Clinical and analytical evaluation of the simultaneous HPLC assay of retinol and alpha-tocopherol

We describe a method for the simultaneous assay of retinol and alpha -tocop herol using normal-phase, high-performance liquid chromatography (HPLC). Ou r normal-phase HPLC method gave better resolution (Rs) of retinol (Rs=1.58) and alpha -tocopherol (Rs=1.40) when compared with the Rs values for alpha -tocopherol and retinol from literature. Also, the alpha -tocopherol conce ntrations obtained by our method agreed well with another normal-phase HPLC method that used fluorometric detection (r=0.951, p<0.001, Sy.x=0.58 mg/L) , The concentrations of retinol in our method agreed well with those determ ined by a reversed-phase HPLC procedure, although the correlation (r=0.646, p<0.001, Sy.x=62 mug/L) was not as good as the method proposed. Our proced ure gave acceptable precision: the within-run CV was 7.7% for alpha -tocoph erol and 5.9% for retinol. The between-day CV was 9.0% for alpha -tocophero l and 6.8% for retinol. The mean recoveries were 97% for alpha -tocopherol and 107% for retinol. Our assays were linear for alpha -tocopherol concentr ations from 0.1 to 30 mg/L and for retinol concentrations from 20 to 2,000 mug/L. In children ages 7 to 12 y, and in adolescents ages 14 to 16 y, the alpha -tocopherol and retinol concentrations in the blood were significantl y lower than the concentrations in normal adults. Individuals over 70 y old also showed a-tocopherol and retinol values that were lower than those of normal adults between ages 30 and 40 y. In female university students, the inter-individual variation of alpha -tocopherol was reduced by dividing the alpha -tocopherol results by their total cholesterol or total lipid concen trations; however, this was not obtained for retinol. In cancer patients un dergoing surgery, the ratio of retinol to retinol-binding protein (RBP) rem ained fairly constant, although the concentrations of both retinol and REP decreased to about one-half the preoperative values after surgery. We concl ude that our normal-phase HPLC method is a stable and reproducible method f or alpha -tocopherol and retinol, and is an easy-to-use analytical tool.