Monitoring of Ubiquinol-10, Ubiquinone-10, Carotenoids, and Tocopherols in Neonatal Plasma Microsamples Using High-Performance Liquid Chromatography with Coulometric Electrochemical Detection

A method for measuring the lipophilic antioxidants α-tocopherol, γ-tocopherol, ubiquinol-10, ubiquinone-10, β-cryptoxanthin, and β-carotene in 5 or 10 μl neonatal plasma is presented. First lipid extraction together with the internal standards γ-tocotrienol, ubiquinone-7, ubiquinol-9, ubiquinone-9, and ethyl β-apo-8′-carotenoate was carried out. Then the components were separated on a RPC2/C18column. Coulometric electrochemical detection was performed using the reduction–oxidation mode (−0.60 V; −0.15 V; +0.60 V). Detection limits for all components were between 21 and 60 fmol, corresponding to 0.004 and 0.012 μmol/liter plasma; the within-day precision (coefficient of variation) was between 3 and 14% for all measured substances. Reference values are given for healthy newborn children from the second to the sixth day of life. Using the presented HPLC technique it is possible to monitor the concentrations of lipophilic antioxidants in plasma of sick immature newborn children in order to study effects of oxidative stress.