Determination of tocopherol and tocopherol acetate concentrations in human feces using high-performance liquid...

A method of sample clarification and high-performance liquid chromatography (HPLC) specifically developed to permit simple and rapid determination of vitamin E (alpha-tocopherol, E) and vitamin E acetate (EA) in feces is reported. Retinol acetate (RA) was used as an internal standard. The vitamins of interest were extracted from an aqueous stool suspension into an organic phase (ethyl acetate-butanol), which was injected directly onto the reversed-phase HPLC system. An isocratic mobile phase of methanol-water (97:3) was employed, with ultraviolet detection at 275 and 285 nm (to permit simultaneous monitoring and absorbance ratio determination). Recoveries of exogenous RA, E, and EA from stool suspensions (relative to water) were 99.0 +/- 7.0, 100.9 +/- 7.0, and 101.2 +/- 13.3%, respectively (n = 10). The organic matrix could be stored at -35 degrees C overnight with no change in E or EA results. Sensitivities for E and EA were 80 and 102 micrograms/g of stool, respectively. Each analysis required nine min. The within-day coefficients of variation were 2.9, 3.6, and 3.0% (n = 7) for RA, E, and EA, respectively. Neither E nor EA were detected in baseline fecal samples from fourteen subjects, but both were present in high but varied concentrations after four weeks supplementation with oral d,l-EA. E but not EA was present in blood samples drawn during periods of oral supplementation with EA. There was poor correlation between fecal levels of E and EA, and the increase in serum levels of E. This method permits rapid, selective, and precise determination of E and EA in human fecal samples.