[Determination of plasma retinol and alpha-tocopherol by HPLC]

Retinol (vit A) and alpha-tocopherol (vit E), the active compounds of vitamins A and E, were assayed by reversed-phase high performance liquid chromatography (HPLC) (C18) with UV absorbance detection at 280 nm. Plasma was deproteinized and liquid-liquid extraction performed with hexane. After evaporation, the residue was dissolved in organic solvents (ether:methanol 25:75, V/V). Standard curves were prepared by adding known amounts of standards to plasma. The use of acetonitrile in the mobile phase (acetonitrile:methanol:water 64.5:33:2.5, V/V) avoided interference peaks, giving a total run time of 8 min. Analyte stability required that samples be treated in the dark. Analytical performance was good: recovery around 100%, detection limits 0.015 mg/l for vit A and 0.030 mg/l for vit E, linear range 2 mg/l for vit A and 20 mg/l for vit E, no recorded interference, and between-run and within-run precision with coefficients of variation < 11%. Analytes were stable at room temperature for 24 h (vit A) and 48 h (vit E) in plasma stored in the dark for one month at -20 degrees C. The standard solution containing both vit A and vit E increased vit A stability. Plasma concentrations (mg/l) for vit A and E were respectively: 0.63 +/- 0.17 and 9.61 +/- 3.1 in adults (n = 29), 0.39 +/- 0.17 et 7.10 +/- 2.41 in children 0 to 15 years (n = 21). This method allows regular monitoring of patients with cystic fibrosis to check for retinol and alpha-tocopherol deficiencies. The usefulness and results of the method are discussed in terms of previous studies.