Abstract
A method for the direct extraction and routine analysis of the vitamin E metabolites γ- and α-carboxyethyl hydroxychroman (γ- and α-CEHC) from human urine has been developed. A relatively small sample volume (5 ml) can be used and, after enzymatic hydrolysis of the conjugated forms and acidification, the metabolites are extracted with diethyl ether. Recovery of α- and γ-CEHC was compared to that of trolox, used as an internal standard, added to 24-h urine collections from vitamin E-unsupplemented volunteers. Various solvent conditions were initially tested; acidification and ether extraction gave the highest recovery. It was found that after addition and extraction from urine, trolox, α- and γ-CEHC are recovered to a similar extent, hence trolox is viable as an internal standard. The samples were analyzed by both GC and HPLC with electrochemical detection (ECD). HPLC-ECD was found to give higher selectivity and higher sensitivity compared to GC or HPLC with UV detection at 290 nm. The HPLC-ECD detection limit was 10 fmol, linearity (r2 > 0.98) was achieved in the range of 40 to 200 fmol, which was found to be optimal for 24-h urines from unsupplemented subjects. Inter-sample variability was typically 2–5%. This greater sensitivity and selectivity means that vitamin E metabolites can be analyzed even in unsupplemented subjects. It is also possible to measure unconjugated forms of the metabolites. Typically these were found to represent ~10% of the total α- and γ-CEHC. This method can be used routinely for the determination of vitamin E metabolites in urine. The new extraction and detection methods described are relatively quick, less laborious, and more cost-effective than previously available methods. —Lodge, J. K., M. G. Traber, A. Elsner, and R. Brigelius-Flohé. A rapid method for the extraction and determination of vitamin E metabolites in human urine.
Highlights
A method for the direct extraction and routine analysis of the vitamin E metabolites ␥- and ␣-carboxyethyl hydroxychroman (␥- and ␣-CEHC) from human urine has been developed
Vitamin E is a mixture of ␣, , ␦, and ␥- tocopherol and tocotrienols, differing in the methyl substitutions on the chromanoxyl ring, and the saturation of the phytyl tail (1). ␣-Tocopherol is the most studied because it has the Abbreviations: CEHC, carboxyethyl hydroxychroman; ECD, electrochemical detection; GC/MS, gas chromatography-mass spectroscopy; TCA, trichloroacetic acid; ␣-TTP, ␣-tocopherol transfer protein
Trolox was added either directly to the urine before extraction or immediately prior to derivatization. This latter protocol yielded a trolox peak that was taken as 100%, any deviations from this value represents a difference in recovery
Summary
From a 24 h urine collection from normal unsupplemented subjects, 5 ml was taken and the internal standard was added (30 g trolox final for GC, 10 g for HPLC-ECD, added in ethanol). A one-step ether extraction was later validated, using 15 ml of diethyl ether In this case the samples could be mixed thoroughly with a vortex mixer for 30 secs. For GC, to remove adventitious water, the extract was resuspended twice in 500 l ethanol and dried under nitrogen These dried samples were resuspended in 500 l of derivatization solution (200 l hexane, 250 l BSTFA, 40 l BSA, and 10 l TMCS) and incubated at 50ЊC for 2 h with constant shaking. To measure unconjugated forms of the metabolites, the urine aliquots were acidified (50 l 6 m HCl, incubated for 5 min on ice) directly extracted with diethyl ether (1 ϫ 15 ml) as described above. A number of standards must be analyzed at the same time as the samples (see below)
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