Analyte identification in biological samples presents significant challenges, mainly due to the high chemical diversity of metabolites, the presence of co-eluting compounds, and isomeric and isobaric analytes within the sample. Differential Mobility Spectrometry (DMS), using single or binary modifiers, has been integrated as an additional separation dimension to Liquid Chromatography–Mass Spectrometry (LC-MS). A binary modifier was found to provide an optimal equilibrium between peak capacity, sensitivity, and selectivity for the analytes present in the sample.

An advantage of DMS is that it enables the construction of reference libraries for analyte identification based on the selected modifier, in combination with specific compensation and separation voltage. When combined with data-independent acquisition techniques, such as SWATH with LC×DMS/MS analysis, the confidence in analyte identification is further enhanced based on the fragmentation of the precursor ions in addition to accurate mass, retention time, and DMS-specific parameters (modifier, compensation voltage, and separation voltage).

While certain analytes generate limited fragmentation through conventional Collision-Induced Dissociation (CID), alternative fragmentation techniques, such as Electron Activated Dissociation (EAD), have provided additional, informative fragmentation patterns. This is particularly useful for detecting chemical modifications like glucuronide or sulfate conjugates, commonly found in steroids. UV-photodissociation (UVPD) at a wavelength of 266 nm has generated fragmentation profiles similar to CID. These three fragmentation techniques can be employed for analyte confirmation and/or as complementary techniques in one single run.

The combination of LC/SWATH×DMS-MS approach as well as multiple fragmentation techniques (CID, EAD, UVPD) establishes a comprehensive analytical workflow, enabling more reliable identification, structural characterization, and quantification of metabolites in complex biological matrices.