Abstract

A sensitive UPLC-MS/MS method for 8 polar phosphate metabolites. • Achieved high-throughput analysis in under 4 min per sample. • Differentiated the distinct energy metabolism trajectories of PC12 and H9C2 cells under hypoxia. • Analyzed the specific metabolic reprogramming in mouse brain tissue. Phosphate-containing metabolites serve as critical regulators of energy homeostasis and signal transduction under hypoxic stress. However, their simultaneous quantification is technically challenging due to high polarity and non-specific adsorption. In this study, a sensitive UHPLC-MS/MS method was established for the simultaneous quantification of eight key phosphate metabolites, including adenosine triphosphate (ATP), phosphoenolpyruvate (PEP) and glucose-1-phosphate (P1G), in biological matrices. By compressing the analytical run time to <4 min per sample, this high-throughput platform demonstrated excellent linearity (r 2  > 0.990) for all analytes. The method satisfied rigorous bioanalytical validation standards, exhibiting intra- and inter-day precision (RSD) of 2.2%–11.5% and accuracy of 87.0%–109.5%. Application of this method to in vitro and in vivo hypoxia models demonstrated its capability to capture distinct, context-dependent metabolic adaptations. Specifically, the platform differentiated the energetic trajectories of neuronal cells and cardiomyocytes under hypoxia and profiled metabolic shifts in mouse brain tissue. This study provides a reliable analytical platform for quantifying highly polar phosphate metabolites, offering a robust tool for monitoring metabolic pool dynamics under physiological and pathological stress.