A rapid method for the extraction and determination of vitamin E metabolites in human urine

A method for the direct extraction and routine analysis of the vitamin E me tabolites gamma- and alpha-carboxyethyl hydroxychroman (gamma- and alpha-CE HC) from human urine has been developed. A relatively small sample volume ( 5 ml) can be used and, after enzymatic hydrolysis of the conjugated forms a nd acidification, the metabolites are extracted with diethyl ether, Recover y of alpha- and gamma-CEHC was compared to that of trolox, used as an inter nal standard, added to 24h 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 add ition and extraction from urine, trolox, alpha- and gamma-CEHC are recovere d to a similar extent, hence trolox is viable as an internal standard. The samples were analyzed by both GC and HPLC with electrochemical detection (E CD), HPLC-ECD was found to give higher selectivity and higher sensitivity c ompared to GC or HPLC with UV detection at 290 nm, The HPLC-ECD detection l imit was 10 fmol, linearity (r(2) > 0.98) was achieved in the range of 40 t o 200 fmol, which was found to he optimal for 24-h urines from unsupplement ed subjects. Inter-sample variability was typically 2-5%. This greater sens itivity and selectivity means that vitamin E metabolites can be analyzed ev en in unsupplemented subjects. It is also possible to measure unconjugated forms of the metabolites. Typically these were found to represent similar t o 10% of the total alpha- and gamma-CEHC.jlr This method can be used routin ely for the determination of vitamin E metabolites in urine. The new extrac tion and detection methods described are relatively quick, less laborious, and more cost-effective than previously available methods.