A Powerful Four-Dimensional Separation Method for Complex Samples


A two-dimensional LC (2D-LC) method was coupled to an ion mobility–high-resolution mass spectrometer (IM–MS), which enables the separation of complex samples in four dimensions [2D-LC, ion mobility (IM) and mass spectrometry (MS)]. This approach works as a continuous multiheart-cutting LC-system, using a long modulation time of four minutes, in comparison to comprehensive two-dimensional liquid chromatography, which allows the complete transfer of most of the first dimension peaks to the second dimension column without fractionation. Hence, each compound delivers only one peak in the second dimension, which simplifies the data handling even when ion mobility as a third and mass spectrometry as a fourth dimension are introduced. The analysis of different complex samples, such as a plant extract from Hediotys diffusa and Scutellaria barbata, a waste water inflow, and a biocoal sample, was shown. The results of the four-dimensional separation method demonstrate that with the same column combination and the same solvents and gradients, that means without method optimization, totally different samples can be separated with outstanding separation power. Each sample was spiked with cyclophosphamide and ifosfamide and the ion suppression was determined by comparison of the peak area in the complex samples and in pure water after analysis of these samples with a 1D-LC and a 2D-LC approach. It is shown that the 2D-LC method allows an external calibration for the spiked compounds in the plant and waste water sample because of the higher separation power in comparison with 1D-LC.

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  1. 1.

    Mukhopadhyay R. IMS/MS: its time has come. Anal Chem. 2008;80:7918–20. doi:10.1021/ac8018608.

    CAS  Article  Google Scholar 

  2. 2.

    Lapthorn C, Pullen F, Chowdhry BZ. Ion mobility spectrometry-mass spectrometry (IMS-MS) of small molecules: separating and assigning structures to ions. Mass Spectrom Rev. 2013;32:43–71. doi:10.1002/mas.21349.

    CAS  Article  Google Scholar 

  3. 3.

    Pringle SD, Giles K, Wildgoose JL, Williams JP, Slade SE, Thalassinos K, et al. An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument. Int J Mass Spectrom. 2007;261:1–12. doi:10.1016/j.ijms.2006.07.021.

    CAS  Article  Google Scholar 

  4. 4.

    May JC, McLean JA. Ion mobility-mass spectrometry: time-dispersive instrumentation. Anal Chem. 2015;87:1422–36. doi:10.1021/ac504720m.

    CAS  Article  Google Scholar 

  5. 5.

    Campuzano I, Bush MF, Robinson CV, Beaumont C, Richardson K, Kim H, Kim HI. Structural characterization of drug-like compounds by ion mobility mass spectrometry: comparison of theoretical and experimentally derived nitrogen collision cross sections. Anal Chem. 2012;84:1026–33. doi:10.1021/ac202625t.

    CAS  Article  Google Scholar 

  6. 6.

    Hofmann J, Struwe WB, Scarff CA, Scrivens JH, Harvey DJ, Pagel K. Estimating collision cross sections of negatively charged N-glycans using traveling wave ion mobility-mass spectrometry. Anal Chem. 2014;86:10789–95. doi:10.1021/ac5028353.

    CAS  Article  Google Scholar 

  7. 7.

    Kanu AB, Dwivedi P, Tam M, Matz L, Hill HH. Ion mobility-mass spectrometry. J Mass Spectrom. 2008;43:1–22. doi:10.1002/jms.1383.

    CAS  Article  Google Scholar 

  8. 8.

    Tao L, McLean JR, McLean JA, Russell DH. A collision cross-section database of singly-charged peptide ions. J Am Soc Mass Spectrom. 2007;18:1232–8. doi:10.1016/j.jasms.2007.04.003.

    CAS  Article  Google Scholar 

  9. 9.

    Kurulugama RT, Darland E, Kuhlmann F, Stafford G, Fjeldsted J. Evaluation of drift gas selection in complex sample analyses using a high performance drift tube ion mobility-QTOF mass spectrometer. Analyst. 2015;140:6834–44. doi:10.1039/c5an00991j.

