Rapid assessment of the coenzyme Q₁₀ redox state using ultrahigh performance liquid chromatography tandem mass spectrometry

An improved method for accurate and rapid assessment of the coenzyme Q₁₀ (CoQ₁₀) redox state using ultrahigh performance liquid chromatography tandem mass spectrometry was described, with particular attention given to the instability of the reduced form of CoQ₁₀ during sample preparation, chromatographic separation and mass spectrometric detection. As highly lipophilic compounds in complex biological matrices, both reduced and oxidized forms of CoQ₁₀ were extracted simultaneously from the tissue samples by methanol which is superior to ethanol and isopropanol. After centrifugation, the supernatants were immediately separated on a C18 column with isocratic elution using methanol containing 2 mM ammonium acetate as a non-aqueous mobile phase, and detected by positive electrospray ionization tandem mass spectrometry in multiple reaction monitoring (MRM) mode. Ammonium acetate as an additive in methanol provided enhanced mass spectrometric responses for both forms of CoQ₁₀, primarily due to stable formation of adduct ions [M + NH₄]⁺, which served as precursor ions in positive ionization MRM transitions. The assay showed a linear range of 8.6-8585 ng mL^-1 for CoQ₁₀H₂ and 8.6-4292 ng mL^-1 for CoQ₁₀. The limits of detection (LODs) were 7.0 and 1.0 ng mL^-1 and limits of quantification (LOQs) were 15.0 and 5.0 ng mL^-1 for CoQ₁₀H₂ and CoQ₁₀, respectively. This rapid extractive and analytical method could avoid artificial auto-oxidation of the reduced form of CoQ₁₀, enabling the native redox state assessment. This reliable method was also successfully applied for the measurement of the CoQ₁₀ redox state in liver tissues of mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin, revealing the down-regulated mitochondrial electron transport chain.