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Adenosine -5′-triphosphate (ATP) plays a valuable role in metabolic activity to produce adequate energy in a biosystem. A high ATP/AMP ratio has a correlation with diabetes that induces suppression of AMP-activated protein kinase (AMPK). Matrix-assisted laser desorption/ionization (MALDI)–mass spectrometry (MS) has outstanding potential in determining the ratio of several types of adenosine phosphates in a sample to rapidly understand the primary energy transfer in metabolism. Although MALDI is viewed as a soft ionization technique for MS analysis, excess photon energy might crack the phosphate bonds leading to misinterpretation of the ATP level. In this work, ionic liquid matrices (ILMs) were employed to reduce fragmentation and increase the detection efficiency during the MALDI process. This study demonstrated for the first time that 2,5-dihydroxybenzoic acid pyridine (DHBP) is one of the most effective matrices for further quantitative analysis of adenosine nucleotides. This systematic screening of ILMs also enhances the fundamental understanding of MALDI. [ABSTRACT FROM AUTHOR]