Reliable Determination of ATP and Its Metabolites by LC-MS Using Blood Collection Tubes with and without Ectonucleotidase Inhibitors

Adenosine triphosphate (ATP) and its breakdown products, including adenosine, play key roles in regulating immune responses. Altered ATP and adenosine levels in blood may reflect the presence or development of various pathologies; however, their rapid metabolism and clearance makes accurate measurement of their concentrations difficult. Not surprisingly, studies simultaneously monitoring ATP and its breakdown products are sparse and show conflicting results, and the workflows used are difficult to implement in clinical routine. Here, we present the simultaneous measurement of ATP and its metabolites in blood samples from healthy donors by combining a liquid chromatography-mass spectrometry-based quantification method with various procedures of blood sampling. We find that ATP and adenosine are best preserved in an ethylenediaminetetraacetic acid (EDTA) blood collection tube containing ectonucleotidase and nucleoside transporter inhibitors. In contrast, inosine and its downstream metabolites are detected in a serum collection tube without inhibitors. Therefore, we propose the use of these two sampling tubes to obtain a faithful determination of ATP and its degradation products. Overall, our approach provides a valuable and reliable tool to monitor changes in the concentration of ATP metabolites that can be easily implemented for biobanking purposes in the context of clinical trials.