A microfluidic chip-based capillary zone electrophoresis-mass spectrometry method for measuring adenosine 5-Triphosphate and its similar nucleotide analogues

Background: Extracellular ATP is involved in disorders that cause inflammation of the airways and cough, thus limiting its release has therapeutic benefits. Standard luminescence -based ATP assays measure levels indirectly through enzyme degradation and do not provide a simultaneous readout for other nucleotide analogues. Conversely, mass spectrometry can provide direct ATP measurements, however, common RPLC and HILIC methods face issues because these molecules are unstable, metal -sensitive analytes which are often poorly retained. These difficulties have traditionally been overcome using passivation or ion -pairing chromatography, but these approaches can be problematic for LC systems. As a result, more effective analytical methods are needed. Results: Here, we introduce a new application that uses microfluidic chip -based capillary zone electrophoresis - mass spectrometry (mu CZE-MS) to measure ATP and its analogues simultaneously in biofluids. The commer- cially available ZipChip Interface and a High -Resolution Bare -glass microchip (ZipChip, HRB, 908 Devices Inc.) coupled to a Thermo Scientific Tribrid Orbitrap, were successfully used to separate and detect various nucleotide standards, as well as ATP, ADP, AMP, and adenosine in plasma and BALF obtained from naive Brown Norway rats. The findings demonstrate that this approach can rapidly and directly detect ATP and its related nucleotide analogues, while also highlighting the need to preserve these molecules in biofluids with chelators like EDTA. In addition, we demonstrate that this mu CZE-MS method is also suitable for detecting a variety of metabolites, revealing additional potential future applications. Significance: This innovative mu CZE-MS approach provides a robust new tool to directly measure ATP and other nucleotide analogues in biofluids. This can enable the study of eATP in human disease and potentially contribute to the creation of ATP -targeting therapies for airway illnesses.