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Abstract
Preanalytical and analytical requirements to attain a precise and accurate determination of fat-soluble nutrients in serum by reversed-phase liquid chromatography and multiwavelength detection are described. Retinol, α-tocopherol, α and β-carotene and lycopene were extracted from serum samples with two volumes of hexane-ethanol (5 : 1 v/v). Analytical recovery of retinol and α-tocopherol ranged from 80–90%, while those of αand β-carotene and lycopene never exceed 50%. Chromatography was performed using a reversed-phase column with a mobile phase consisting of ethanol-acetonitrile 1 : 1 with 0.1 mL diethylamine per liter of solvent. Limits of quantitation were 5 ng/mL for retinol, 200 ng/mL for α-tocopherol, and 5 ng/mL for α, β-carotene, and lycopene, respectively. This method has been used in large-scale determinations of fat-soluble nutrients, never needing dilution of samples or being under limit of quantitation.