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.
Department of Medico-Surgical Sciences and Biotechnologies, Laboratory of Vascular Biology and Mass Spectrometry, Sapienza University of Rome, Latina, Italy.
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Zerbinati
Department of Internal Medicine, Laboratory of Clinical Biochemistry and Nutrition, University of Perugia, Perugia, Italy.
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Galli
Department of Medico-Surgical Sciences and Biotechnologies, Laboratory of Vascular Biology and Mass Spectrometry, Sapienza University of Rome, Latina, Italy.
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Regolanti
Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy.
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Poli
Department of Medico-Surgical Sciences and Biotechnologies, Laboratory of Vascular Biology and Mass Spectrometry, Sapienza University of Rome, Latina, Italy. Electronic address: luigi.iuliano@uniroma1.it.
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Iuliano
D000328:Adult; D000975:Antioxidants / Q000378:metabolism*; D001786:Blood Glucose / Q000378:metabolism; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood; D008077:Lipoproteins, LDL / Q000097:blood; D008297:Male; D008875:Middle Aged; D015588:Observer Variation; D011865:Radioisotope Dilution Technique; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014280:Triglycerides / Q000097:blood; D014297:Trimethylsilyl Compounds / Q000737:chemistry; D014316:Tritium; D024502:alpha-Tocopherol / Q000097:blood*; D024504:gamma-Tocopherol / Q000097:blood*
D000975:Antioxidants; D001786:Blood Glucose; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D014280:Triglycerides; D014297:Trimethylsilyl Compounds; D014316:Tritium; D024504:gamma-Tocopherol; D024502:alpha-Tocopherol
Antioxidants; Lipid peroxidation; Method; Oxidative stress; Vitamin E