Integrated Redox Profiling: Simultaneous Determination of Ubiquinol-10, Ubiquinone-10, and Alpha-Lipoic Acid in Serum...

Background: Coenzyme Q₁₀ and Alpha-lipoic acid are two essential antioxidants involved in numerous physiological processes, including cellular energy production and the mitigation of oxidative stress. Their accurate quantification is critical for understanding their biological roles and therapeutic potential. Herein, an RPLC-MS/MS method for the rapid and simultaneous determination of ubiquinone-10 (CoQ₁₀), the reduced form ubiquinol-10 (CoQ₁₀H₂), and Alpha-lipoic acid (ALA) in human serum was developed and validated. Methods: Chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 column (2.1 mm × 150 mm, i.d. 1.7 μm). Detection was performed on a SCIEX Triple Quad 6500+ system, applying multiple reaction monitoring (MRM). Single-phase protein precipitation was selected as the sample preparation protocol, providing satisfactory recovery for the analytes. Results: The method was linear over the concentration of 53.8-613 ng/mL for CoQ₁₀H₂, 23.1-263 ng/mL for CoQ₁₀ and 7.7-87.6 ng/mL for ALA. Intra- and inter-day accuracy was found to be between 81.8 and 109% and 84.4 to 106%, respectively, for all analytes, while intra- and inter-day precision was found to vary from 0.8% to 9.9% %RSD and 2.0% to 7.7% %RSD, respectively. A limit of quantitation (LOQ) of 4.2 ng/mL was found for CoQ₁₀H₂, 1.7 ng/mL for CoQ₁₀ and 0.7 ng/mL for ALA. Conclusions: The developed LC-MS/MS method enables rapid, sensitive and simultaneous quantification of CoQ₁₀H₂, CoQ_10, and ALA in human serum with satisfactory accuracy, precision and sensitivity. The method is suitable for bioanalytical applications and was successfully applied to the analysis of 10 real samples obtained from healthy volunteers.