23] An HPLC method for the simultaneous determination of retinol and α-tocopherol in plasma or serum

Abstract

This chapter describes the high-performance liquid chromatography (HPLC) method that offers a rapid, sensitive, simple, specific, precise, and nondestructive microprocedure for the simultaneous determination of retinol and α-tocopherol in plasma or serum. An isocratic HPLC system is capable of reproducing the conditions including a UV detector and a 10-mV, 10-cm recorder, intergrating recorder or data handling system. Step requiring hexane should be carried out in a hood. Alternatively, heptane may be used for extraction. Either plasma or serum can be used as sample. Specimens collected after an overnight fast are preferred. Direct exposure to natural illumination should be avoided. Retinol and α-tocopherol in serum are stable to repeated freezing (-20 °) and thawing (17 cycles over a period of 5 weeks). Sample sizes of 100–400 μl do not affect the linearity of the assay, provided the proportion of ethanol to plasma in the initial precipitation of proteins is not changed. Injection volumes of 30–90 μl do not affect linearity of the assay. It is necessary to use α-tocopheryl acetate as internal standard when assaying samples taken from plastic blood collection bags. Some phthalate esters (used as plasticizers) are extracted by the plasma and either cochromatograph or interfere with quantification of retinyl acetate. In addition, tocol has been used successfully as a single internal standard in a simultaneous assay. No interfering peaks are present in plasma with retention times of the two internal standards. In addition using plasma from a vitamin A deficient rat and a β- lipoproteinemic human, it has been shown that no peaks occur in plasma which interfere with retinol and tocopherol, respectively.