Gas chromatography-mass spectrometry microanalysis of alpha- and gamma-tocopherol in plasma and whole blood.

Background: Assessing vitamin E status in humans is critical for nutritional evaluation and verification of clinical and biological compliance of supplemented subjects. An accurate analytical method for measuring the two main vitamin E isoforms, i.e. α- and γ-tocopherol (α- and γ-TOH) in small volumes of plasma can facilitate the application of this analysis to clinical trials and in situations where a limited amount of sample is available.

Methods: We have developed a micro method, which uses only 5 μL plasma, based on isotope dilution, trimethylsilation and GC-MS. The method was validated according to the guidelines of the International Conference on Harmonization of analytical procedures. The method was also applied to 5 μL of whole blood for the potential use in conditions were the availability of specimens is limited.

Results: Accurate quantitation of α-TOH and γ-TOH was achieved at levels ≥ 0.417 μM and ≥ 0.007 μM, respectively. Within-day coefficient of variation was 1.31% and 4.70% for α-TOH and γ-TOH, respectively. Between-day coefficient of variation was 1.32% and 2.88% for α-TOH and γ-TOH, respectively. Recovery, assessed at three concentration levels, ranged 98-103% and 100-102% for α-TOH and γ-TOH, respectively. The method allowed the detection of α-TOH and γ-TOH in 5 μL whole blood and in membranes of red blood cells washed from 5 μL of blood as well. The analytical performance was assessed in plasma from a cohort of Italian healthy subjects (n = 205). The mean plasma concentrations were 28.01 ± 6.31 and 0.68 ± 0.48 μM (mean ± SD) for α-TOH and γ-TOH, respectively. Alpha-TOH correlated with total cholesterol (r = 0.617, p < 0.0001) and triglycerides (r = 0.420, p < 0.0001) while γ-TOH correlated modestly with total cholesterol (r = 0.213, p < 0.0001) but not with triglycerides. γ-TOH, but not α-TOH, was significantly lower in smokers than in non-smokers (0.72 ± 0.50 vs. 0.56 ± 0.37, μM, mean ± SD, p = 0.017). Given the high sensitivity, the method allowed to be applied to 5 μM whole blood without specific modification.

Conclusions: This micro-method represents an analytical advancement in α- and γ-TOH assay that is available to accurately verify the nutritional status and compliance after supplementation in large-scale settings, and to measure the two vitamers in conditions where sample availability is limited.