Comparative evaluation of comprehensive offline 2D-LC strategies coupled to MS for untargeted metabolomic studies of human urine

Out of the broad selection of analytical methods applied in metabolomic studies, liquid chromatography coupled to mass spectrometry (LC-MS) has the highest coverage potential. In that regard, the quality of the separation process is crucial for the analytical outcome. Reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) are widely applied, and the potential of various setups to combine these modes for more complementary data has been deeply explored. In our previous study on the orthogonality of LC conditions in the field of metabolomics, combinations of a mixed-mode phase with parallel RP and ion-exchange (IEX) properties and several HILIC columns exhibited the widest compound distributions in a two-dimensional (2D) separation space. For further performance evaluation, an offline comprehensive 2D-LC-TOF-MS (LC×LC-TOF-MS) system was set up with the mixed RP/IEX mode in the first dimension (¹D) and HILIC mode in the second dimension (²D). The transfer of fractions to the HILIC column and the effect of offline fraction preparation procedures (dilution and evaporation approaches) were comparatively investigated by using reference substances. In addition, the separation performance of the offline LC×LC-TOF-MS system with and without offline fraction preparation was assessed in comparison to other common LC-TOF-MS strategies (direct flow injection DFI, 1D-LC, serial coupling LC) by the number of detectable features in a human urine sample. In conclusion, the direct transfer of 5 µL fraction volumes without offline treatment was the most promising approach for future application in untargeted metabolomic studies for marker identification from human urine.