Validating Quantitative Untargeted Lipidomics Across Nine Liquid Chromatography-High-Resolution Mass Spectrometry Platforms

Anal Chem. 2017 Nov 21;89(22):12360-12368. doi: 10.1021/acs.analchem.7b03404. Epub 2017 Nov 7.


Liquid chromatography-mass spectrometry (LC-MS) methods are most often used for untargeted metabolomics and lipidomics. However, methods have not been standardized as accepted "best practice" documents, and reports lack harmonization with respect to quantitative data that enable interstudy comparisons. Researchers use a wide variety of high-resolution mass spectrometers under different operating conditions, and it is unclear if results would yield different biological conclusions depending on the instrument performance. To this end, we used 126 identical human plasma samples and 29 quality control samples from a nutritional intervention study. We investigated lipidomic data acquisitions across nine different MS instruments (1 single TOF, 1 Q/orbital ion trap, and 7 QTOF instruments). Sample preparations, chromatography conditions, and data processing methods were kept identical. Single-point internal standard calibrations were used to estimate absolute concentrations for 307 unique lipids identified by accurate mass, MS/MS spectral match, and retention times. Quantitative results were highly comparable between the LC-MS platforms tested. Using partial least-squares discriminant analysis (PLS-DA) to compare results between platforms, a 92% overlap for the most discriminating lipids based on variable importance in projection (VIP) scores was achieved for all lipids that were detected by at least two instrument platforms. Importantly, even the relative positions of individual samples on the PLS-DA projections were identical. The key for success in harmonizing results was to avoid ion saturation by carefully evaluating linear dynamic ranges using serial dilutions and adjusting the resuspension volume and/or injection volume before running actual study samples.

Publication types

  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • Chromatography, High Pressure Liquid / standards*
  • Humans
  • Lipids / blood*
  • Mass Spectrometry / standards*
  • Metabolomics / methods*
  • Metabolomics / standards*
  • Quality Control


  • Lipids