Simultaneous measurement of seven carotenoids, retinol and alpha-tocopherol in serum by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative measurement in serum of seven carotenoids (lutein, zeaxanthin, canthaxanthin, beta-cryptoxanthin, lycopenes, alpha-carotene and beta-carotene), retinol, alpha-tocopherol and two internal standards (tocol and echinenone) has been developed. The geometric isomers, lutein and zeaxanthin, were completely separated as well as at least nine unidentified carotenoids. All compounds were resolved on an Adsorbosphere HS C-18 (3 mu m) column (250 x 4.6 mm I.D.) with a step gradient, 7.1 min after injection, from acetonitrile-methanol (60:40, v/v) containing 0.05% acetic acid to acetonitrile-methanol-dichloromethane (45.6:30.4:24, v/v) containing 0.04% acetic acid, in a total run time of 23 min. Chromatograms at four different wavelengths (292, 325, 450 and 473 nm) and spectra were monitored with a diode array detector. Because of its specificity and sensitivity for a large number of carotenoids, this procedure may be of interest for nutritional and epidemiological studies. Its speed and robustness make it suitable for analyses on large numbers of subjects.