    CAS  Article  Google Scholar 

  10. 10.

    Counterman AE, Valentine SJ, Srebalus CA, Henderson SC, Hoaglund CS, Clemmer DE. High-order structure and dissociation of gaseous peptide aggregates that are hidden in mass spectra. J Am Soc Mass Spectrom. 1998;9:743–59. doi:10.1016/S1044-0305(98)00052-X.

    CAS  Article  Google Scholar 

  11. 11.

    Valentine SJ, Counterman AE, Clemmer DE. A database of 660 peptide ion cross sections: use of intrinsic size parameters for bona fide predictions of cross sections. J Am Soc Mass Spectrom. 1999;10:1188–211. doi:10.1016/S1044-0305(99)00079-3.

    CAS  Article  Google Scholar 

  12. 12.

    Bush MF, Hall Z, Giles K, Hoyes J, Robinson CV, Ruotolo BT. Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal Chem. 2010;82:9557–65. doi:10.1021/ac1022953.

    CAS  Article  Google Scholar 

  13. 13.

    Paglia G, Williams JP, Menikarachchi L, Thompson JW, Tyldesley-Worster R, Halldorsson S, et al. Ion mobility derived collision cross sections to support metabolomics applications. Anal Chem. 2014;86:3985–93. doi:10.1021/ac500405x.

    CAS  Article  Google Scholar 

  14. 14.

    Paglia G, Angel P, Williams JP, Richardson K, Olivos HJ, Thompson JW, et al. Ion mobility-derived collision cross section as an additional measure for lipid fingerprinting and identification. Anal Chem. 2015;87:1137–44. doi:10.1021/ac503715v.

    CAS  Article  Google Scholar 

  15. 15.

    May JC, Goodwin CR, Lareau NM, Leaptrot KL, Morris CB, Kurulugama RT, et al. Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer. Anal Chem. 2014;86:2107–16. doi:10.1021/ac4038448.

    CAS  Article  Google Scholar 

  16. 16.

    Liu X, Valentine SJ, Plasencia MD, Trimpin S, Naylor S, Clemmer DE. Mapping the human plasma proteome by SCX-LC-IMS-MS. J Am Soc Mass Spectrom. 2007;18:1249–64. doi:10.1016/j.jasms.2007.04.012.

    CAS  Article  Google Scholar 

  17. 17.

    Stephan S, Jakob C, Hippler J, Schmitz OJ. A novel four-dimensional analytical approach for analysis of complex samples. Anal Bioanal Chem. 2016;408:3751–9. doi:10.1007/s00216-016-9460-9.

    CAS  Article  Google Scholar 

  18. 18.

    Stephan S, Hippler J, Kohler T, Deeb AA, Schmidt TC, Schmitz OJ. Contaminant screening of wastewater with HPLC-IM-qTOF-MS and LC + LC-IM-qTOF-MS using a CCS database. Anal Bioanal Chem. 2016;408:6545–55. doi:10.1007/s00216-016-9820-5.

    CAS  Article  Google Scholar 

  19. 19.

    Li D, Schmitz OJ. Use of shift gradient in the second dimension to improve the separation space in comprehensive two-dimensional liquid chromatography. Anal Bioanal Chem. 2013;405:6511–7. doi:10.1007/s00216-013-7089-5.

    CAS  Article  Google Scholar 

  20. 20.

    Stoll DR, Talus ES, Harmes DC, Zhang K. Evaluation of detection sensitivity in comprehensive two-dimensional liquid chromatography separations of an active pharmaceutical ingredient and its degradants. Anal Bioanal Chem. 2015;407:265–77. doi:10.1007/s00216-014-8036-9.

    CAS  Article  Google Scholar 

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Correspondence to Oliver J. Schmitz.

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Stephan, S., Hippler, J., Köhler, T. et al. A Powerful Four-Dimensional Separation Method for Complex Samples. J. Anal. Test. 1, 1 (2017). https://doi.org/10.1007/s41664-017-0004-x

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  • LC + LC
  • Ion mobility
  • IM-qTOF-MS
  • 2D-LC
  • CCS