A novel application of LC-MS/MS accurately quantifies the labile redox pools of cellular coenzymes Q₉ and Q₁₀

The antioxidant coenzyme Q (CoQ) plays an essential role in the electron transport chain (ETC). CoQ cycles between oxidized and reduced forms. Redox balance changes in the CoQ pool are associated with altered mitochondrial function. Thus, determining the CoQ redox pool is important for investigating cellular redox regulation. Existing quantification methods inadequately account for artefactual sample oxidation. To overcome these limitations, we found that a reduced stable isotope-labeled internal standard (IS) can correct for oxidation of extracted CoQ₉ and CoQ₁₀. The reduced IS oxidizes at the same rate as both CoQ isoforms. Employing this correction factor rescues artefactual oxidation of the CoQ redox pool when measured by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). We validated our method within murine and human cellular systems. Statin treatment dose-dependently decreased total CoQ, while directional perturbation of the mitochondrial ETC altered the CoQ redox pool as expected. The pro-oxidant tert-butyl hydroperoxide partially oxidized the cellular CoQ redox pool. Finally, we found that primary murine macrophages deficient in PON2, a mitochondrial antioxidant enzyme, contain a partially oxidized CoQ₉ redox pool. These results were revealed only after correcting for sample oxidation. Whereas prior LC-MS/MS methods for measuring the CoQ redox pool inadequately account for artefactual oxidation, the presented method rescues this error and potentiates accurate measurement of murine and human CoQ redox pools.