Abstract. Abnormalities in mitochondrial metabolism and regulation of energy balance contribute to human diseases. The consequences of high fat and other nutrient intake, and the resulting acquired mitochondrial dysfunction, are essential to fully understand common disorders, including obesity, cancer, and atherosclerosis. To simultaneously and noninvasively measure and quantify indirect markers of mitochondrial function, we have developed a method based on gas chromatography coupled to quadrupole-time of flight mass spectrometry and an electron ionization interface, and validated the system using plasma from patients with peripheral artery disease, human cancer cells, and mouse tissues. This approach was used to increase sensibility in the measurement of a wide dynamic range and chemical diversity of multiple intermediate metabolites used in energy metabolism. We demonstrate that our targeted metabolomics method allows for quick and accurate identification and quantification of molecules, including the measurement of small yet significant biological changes in experimental samples. The apparently low process variability required for its performance in plasma, cell lysates, and tissues allowed a rapid identification of correlations between interconnected pathways. Our results suggest that delineating the process of energy generation by targeted metabolomics can be a valid surrogate for predicting mitochondrial dysfunction in biological samples. Importantly, when used in plasma, targeted metabolomics should be viewed as a robust and noninvasive source of biomarkers in specific pathophysiological scenarios.

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