High-performance liquid chromatography of the unsaponifiable from samples of marine and freshwater fish: fractionation and identification of retinol (vitamin A 1) and dehydroretinol (vitamin A 2) isomers

Abstract

Retinol (vitamin A 1) and dehydroretinol (vitamin A 2) geometric isomers, obtained in photosynthetic mixtures or from natural samples, were simultaneously fractionated by means of isocratic normal phase high-performance liquid chromatography. The use of low percentages of isopropanol (0.4–1.1%) as a modifier in hexane was studied, and the resolution of both pairs of retinols (9- cis/all- trans and 13- cis/11- cis) whose baseline separation had not previously been achieved was obtained. The proposed chromatographic method therefore allows the complete separation of all six of the most commonly occurring retinols in natural samples, including the above four mono cis-retinols plus the two 9,13- and 11,13-di- cis-retinols Photoisomerization of retinal and of retinol in different solvent systems and during the extraction process was also studied, before analyzing the unsaponifiable from fresh and marine water fish, which constitute rich natural sources of both vitamin A 1 and A 2. Vitamin A 2 was found to be present in different relative percentages depending on the analytical matrix. Four cis isomers (9- cis, 11- cis-, 13- cis- and 9,13-di- cis-retinol or corresponding dehydroretinols) were found to occur naturally together with the main all- trans form, confirming the need to separate geometric isomers in every dosage of vitamin A-containing compounds.