SIMULTANEOUS DETERMINATION OF RETINOL, ALPHA-TOCOPHEROL AND BETA-CAROTENE IN SERUM BY ISOCRATIC HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A simultaneous determination of retinol, alpha-tocopherol and beta-carotene in serum by high-performance liquid chromatography is described. Total analysis time is 13 min. A reversed-phase (Ultrasphere ODS, 5-mu-m) column is used with a mobile phase of acetonitrile-methanol-dichloromethane (70:10:20, v/v/v) and a flow-rate of 1.2 ml/min. Retinol is monitored at 325 nm, alpha-tocopherol at 292 nm and beta-carotene at 450 nm. Serum is deproteinized with ethanol containing the internal standard (alpha-tocopherol acetate), then extracted with hexane. The evaporated organic layer is reconstituted with the mobile phase and injected. The choice of the eluent is discussed, as well as the choice of an internal standard and the need for an antioxidant during the extraction step. Sixteen different eluents are compared in terms of analysis time and selectivity. The linear concentration ranges (retinol 0.016-13.7-mu-M, alpha-tocopherol 0.18-91.8-mu-M, beta-carotene 0.05-5.75-mu-M), within-run coefficients of variation (retinol < 7%; alpha-tocopherol < 8%, beta-carotene < 7%), between-run coefficients of variation (retinol < 13%, alpha-tocopherol < 9%, beta-carotene < 8%) and recoveries (retinol > 95%, alpha-tocopherol > 91 %, beta-carotene > 80%) are suitable for clinical investigations. Serum reference values-were found to be 2.47 +/- 0.61-mu-M (retinol), 30.5 +/- 6.8-mu-M (alpha-tocopherol) and 0.91 +/- 0.55-mu-M (beta-carotene). A significant difference (p < 0.001) between males and females was found for retinol.