Analysis of adenosine phosphates in HepG-2 cell by a HPLC–ESI-MS system with porous graphitic carbon as stationary phase

Abstract

A high performance liquid chromatography coupled with electrospray ionization/mass spectrometry method was developed for the determination of adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), and adenosine 5′-triphosphate (ATP) in the extract of HepG-2 cells. The chromatographic conditions were optimized by using porous graphitic carbon as the stationary phase for the retention and separation of the AMP, ADP and ATP. Negative-ion mode ESI-MS in basic mobile phase was applied to improve the method sensitivity. An external calibration method with linear ranges from 0.22 to 57.80μM for AMP, from 0.59 to 117.37μM for ADP, and from 0.49 to 98.81μM for ATP was used for quantitative analysis. The levels of ATP, ADP, and AMP in HepG-2 cells treated with benzo[a]pyrene with different time periods were determined. Total adenine nucleotides and the energy charge potential were calculated for the investigation of the effect of benzo[a]pyrene on cell energy metabolism.