Analysis of retinoids and carotenoids: problems resolved and unsolved.

Progress in nutritional biochemistry has always depended on progress in analysis of nutrients. Animal growth assays were fundamentally important in the discovery and initial isolation of the fat-soluble vitamins. Chromatography, initially introduced by Tswett for separation of plant pigments (including carotenoids), quickly became indispensable for separation of carotenoids and vitamin A compounds; the early open-column methods were eventually superseded by more efficient HPLC techniques, and reversed-phase HPLC has become the current method of choice for analysis of retinoids and carotenoids in biological tissues. Detection and quantitation of retinoids and carotenoids most often has depended on their unparalleled spectral properties; the conjugated polyene structures of these compounds give them unique light absorption spectra and high molar absorptivities, and hence outstanding lower limits of detection. Other techniques, such as gas chromatography (GC) and mass spectroscopy (coupled with GC and HPLC), immunoassays, supercritical fluid chromatography, and capillary electrophoresis, have proven useful in certain applications. Analysis of retinoid-binding proteins has been mostly by conventional protein methods, although the fluorescence of the retinol ligand has been useful in some instances to provide a highly specific assay. Current challenges in retinoid and carotenoid analysis include the resolution of stereoisomers, and quantitation of these compounds at ultratrace levels in biological tissues. Possible new approaches include accelerator mass spectroscopy, and use of gene expression assays to assess vitamin A status.