Artifactual FA dimers mimic FAHFA signals in untargeted metabolomics pipelines

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6 Scopus citations


FA esters of hydroxy FAs (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Because of their low molar abundance, FAHFAs are typically quantified using highly sensitive LC-MS/MS methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point but could lead to misidentification. To address this possibility, we analyzed human serum with a commonly applied high-resolution LC-MS untargeted metabolomics platform. We found that many chemical features are putatively assigned to the FAHFA lipid class based on exact mass and fragmentation patterns matching spectral databases. Careful validation using authentic standards revealed that many investigated signals provisionally assigned as FAHFAs are in fact FA dimers formed in the LC-MS pipeline. These isobaric FA dimers differ structurally only by the presence of an olefinic bond. Furthermore, stable isotope-labeled oleic acid spiked into human serum at subphysiological concentrations showed concentration-dependent formation of a diverse repertoire of FA dimers that analytically mimicked FAHFAs. Conversely, validated FAHFA species did not form spontaneously in the LC-MS pipeline. Together, these findings underscore that FAHFAs are endogenous lipid species. However, nonbiological FA dimers forming in the setting of high concentrations of FFAs can be misidentified as FAHFAs. Based on these results, we assembled a FA dimer database to identify nonbiological FA dimers in untargeted metabolomics datasets.

Original languageEnglish (US)
Article number100201
JournalJournal of lipid research
Issue number5
StatePublished - May 2022

Bibliographical note

Funding Information:
The authors thank the participants for their participation in the acute exercise study. They also thank André d’Avignon for helpful discussions. This work was supported by the National Institutes of Health (grant numbers: DK091538, AG069781, and DK098203).

Publisher Copyright:


  • LC-MS/MS
  • adipose tissue
  • insulin resistance
  • isobaric FA dimers
  • lipidomics
  • lipids
  • lipokines
  • obesity
  • olefinic bond
  • spectral database
  • Chromatography, Liquid/methods
  • Metabolomics
  • Humans
  • Tandem Mass Spectrometry/methods
  • Fatty Acids/chemistry
  • Esters/chemistry

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural


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