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

• Published in: Talanta (Oxford) Vol. 170; pp. 552 - 561
• Main Authors: Giusepponi, Danilo, Torquato, Pierangelo, Bartolini, Desirée, Piroddi, Marta, Birringer, Marc, Lorkowski, Stefen, Libetta, Carmelo, Cruciani, Gabriele, Moretti, Simone, Saluti, Giorgio, Galli, Francesco, Galarini, Roberta
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2017
• Subjects: Adult; alpha-tocopherol; bioactive compounds; blood serum; Chromatography, Liquid - methods; cytochrome P-450; enzymatic hydrolysis; Female; gamma-tocopherol; Human plasma/serum; Humans; ionization; isotope dilution technique; kidney diseases; Limit of Detection; Liquid-chromatography tandem-mass spectrometry (LC-MS/MS); Long chain metabolites; Male; metabolites; patients; quantitative analysis; Renal Insufficiency, Chronic - blood; Renal Insufficiency, Chronic - metabolism; reversed-phase liquid chromatography; Short chain metabolites; tandem mass spectrometry; Tandem Mass Spectrometry - methods; Tocopherols - analysis; Tocopherols - blood; Tocopherols - metabolism; Vitamin E; Vitamin E - analysis; Vitamin E - blood; Vitamin E - metabolism; Vitamins - analysis; Vitamins - blood; Vitamins - metabolism; Short chain metabolites; Vitamin E; Human plasma/serum; Long chain metabolites; Liquid-chromatography tandem-mass spectrometry (LC-MS/MS)
• ISSN: 0039-9140; 1873-3573; 1873-3573
• DOI: 10.1016/j.talanta.2017.04.030

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 (α-tocopherol, α-T and γ-tocopherol, γ-T) and its cytochrome P-450 metabolites: 13′-hydroxychromanol (α-13'-OH), 13′-carboxychromanol (α-13'-COOH) and carboxyethyl hydroxychromanols (α-CEHC and γ-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). α-T and γ-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 α-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 (α-13'-OH and α-13'-COOH) were further investigated using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). [Display omitted] •First fully validated method for vitamin E metabolites in serum and plasma.•Good method accuracies with low detection limits (0.05–0.1µgL−1).•Successful application on samples of supplemented subjects and dialyzed patients.•Evidence for new potential biomarkers.