The analysis of cis-trans isomeric retinols by high-speed liquid chromatography.

Since cis_??_traps isomerization of retinal and retinal_??_retinol transfor mation in vivo are essential for the visual process, the establishment of a method for separation and determination of both retinal and retinol isomers should be the most important task in the study of problems concerned with vision. In our recent reports, it was concluded that modern high-speed liquid chromatography (HSLC) is uniquely suited to the simultaneous determination of a wide range of labile conjugated polyenes such as cis-traps retinal isomers (1) or ergocalciferol and its related compounds (2). As part of our progress, we now want to report on the first example of HSLC analysis of cis-traps isomeric retinols in the presence or absence of retinal isomers, though some success had been obtained previously on a qualitative separation of retinyl acetate isomers (3). A Shimadzu-DuPont 830 Liquid Chromatograph, equipped with a UV-202 spectrophotometer, was used throughout the study. From preliminary trials, the preferred operating conditions and relative retention times (RRT) were es tablished as shown in Table 1, and the simultaneous determination of geometric isomers of retinol in a mixture is now feasible. In our previous isomerization experiments of all-trans retinal in aprotic solvents, it was recognized that the in vitro formation of the most important 11-cis isomer increased rapidly with an increase in the dielectric constant of the solvent (1). However, our HSLC analysis indicated that this was not the case in retinol. Thus, the expected 11-cis retinol was hardly detected in a photo-stationary mixture even when all-traps retinol was irradiated in acetonitrile which is a typical aprotic solvent and is known to be the most suitable media for obtaining the hindered 11-cis isomer from the corresponding all-traps aldehyde. The fact that 11-cis retinol can not be produced in vitro from the all-trans alcohol by conventional isomerization techniques was also verified by the experiment employing an am phiprotic solvent such as alcohol. A percentage composition of a mixture derived from the all-traps compound by irradiation with a 30W fluorescent lamp for 30min