SIMULTANEOUS ANALYSIS OF INDIVIDUAL CAROTENOIDS, RETINOL, RETINYL ESTERS, AND TOCOPHEROLS IN SERUM BY ISOCRATIC NONAQUEOUS REVERSED-PHASE HPLC

A sensitive high-performance reversed-phase liquid chromatographic assay which utilizes isocratic elution and detection by two detectors has been developed to measure simultaneously several carotenoids retinol, retinyl esters, and alpha- and gamma-tocopherols in serum. Although 100 mul of serum or plasma is used routinely, as little as 10 mul can be analyzed. Typical lower limits of detection (5:1 signal/noise ratio) are 0.2 pmol for retinol, 6 pmol for alpha-tocopherol, 1 pmol for beta-carotene, and 2 pmol for retinyl palmitate. Although lutein and zeaxanthin were only partially resolved from each other, all other major serum carotenoids including alpha-carotene from beta-carotene and alpha-cryptoxanthin from beta-cryptoxanthin were completely separated. Retinyl hexanoate and beta-apo-8'-carotenyl decanoate are convenient, readily prepared internal standards for this assay. Relative retention indices, based on elution times of alkylphenone standards, proved useful both for characterization of compounds and for comparison between columns.