Advancements in Adenine Nucleotides Extraction and Quantification from a Single Drop of Human Blood

Adenine nucleotides (ANs)-adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP)-are essential for energy transfer and the supply of countless processes within cellular metabolism. Their concentrations can be expressed as adenylate energy charge (AEC), a measure of cellular metabolic energy that directly correlates with the homeostasis of the organism. AEC index has broad diagnostic potential, as reduced ATP levels are associated to various conditions, such as inflammatory diseases, metabolic disorders, and cancer. We introduce a novel methodology for rapid isolation, purification, and quantification of ANs from a single drop of capillary blood. Of all the stationary phases tested, activated carbon proved to be the most efficient for the purification of adenine nucleotides, using an automated micro-solid phase extraction (µ-SPE) platform. An optimized µ-SPE method, coupled with RP-HPLC and a run time of 30 min, provides a reliable analytical framework for adenine nucleotide analysis of diverse biological samples. AN concentrations measured in capillary blood samples were 1393.1 µM, 254.8 µM, and 76.9 µM for ATP, ADP, and AMP molecules aligning with values reported in the literature. Overall, this study presents a streamlined and precise approach for analyzing ANs from microliters of blood, offering promising applications in clinical diagnostics.