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.
Department of Laboratory Medicine, Ohashi Hospital, Toho University School of Medicine, Meguro, Tokyo, Japan. ihara@med.toho-u.ac.jp
H
H
Ihara
H
H
Ishigaki
N
N
Shino YHashizume
M
M
Takase
J
J
Nagao
Y
Y
Sumiyama
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003110:Colonic Neoplasms / Q000601:surgery; D005260:Female; D005470:Fluorometry / Q000379:methods; D006801:Humans; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D012177:Retinol-Binding Proteins / Q000032:analysis; D013274:Stomach Neoplasms / Q000601:surgery; D013997:Time Factors; D014801:Vitamin A / Q000097:blood*; D014810:Vitamin E / Q000097:blood*
D012177:Retinol-Binding Proteins; D014801:Vitamin A; D014810:Vitamin E