Determination of retinol, alpha-tocopherol, and beta-carotene in serum by liquid chromatography with absorbance and electrochemical detection.

We describe a method for the determination of retinol, alpha-tocopherol, and beta-carotene in serum, using a liquid-chromatographic separation with wavelength-programmed ultraviolet/visible absorbance and amperometric electrochemical detection with a glassy carbon electrode. After protein denaturation and addition of an internal standard, tocol, 250-microL samples are twice extracted with hexane. The reversed-phase, gradient-elution chromatographic separation provides baseline resolution of: the all-trans isomer of retinol from the cis isomers, alpha- from gamma-tocopherol, and all-trans-beta-carotene from alpha-carotene and from cis-beta-carotene isomers. The linearity of response and the detection limits for the two detectors for the three analytes are measured. A comparison of the values obtained for serum extracts shows good agreement between the absorbance and electrochemical detectors.