Highly sensitive and selective determination of redox states of coenzymes Q₉ and Q₁₀ in mice tissues: Application of orbitrap mass spectrometry

Coenzyme Q (CoQ) is a redox active molecule that plays a fundamental role in mitochondrial energy generation and functions as a potent endogenous antioxidant. Redox ratio of CoQ has been suggested as a good marker of mitochondrial dysfunction and oxidative stress. Nevertheless, simultaneous measurement of redox states of CoQ is challenging owing to its hydrophobicity and instability of the reduced form. In order to improve the analytical methodology, paying special attention to this instability, we developed a highly sensitive and selective high-resolution/accurate-mass (HR/AM) UHPLC-MS/MS method for the rapid determination of redox states of CoQ₉ and CoQ₁₀ by ultra-performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry. CoQs were extracted using hexane with the addition of butylated hydroxytoluene to limit oxidation during sample preparation. Chromatographic separation of the analytes was achieved on a Kinetex C₁₈ column with the isocratic elution of 5 mM ammonium formate in 2-propanol/methanol (60:40) within 4 min. A full MS/all ion fragmentation (AIF) acquisition mode with mass accuracy < 5 ppm was used for detection and determination of redox states of CoQ₉ and CoQ₁₀ in healthy mice tissues using reduced and oxidized CoQ₄ as internal standards. The validated method showed good linearity (r² ≥ 0.9991), intraday, inter-day precision (CVs ≤ 11.9%) and accuracy (RE ≤±15.2%). In contrast to existing methods, the current method offers enhanced sensitivity (up to 52 fold) with LOD and LOQ ranged from 0.01 to 0.49 ng mL^-1 and 0.04-1.48 ng mL^-1, respectively. Moreover, we evaluated various diluents to investigate bench top stability (at 4 °C) of targeted analytes in tissue samples during LC-MS assay up to 24 h. Ethanol was determined to be an optimum diluent without any significant oxidation of reduced CoQ up to 24 h. The developed method offers a rapid, highly sensitive and selective strategy for the measurement of redox states of CoQs in clinical studies.