High-Resolution Mass Spectrometry Quantification: Impact of Differences in Data Processing of Centroid and Continuum Data

  • L. Vereyken
  • L. Dillen
  • R. J. Vreeken
  • F. CuyckensEmail author
Critical Insight


High-resolution mass spectrometry (HRMS) in full scan mode acquires all ions present in the sample of interest offering a lot of qualitative information. This, in combination with the improved performance of the new generation HRMS systems, triggers more (bio) analysts to switch from triple quad MS systems to HRMS for quantitative analysis. Quantitative processing of HRMS data is performed based on narrow mass extraction windows rather than on nominal mass product ion chromatograms (SRM or MRM). Optimal processing of HRMS data requires different considerations and software tools and can have an impact on data processing and final results. The selection of centroid versus continuum/profile data for processing, selection of the optimal narrow mass extraction window, using theoretical versus measured accurate mass for the extraction of the ion chromatograms as well as differences in calculations and data handling residing in the different vendor software packages are tackled in the presented manuscript. These differences are illustrated on HRMS data acquired for the same plasma samples on three different platforms, i.e., a Sciex QToF, a Waters QToF, and a Thermo Orbitrap system, and processed in four different software packages, i.e., Sciex Analyst® TF, Waters Masslynx, Waters Unifi, and Thermo Xcalibur. The impact of these differences on quantitative HRMS performance was evaluated on calibration curves of eight small molecule compounds in plasma using four different ways of processing. Simple guidelines are provided for the selection of an optimal mass extraction window for continuum and centroided data.

Graphical Abstract


High-Resolution Mass Spectrometry Mass Extraction Window Profile data Continuum data Centroid data Quantification Q-Tof Orbitrap 



The authors thank Lyle Burton (ABSciex) for developing and providing the research tool to transform continuum raw data files to centroid data files post-acquisition. We thank Anne Van Vlaslaer, Ils Pijpers, and Emmanuel Njumbe Ediage (Janssen R&D) for the analysis of the calibration curves in plasma and Ronald de Vries for fruitful discussions.

Supplementary material

13361_2018_2101_MOESM1_ESM.docx (15 kb)
Supplementary Table 1 Maximum MEW calculations based on measured resolution and mass. *the differences between the system resolution and measured resolution can be mainly explained by the mass dependency of mass resolution on QTof and Orbitrap instruments, experiments on the TripleTof® were performed with an aged detector that had a clear detrimental impact on MS resolution. (DOCX 14 kb)
13361_2018_2101_MOESM2_ESM.docx (33 kb)
Supplementary Table 2 Results of calibration curves for acetaminophen, tolbutamide, norethindrone, midazolam, abiraterone, prednisone, loperamide, and simeprevir n = 5) acquired on a Waters Synapt G2-S in continuum mode and processed after post-acquisition centroiding of the same data A mass accuracy (MA) of 2 mDa is applied according to the specifications of the instrument. Data are processed in four different ways as discussed in the manuscript: (i) smoothed centroid data MEW = 2 × MA or smallest MEW with no loss of data points using unsmoothed data in TargetLynx, (ii) continuum data MEW = FWHM + 2 × MA in TargetLynx, (iii) continuum data 2D processing in Unifi, and (iv) continuum data 3D processing in Unifi, where the selection of the MEW has no impact on the XIC generation. Results obtained with other MEWs of 10 mDa, 20 mDa and the max MEW are given for comparison. Exact nominal mass has been calculated with MassLyx. (DOCX 32 kb)
13361_2018_2101_MOESM3_ESM.docx (53 kb)
Supplemental Figure 1 Comparison of different MEWs (0.5–10–20–33-max MEW (34 mDa)) for simeprevir data (at a concentration of 5 ng/mL) acquired on the Synapt G2-S. Data were centroided post-acquisition. This illustrates the difference in peak evaluation using unsmoothed (a) and smoothed data (b): the loss of data points is only visible with unsmoothed data, while the entire chromatographic peak is lost when the MEW is chosen to small (DOCX 53 kb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Janssen Research and DevelopmentBeerseBelgium

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