Concurrent determination of fat-soluble vitamins and related compounds in plasma using HPLC with electrochemical...

Fat-soluble vitamins and related compounds play an important role in numerous physiological processes, including oxidative metabolism and the direct or indirect prevention of oxidative stress (OS). Monitoring these compounds in biological matrices is useful during in vivo studies investigating OS-related pathologies. We validated a HPLC-ECD method for the simultaneous determination of α- and γ-tocopherol, β-carotene, coenzyme Q₁₀ and retinol in human plasma. Vitamins were separated using liquid-liquid extraction. A gradient, reversed-phase HPLC method with detector potentials set at 500, 800 and -550 mV over a 45 min runtime was optimized. The detector response was linear for all analytes, with an estimated LOD of 25.5 ng/mL and estimated LOQ of 84.9 ng/mL for retinol, and LOD ≤15.0 ng/mL/LOQ ≤50.0 ng/mL for all other analytes. Intra- and inter-day variations ranged between 2.60 - 5.15% and 3.23 - 12.96%, respectively. Mean recoveries remained within the 90-110% range. A low plasma volume of only 100 μL was sufficient for analysis, which is an improvement over previous combined methods. This is the first time these five analytes were quantified simultaneously using HPLC-ECD as an alternative for HPLC-MS. This will facilitate their incorporation into biomarker panels in future OS research.