Background: Our study presented a novel LC-MS/MS method for the simultaneous quantification of α-tocopherol (α-TOH) and its phase II metabolites, α-13'-COOH and α-13'-OH, in human serum using deuterium-labeled internal standards (d6-α-TOH, d6-α-13'-COOH, d6-α-13'-OH). Methods: The method addresses the analytical challenge posed by the significantly different concentration ranges of α-TOH (µmol/L) and its metabolites (nmol/L). Previous methods quantified these analytes separately, which caused an increase in workflow complexity. Results: Key features include the synthesis of stable isotope-labeled standards and the use of a pentafluorophenyl-based core-shell chromatography column for baseline separation of both α-TOH and its metabolites. Additionally, solid phase extraction (SPE) with a HybridSPE® material provides a streamlined sample preparation, enhancing analyte recovery and improving sensitivity. By utilizing deuterium-labeled standards, the method compensates for matrix effects and ion suppression. This new approach achieves precise and accurate measurements with limits of detection (LOD) and quantification (LOQ), similar to previous studies. Calibration, accuracy, and precision parameters align well with the existing literature. Conclusions: Our method offers significant advantages in the simultaneous analysis of tocopherol and its metabolites despite concentration differences spanning up to three orders of magnitude. In contrast to earlier studies, which required separate sample preparations and analytical techniques for tocopherol and its metabolites, our approach streamlines this process. The use of a solid-phase extraction procedure allows for parallel sample preparation. This not only enhances efficiency but also significantly accelerates pre-analytical workflows, making the method highly suitable for large-scale studies.
Keywords: liquid-chromatography tandem-mass spectrometry (LC-MS/MS); long chain metabolites; stable isotope dilution analysis; vitamin E.