Gas chromatography mass spectrometry analysis of carboxyethyl-hydroxychroman metabolites of alpha- and gamma-tocopherol in human plasma

Alpha- and gamma-tocopherol (alpha- and gamma-T, respectively) metabolite analysis is of key relevance in the study of vitamin E metabolism. Whilst there is information on urinary excretion of the two major metabolites of these vitamin E homologues, namely the 2,5,7,8-tetramethyl-2-(beta-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), their concentration and response to supplements in plasma remains poorly investigated. In this study we describe a gas chromatography-mass spectrometry (GC/MS)-based assay to measure both alpha- and gamma-T and their corresponding CEHC metabolites in human plasma. As an example of the application of this method we report data obtained following the supplemention of two healthy volunteers with 100 mg of deuterium-labeled gamma-T acetate (d(2)-gamma-TAC). Under routine analytical conditions a good linearity in the range 0.0025--1 microM was observed for both the alpha- and gamma-CEHC deuterated standards. In plasma samples, the detection limit for alpha- and gamma-CEHC was 2.5 and 5 nmol/l, respectively. The minimum amount of plasma required for the assay was 500 microl. The plasma concentrations of alpha-CEHC and gamma-CEHC in unsupplemented healthy subjects were 12.6 +/-7.5 and 160.7 +/- 44.9 nmol/l, respectively. In the two volunteers supplemented with 100 mg of d(2)-gamma-TAC, plasma d(2)-gamma-T concentrations increased 250 to 450-fold 6 h postsupplementation. Plasma and urinary d(2)-gamma-CEHC concentrations increased 20 to 40-fold 9--12 h postsupplementation. Interestingly, the acute increase in d(2) gamma-T did not significantly affect the baseline plasma concentrations of d(0)-gamma-T and only slight lowered alpha-T concentrations. Likewise, plasma alpha-CEHC levels were not influenced and urinary excretion of alpha-CEHC were unaltered. This GC/MS method provides a versatile and accurate mean for assessing carboxyethyl-hydroxychroman metabolites of vitamin E in plasma.