Targeted metabolomics identifies reliable and stable metabolites in human serum and plasma samples

PLoS One. 2014 Feb 24;9(2):e89728. doi: 10.1371/journal.pone.0089728. eCollection 2014.


Background: Information regarding the variability of metabolite levels over time in an individual is required to estimate the reproducibility of metabolite measurements. In intervention studies, it is critical to appropriately judge changes that are elicited by any kind of intervention. The pre-analytic phase (collection, transport and sample processing) is a particularly important component of data quality in multi-center studies.

Methods: Reliability of metabolites (within-and between-person variance, intraclass correlation coefficient) and stability (shipment simulation at different temperatures, use of gel-barrier collection tubes, freeze-thaw cycles) were analyzed in fasting serum and plasma samples of 22 healthy human subjects using a targeted LC-MS approach.

Results: Reliability of metabolite measurements was higher in serum compared to plasma samples and was good in most saturated short-and medium-chain acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids and hexose. The majority of metabolites were stable for 24 h on cool packs and at room temperature in non-centrifuged tubes. Plasma and serum metabolite stability showed good coherence. Serum metabolite concentrations were mostly unaffected by tube type and one or two freeze-thaw cycles.

Conclusion: A single time point measurement is assumed to be sufficient for a targeted metabolomics analysis of most metabolites. For shipment, samples should ideally be separated and frozen immediately after collection, as some amino acids and biogenic amines become unstable within 3 h on cool packs. Serum gel-barrier tubes can be used safely for this process as they have no effect on concentration in most metabolites. Shipment of non-centrifuged samples on cool packs is a cost-efficient alternative for most metabolites.

Publication types

  • Observational Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Amino Acids / blood
  • Chromatography, Liquid
  • Drug Stability*
  • Female
  • Freezing
  • Hexoses / blood
  • Humans
  • Male
  • Metabolome / physiology*
  • Metabolomics
  • Middle Aged
  • Phospholipids / blood
  • Plasma / chemistry*
  • Reproducibility of Results*
  • Serum / chemistry*
  • Specimen Handling*
  • Tandem Mass Spectrometry
  • Temperature


  • Amino Acids
  • Hexoses
  • Phospholipids

Grant support

This study was supported in part by a grant (01GI0925) from the German Federal Ministry of Education and Research (BMBF) (URL: to the German Center for Diabetes Research (DZD e.V.). The work leading to this publication has received support from the Innovative Medicines Initiative Joint Undertaking (URL: under grant agreement n°115317 (DIRECT), resources of which are composed of financial contribution from the Helmholtz association (URL:, the European Union’s Seventh Framework Programme (URL: (FP7/2007–2013) and EFPIA companies’ (URL: in kind contribution. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.