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Mass Accuracy and Isotopic Abundance Measurements for HR-MS Instrumentation: Capabilities for Non-Targeted Analyses


The development of automated non-targeted workflows for small molecule analyses is highly desirable in many areas of research and diagnostics. Sufficient mass and chromatographic resolution is necessary for the detectability of compounds and subsequent componentization and interpretation of ions. The mass accuracy and relative isotopic abundance are critical in correct molecular formulae generation for unknown compounds. While high-resolution instrumentation provides accurate mass information, sample complexity can greatly influence data quality and the measurement of compounds of interest. Two high-resolution instruments, an Orbitrap and a Q-TOF, were evaluated for mass accuracy and relative isotopic abundance with various concentrations of a standard mixture in four complex sample matrices. The overall average ± standard deviation of the mass accuracy was 1.06 ± 0.76 ppm and 1.62 ± 1.88 ppm for the Orbitrap and the Q-TOF, respectively; however, individual measurements were ± 5 ppm for the Orbitrap and greater than 10 ppm for the Q-TOF. Relative isotopic abundance measurements for A + 1 were within 5% of the theoretical value if the intensity of the monoisotopic peak was greater than 1E7 for the Orbitrap and 1E5 for the Q-TOF, where an increase in error is observed with a decrease in intensity. Furthermore, complicating factors were found in the data that would impact automated data analysis strategies, including coeluting species that interfere with detectability and relative isotopic abundance measurements. The implications of these findings will be discussed with an emphasis on reasonable expectations from these instruments, guidelines for experimental workflows, data analysis considerations, and software design for non-targeted analyses.

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  1. Drexler, D.M., Reily, M.D., Shipkova, P.A.: Advances in mass spectrometry applied to pharmaceutical metabolomics. Anal. Bioanal. Chem. 399, 2645–2653 (2011)

    CAS  Article  Google Scholar 

  2. Hernández, F., Sancho, J.V., Ibáñez, M., Abad, E., Portolés, T., Mattioli, L.: Current use of high-resolution mass spectrometry in the environmental sciences. Anal. Bioanal. Chem. 403, 1251–1264 (2012)

    Article  Google Scholar 

  3. Kaufmann, A.: The current role of high-resolution mass spectrometry in food analysis. Anal. Bioanal. Chem. 403, 1233–1249 (2012)

    CAS  Article  Google Scholar 

  4. Kind, T., Fiehn, O.: Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. BMC Bioinforma. 8, 105 (2007)

    Article  Google Scholar 

  5. Bristow, A.W., Webb, K.S.: Intercomparison study on accurate mass measurement of small molecules in mass spectrometry. J. Am. Soc. Mass Spectrom. 14, 1086–1098 (2003)

    CAS  Article  Google Scholar 

  6. Bristow, T., Constantine, J., Harrison, M., Cavoit, F.: Performance optimisation of a new-generation orthogonal-acceleration quadrupole-time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 22, 1213–1222 (2008)

    CAS  Article  Google Scholar 

  7. Cortés-Francisco, N., Flores, C., Moyano, E., Caixach, J.: Accurate mass measurements and ultrahigh-resolution: evaluation of different mass spectrometers for daily routine analysis of small molecules in negative electrospray ionization mode. Anal. Bioanal. Chem. 400, 3595–3606 (2011)

    Article  Google Scholar 

  8. Croley, T., White, K., Callahan, J., Musser, S.: The chromatographic role in high resolution mass spectrometry for non-targeted analysis. J. Am. Soc. Mass Spectrom. 23, 1569–1578 (2012)

    CAS  Article  Google Scholar 

  9. Kaufmann, A., Walker, S.: Accuracy of relative isotopic abundance and mass measurements in a single-stage Orbitrap mass spectrometer. Rapid Commun. Mass Spectrom. 26, 1081–1090 (2012)

