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

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 20 microM for AMP, 2 to 20 microM for ADP, and 2.5 to 20 microM 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 120 microg/ml cause a gradual increased in energy charge potential of the cells.