Determination of tocopherols and their metabolites by liquid-chromatography coupled with tandem mass spectrometry in human plasma and serum

Several studies are increasingly underlying the biological role of vitamin E metabolites as bioactive compounds with anti-inflammatory, anti-proliferative and anti-atherogenic activity. A quantitative method for the simultaneous determination in human plasma and serum of vitamin E (alpha-tocopherol, alpha-T and gamma-tocopherol, gamma-T) and its cytochrome P-450 metabolites: 13'-hydroxychromanol (alpha-13'-OH), 13'-carboxychromanol (alpha-13'COOH) and carboxyethyl hydroxychromanols (alpha-CEHC and gamma-CEHC), was developed and validated. After enzymatic hydrolysis and deproteinization, the metabolites were extracted with a mixture of hexane/ methyl tertiary butyl ether (2/1, v/v). The separation was achieved by reversed phase chromatography and the analytes detected by a triple quadrupole mass analyser using electrospray ionization in positive mode (LC-MS/MS). alpha-T and gamma-T were extracted separately without enzymatic hydrolysis. The analytes were quantified with the isotopic dilution method. After an extensive validation study (three levels in three different occasions for a total of 54 experiments), the procedure was successfully applied to the analysis of sera of healthy volunteers (before and after supplementation with alpha-T) and plasma of patients affected by chronic kidney disease. Finally, the structures of three unknown compounds found in blood and related to the long chain metabolites (alpha-13'-OH and alpha-13'-COOH) were further investigated using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS).