in fraction I was found in O₁₀ compounds, where N₂O_x class species with lower concentrations were relatively enriched in this fraction. The CHO class species dominated by O₉ and O₈ classes were found in fraction II and III, respectively. It shows that the preliminary separation of marine DOM with highly uniform molecular composition can be achieved by using UHPLC. The UHPLC-Orbitrap MS/MS results showed that the parent ions of the homologous series (such as differing CH₂) in different fractions were fragmented in a similar manner. However, the differences in the species and abundance of fragment ions produced by the same parent ion at different retention times suggest that the same molecular formula exists as different isomers in different fractions of marine DOM. MS/MS fragment ions are expected to be used as biomarkers to determine the structure and formation mechanism of marine DOM, further indicating the characteristics and origin of DOM samples.