Citation: ZHU Huiyu, WU Danni, WANG Hailin. Quantification of intracellular adenosine 5'-triphosphate and its metabolites by high performance liquid chromatography analysis[J]. Chinese Journal of Chromatography, ;2017, 35(1): 54-58. doi: 10.3724/SP.J.1123.2016.08031 shu

Quantification of intracellular adenosine 5'-triphosphate and its metabolites by high performance liquid chromatography analysis

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China

Received Date: 26 August 2016

Fund Project: National Natural Science Foundation of China (No. 21327006).

This study was aimed to provide insight regarding the intracellular metabolites of adenosine 5'-triphosphate (ATP) and whether 2-tert-butyl-1,4-benzoquinone (TBBQ) affects cell metabolites. A rapid high performance liquid chromatography (HPLC) protocol was developed for the separation and quantitation of ATP and its metabolites (adenosine diphosphate (ADP) and adenosine monophosphate (AMP)) in cells. Chromatographic separation was performed using a Shimadzu HPLC system equipped with an Agela Venusil MP C18 column; isocratic elution was adopted. The mobile phase comprised solvent A (50 mmol/L disodium hydrogen phosphate and 15 mmol/L trimethylamine (TEA); pH adjusted to 7.88 using acetic acid (HAc)) and solvent B (methanol). The correlation coefficients of the three analytes were very high (R²≥0.9996), and the contents of the three metabolites in the MRC-5 cells were within the linear ranges (0.1-100 μ mol/L). The limits of detection for the detected three compounds were low. Samples were extracted from cells (after exposure and non-exposure to quinones) using 80% (v/v) methanol aqueous solution. The method developed in this study was successfully applied to detect ATP, ADP and AMP in MRC-5 cells, and the results demonstrated that ATP, ADP, AMP levels in cells were affected by TBBQ, but the relations between the concentration of TBBQ and the level of ATP, ADP and AMP were complex.
- high performance liquid chromatography (HPLC),
- adenosine 5'-triphosphate (ATP),
- quinones,
- metabolites,
- intracellular
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