Analytical and Bioanalytical Chemistry

, Volume 402, Issue 3, pp 1183–1198

Monitoring metabolites consumption and secretion in cultured cells using ultra-performance liquid chromatography quadrupole–time of flight mass spectrometry (UPLC–Q–ToF-MS)

  • Giuseppe Paglia
  • Sigrún Hrafnsdóttir
  • Manuela Magnúsdóttir
  • Ronan M. T. Fleming
  • Steinunn Thorlacius
  • Bernhard Ø. Palsson
  • Ines Thiele
Original Paper

Abstract

Here we present an ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method for extracellular measurements of known and unexpected metabolites in parallel. The method was developed by testing 86 metabolites, including amino acids, organic acids, sugars, purines, pyrimidines, vitamins, and nucleosides, that can be resolved by combining chromatographic and m/z dimensions. Subsequently, a targeted quantitative method was developed for 80 metabolites. The presented method combines a UPLC approach using hydrophilic interaction liquid chromatography (HILIC) and MS detection achieved by a hybrid quadrupole–time of flight (Q–ToF) mass spectrometer. The optimal setup was achieved by evaluating reproducibility and repeatability of the analytical platforms using pooled quality control samples to minimize the drift in instrumental performance over time. Then, the method was validated by analyzing extracellular metabolites from acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) treated with direct (A-769662) and indirect (AICAR) AMP activated kinase (AMPK) activators, monitoring uptake and secretion of the targeted compound over time. This analysis pointed towards a perturbed purine and pyrimidine catabolism upon AICAR treatment. Our data suggest that the method presented can be used for qualitative and quantitative analysis of extracellular metabolites and it is suitable for routine applications such as in vitro drug screening.

Figure

UPLC-MS analysis of extracellular metabolites from acute lymphoblastic leukemia cell lines treated with AMP activated kinase (AMPK) activators points out that purine catabolism is affected upon AICAR treatment.

Keywords

Metabolomics Mass spectrometry UPLC HILIC Exo-metabolome 

Supplementary material

216_2011_5556_MOESM1_ESM.pdf (1.1 mb)
ESM 1(PDF 1.08 mb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Giuseppe Paglia
    • 1
  • Sigrún Hrafnsdóttir
    • 1
  • Manuela Magnúsdóttir
    • 1
  • Ronan M. T. Fleming
    • 1
  • Steinunn Thorlacius
    • 1
  • Bernhard Ø. Palsson
    • 1
  • Ines Thiele
    • 2
  1. 1.Center for Systems BiologyUniversity of IcelandReykjavik 101Iceland
  2. 2.Center for Systems Biology, Faculty of Industrial Engineering, Mechanical Engineering & Computer ScienceUniversity of IcelandReykjavik 101Iceland

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