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To gain insight into the chemical composition and characteristics of petroleum, its characterization and structural identification at the molecular level is essential. To this aim, mass spectrometry techniques, especially ultrahigh-resolution mass spectrometry and its coupling with other complementary tools such as hydrogen–deuterium exchange, tandem mass spectrometry are important analytical methods. In particular, the analytical power of ion mobility (IM) coupled with high-resolution mass spectrometry (IMMS) in structural characterization has expanded its scope from biomolecules to petroleum since 2009, providing improved peak capacities and more accurate structural assignments in petroleum samples.