Rapid and Micro-Method for the Determination of Tocopherols in Liver

Abstract

A high speed liquid chromatographic (HSLC) method with a spectrofluorometer was satisfactorily applied for the determination of α, β, γ, and δ-tocopherols in liver. It was found that tocopherols in liver were almost in free forms. Therfore, the n-hexane extracts of liver homogenate were possible to inject directly into the column without any pretreatment. Experimental procedure was as follows : The tocopherols were extracted from 2 ml of liver homogenate (liver ca.0.2g) with 5.0 ml of n-hexane after addition of 2.0 ml of ethanol containing tocol 2〜4 μg as an internal standard.Four ml of the n-hexane extracts was evaporated under N_2 gas at 40℃. The residue was dissolved in 100μl of n-hexane. Twenty μl of the solution was ipjected into the HSLC column. The eluted tocopherols were determined separately by using a fluorometer (Ex.298 nm, Em.325 nm). Fat soluble substances other than tocopherols in liver sometimes interfere the determination by commonly used method, while they did not in this method. The HSLC method have found to offer more rapid and accurate techinique than conventional method for determinig α, β, γ, and δ-tocopherols in liver.