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-tocopherol using normal-phase, high-performance liquid chromatography (HPLC). Our 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 a-tocopherol and retinol from literature. Also, the alpha-tocopherol concentrations 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 determined by a reversed-phase HPLC procedure, although the correlation (r=0.646, p<.001, Sy.x=62 microg/L) was not as good as the method proposed. Our procedure gave acceptable precision: the within-run CV was 7.7% for alpha-tocopherol and 5.9% for retinol. The between-day CV was 9.0% for alpha-tocopherol and 6.8% for retinol. The mean recoveries were 97% for alpha-tocopherol and 107% for retinol. Our assays were linear for alpha-tocopherol concentrations from 0.1 to 30 mg/L and for retinol concentrations from 20 to 2,000 microg/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 significantly lower than the concentrations in normal adults. Individuals over 70 y old also showed alpha-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 concentrations; however, this was not obtained for retinol. In cancer patients undergoing surgery, the ratio of retinol to retinol-binding protein (RBP) remained fairly constant, although the concentrations of both retinol and RBP decreased to about one-half the preoperative values after surgery. We conclude that our normal-phase HPLC method is a stable and reproducible method for alpha-tocopherol and retinol, and is an easy-to-use analytical tool.