Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography-electrospray ionization mass spectrometry

Tianxiu Qian, Zongwei CAI*, M. S. Yang

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

68 Citations (Scopus)

Abstract

A method using ion-pairing liquid chromatography-mass spectrometry (MS) was developed for analyzing adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), and adenosine 5′-triphosphate (ATP) in cellular extracts. Dimethylhexylamine (DMHA) was used as ion-pairing agent to retain and separate the analytes on a reversed-phase microbore column with a gradient program. Positive-ion electrospray ionization-MS was applied for the detection because of the use of the ion-pairing agent. Adduct ions of DMHA with AMP, ADP, and ATP were found to be the most intensive peaks and thus selected as quantitative ions. An external calibration method with linear ranges from 0. 1 to 20μM for AMP, 2 to 20μM for ADP, and 2.5 to 20μM for ATP was used for the quantitation. The method was applied to determine concentrations of AMP, ADP, and ATP in extracts of cultured rat C6 glioma cells that were pretreated with various concentrations of Zn. The detected levels of the adenosine nucleotides have been used to calculate total adenosine nucleotide and energy charge potential. Changes in cellular energy status upon exposure to increasing concentration of Zn in the culture medium were analyzed. The results indicated that the addition of Zn in a range of 40 to 120μg/ml cause a gradual increased in energy charge potential of the cells.

Original languageEnglish
Pages (from-to)77-84
Number of pages8
JournalAnalytical Biochemistry
Volume325
Issue number1
DOIs
Publication statusPublished - 1 Feb 2004

Scopus Subject Areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

User-Defined Keywords

  • Adenosine nucleotides
  • Energy charge potential
  • Ion-pairing LC-MS

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