Determination of vitamins A and E in serum and plasma using a simplified clarification method and high-performance...

A method of sample clarification and high-performance liquid chromatography specifically developed to permit precise and rapid determination of vitamin A (retinol) and vitamin E (alpha-tocopherol) in serum and plasma is reported. Serum proteins were denatured by the addition of acetonitrile containing alpha-tocopherol acetate, the internal standard; the vitamins were subsequently extracted into an organic matrix consisting of ethyl acetate-butanol (1:1); no solvent evaporation step was required. The three vitamins of interest were eluted from a reversed-phase C18 column with an isocratic mobile phase methanol-water (95:5); detection was accomplished by measuring ultraviolet absorption at 280 nm. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from spiked aqueous samples averaged 100.0, 100.0 and 98.8%, respectively. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from plasma and serum relative to water were 102.6, 96.9 and 96.5%, respectively. Retinol and alpha-tocopherol were stable in the extraction matrix for up to 3.5 h, and were stable in heparinized plasma stored at room temperature for two days. Oxalate, citrate and EDTA caused significant losses of retinol and alpha-tocopherol, while vitamin levels in serum and heparinized plasma were similar. Limits of detection for retinol and alpha-tocopherol were 60 ng/ml and 0.9 micrograms/ml, respectively. Each run required 12 min. Same-day coefficients of variation were 3.5 and 3.6% for retinol and alpha-tocopherol, respectively (n = 11). Between-day coefficients of variation for retinol and alpha-tocopherol were 4.8 and 5.5%, respectively (n = 5). This method permits simple, rapid, sensitive, selective and precise determination of retinol and alpha-tocopherol using 0.5 ml of serum or heparinized plasma.