Gas chromatography mass spectrometry analysis of vitamin E and its oxidation products

To facilitate studies of vitamin E (alpha-tocopherol) antioxidant actions in tissues, we have developed a stable isotope dilution capillary gas chromatography-mass spectrometry assay for alpha-tocopherol and its three principal oxidation products, alpha-tocopherolquinone, 5,6-epoxy-alpha-tocopherolquinone, 2,3-epoxy-alpha-tocopherolquinone, and for alpha-tocopherolhydroquinone, a reduction product of alpha-tocopherolquinone. Deuterium-labeled internal standards (5,7-[H-2(3)-methyl]-alpha-tocopherol, 2,6-[H-2(3)-methyl]-alpha-tocopherolquinone, 2,6-[H-2(3)-methyl]-5,6-epoxy-alpha-tocopherolquinone, 2,6-[H-2(3)-methyl]-2,3-epoxy-alpha-tocopherolquinone, and 2-[H-2(3)-methyl]-alpha-tocopherolhydroquinon are added to cell or tissue homogenates, The products then are extracted and converted to O-trimethylsilyl derivatives, Products are analyzed by capillary gas chromatography with on column injection and detected by selected ion monitoring of characteristic fragment ions in electron ionization mode, Standard curves were linear from 25 fmol to 2 pmol for products and from 25 fmol to 4 pmol for alpha-tocopherol, The use of H-2(6)- and H-2(3)-internal standards for alpha-tocopherolquinone and alpha-tocopherolhydroquinone permits simultaneous analysis of both products despite possible redox interconversion during sample workup, alpha-Tocopherol oxidized in microsomes treated with azo-bis(amidinopropane HCl) was quantitatively accounted for as the epoxyquinones, alpha-tocopherolquinone, and alpha-tocopherolhydroquinone. However, over half of the oxidation products were present in microsomes as acid-labile tocopherone precursors, This method permits comprehensive assessment of vitamin E status in tissues and quantitative studies of vitamin E antioxidant actions and turnover. (C) 1996 Academic Press, Inc.