Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

Abstract

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13- cis, 9- cis, all- trans, all- trans-4-oxo, 9- cis-4-oxo, 13- cis-4-oxo), three retinal isomers (13- cis-, 9- cis-, and all- trans) and two retinol isomers (13- cis- and all- trans). Human serum samples were subjected to this HPLC analysis and at least, all- trans retinol, 13- cis retinol, and all- trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all- trans retinoic acid in human sera.