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

Danilo Giusepponi, Pierangelo Torquato, Desirée Bartolini, Marta Piroddi, Marc Birringer, Stefen Lorkowski, Carmelo Libetta, Gabriele Cruciani, Simone Moretti, Giorgio Saluti, Francesco Galli, Roberta Galarini

Research output: Contribution to journalArticlepeer-review

Abstract

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).

Original languageEnglish
Pages (from-to)552-561
Number of pages10
JournalTalanta
Volume170
DOIs
Publication statusPublished - Aug 1 2017

Keywords

  • Human plasma/serum
  • Liquid-chromatography tandem-mass spectrometry (LC-MS/MS)
  • Long chain metabolites
  • Short chain metabolites
  • Vitamin E

ASJC Scopus subject areas

  • Chemistry(all)

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