Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Nov 1.
doi: 10.1007/s00216-025-06195-2. Online ahead of print.

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

Maria Grübner  1   2 , Andreas Dunkel  3   4 , Frank Steiner  2 , Thomas Hofmann  5
Affiliations

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

Maria Grübner et al. Anal Bioanal Chem. .

Abstract

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.

Keywords: Comprehensive offline two-dimensional LC; Evaporation; Fractionation; HILIC; Metabolomics; Mixed-mode chromatography.

PubMed Disclaimer

Conflict of interest statement

Declarations. Conflict of interest: The authors declare the following competing financial interest: Maria Grübner and Frank Steiner are employed by Thermo Fisher Scientific, the manufacturer of the UHPLC system and columns used for this work. Andreas Dunkel and Thomas Hofmann have no relevant financial or non-financial interests to disclose.

References

    1. Spagou K, Tsoukali H, Raikos N, Gika H, Wilson ID, Theodoridis G. Hydrophilic interaction chromatography coupled to MS for metabonomic/metabolomic studies. J Sep Sci. 2010;33:716–27. https://doi.org/10.1002/jssc.200900803 . - DOI - PubMed
    1. Wolfender J-L, Marti G, Thomas A, Bertrand S. Current approaches and challenges for the metabolite profiling of complex natural extracts. J Chromatogr A. 2015;1382:136–64. https://doi.org/10.1016/j.chroma.2014.10.091 . - DOI - PubMed
    1. Wishart DS, Guo A, Oler E, Wang F, Anjum A, Peters H, Dizon R, Sayeeda Z, Tian S, Lee BL, Berjanskii M, Mah R, Yamamoto M, Jovel J, Torres-Calzada C, Hiebert-Giesbrecht M, Lui VW, Varshavi D, Varshavi D, Allen D, Arndt D, Khetarpal N, Sivakumaran A, Harford K, Sanford S, Yee K, Cao X, Budinski Z, Liigand J, Zhang L, Zheng J, Mandal R, Karu N, Dambrova M, Schiöth HB, Greiner R, Gautam V. HMDB 5.0: the human metabolome database for 2022. Nucleic Acids Res. 2022;50(D1):D622–31. https://doi.org/10.1093/nar/gkab1062 . - DOI - PubMed
    1. Roca M, Alcoriza MI, Garcia-Cañaveras JC, Lahoz A. Reviewing the metabolome coverage provided by LC-MS: focus on sample preparation and chromatography-a tutorial. Anal Chim Acta. 2021;1147:38–55. https://doi.org/10.1016/j.aca.2020.12.025 . - DOI - PubMed
    1. Ortmayr K, Causon TJ, Hann S, Koellensperger G. Increasing selectivity and coverage in LC-MS based metabolome analysis. TrAC Trends Anal Chem. 2016;82:358–66. https://doi.org/10.1016/j.trac.2016.06.011 . - DOI

LinkOut - more resources