    CAS  Article  Google Scholar 

  10. Lee, K.A., Farnsworth, C., Yu, W., Bonilla, L.E.: 24-hour lock mass protection. J. Proteome Res. 10, 880–885 (2010)

    Article  Google Scholar 

  11. Lommen, A., Gerssen, A., Oosterink, J.E., Kools, H.J., Ruiz-Aracama, A., Peters, R.J., Mol, H.G.: Ultra-fast searching assists in evaluating sub-ppm mass accuracy enhancement in U-HPLC/Orbitrap MS data. Metabolomics 7, 15–24 (2011)

    CAS  Article  Google Scholar 

  12. Abate, S., Ahn, Y.G., Kind, T., Cataldi, T.R., Fiehn, O.: Determination of elemental compositions by gas chromatography/time-of-flight mass spectrometry using chemical and electron ionization. Rapid Commun. Mass Spectrom. 24, 1172–1180 (2010)

    CAS  Article  Google Scholar 

  13. Pelander, A., Decker, P., Baessmann, C., Ojanperä, I.: Evaluation of a high resolving power time-of-flight mass spectrometer for drug analysis in terms of resolving power and acquisition rate. J. Am. Soc. Mass Spectrom. 22, 379–385 (2011)

    CAS  Article  Google Scholar 

  14. Mol, H.G., Zomer, P., de Koning, M.: Qualitative aspects and validation of a screening method for pesticides in vegetables and fruits based on liquid chromatography coupled to full scan high resolution (Orbitrap) mass spectrometry. Anal. Bioanal. Chem. 403, 2891–2908 (2012)

    CAS  Article  Google Scholar 

  15. Weber, R.J., Southam, A.D., Sommer, U., Viant, M.R.: Characterization of isotopic abundance measurements in high resolution FT-ICR and Orbitrap mass spectra for improved confidence of metabolite identification. Anal. Chem. 83, 3737–3743 (2011)

    CAS  Article  Google Scholar 

  16. Xu, Y., Heilier, J.F., Madalinski, G., Genin, E., Ezan, E., Tabet, J.C., Junot, C.: Evaluation of accurate mass and relative isotopic abundance measurements in the LTQ-Orbitrap mass spectrometer for further metabolomics database building. Anal. Chem. 82, 5490–5501 (2010)

    CAS  Article  Google Scholar 

  17. Erve, J.C., Gu, M., Wang, Y., DeMaio, W., Talaat, R.E.: Spectral accuracy of molecular ions in an LTQ/Orbitrap mass spectrometer and implications for elemental composition determination. J. Am. Soc. Mass Spectrom. 20, 2058–2069 (2009)

    CAS  Article  Google Scholar 

  18. Jiang, W., Erve, J.C.: Spectral accuracy of a new hybrid quadrupole time-of-flight mass spectrometer: application to ranking small molecule elemental compositions. Rapid Commun. Mass Spectrom. 26, 1014–1022 (2012)

    CAS  Article  Google Scholar 

  19. Keller, B.O., Sui, J., Young, A.B., Whittal, R.M.: Interferences and contaminants encountered in modern mass spectrometry. Anal. Chim. Acta. 627, 71–81 (2008)

    CAS  Article  Google Scholar 

  20. Wenger, C.D., McAlister, G.C., Xia, Q., Coon, J.J.: Sub-part-per-million precursor and product mass accuracy for high-throughput proteomics on an electron transfer dissociation-enabled Orbitrap mass spectrometer. Mol. Cell. Proteom. 9, 754–763 (2010)

    CAS  Article  Google Scholar 

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The authors thank Samantha Farris for assistance in data analysis.

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Correspondence to Ann M. Knolhoff.

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Knolhoff, A.M., Callahan, J.H. & Croley, T.R. Mass Accuracy and Isotopic Abundance Measurements for HR-MS Instrumentation: Capabilities for Non-Targeted Analyses. J. Am. Soc. Mass Spectrom. 25, 1285–1294 (2014).

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Key words

  • Non-targeted analysis
  • Mass accuracy
  • Relative isotopic abundance
  • High-resolution mass spectrometry