Chromatographic and electrophoretic analysis of biomedically important retinoids

The determination of retinol (vitamin A) and its metabolites, as well as synthetic retinoids, in biological samples is a challenging task due to the sensitivity of these compounds to light, heat and oxygen, high protein binding, separation of geometric isomers and determination of low endogenous levels. Numerous procedures for sample preparation have been published for biological fluids and tissues, consisting of solvent extraction, solid-phase extraction (off-line) and HPLC with column switching (on-line solid-phase extraction). The last-mentioned technique has several advantages, including a high degree of automation, no evaporation of extraction solvents, protection from light and higher sensitivity. Due to the favourable UV characteristics of most retinoids, HPLC with UV detection is most often employed, and photodiode array detection is becoming more and more popular. Fluorescence and electrochemical detection have found only a limited field of application, but the use of LC-MS resulted in a few highly sensitive methods. Reconsideration of GC through the use of better deactivated columns and cold on-column injection and evaluation of new promising separation methods, such as supercritical fluid chromatography and capillary electrophoresis, have shown preliminary encouraging results, but appear to reach the required sensitivity only by coupling to MS. Therefore, HPLC with UV detection is still the method of choice for highly sensitive and selective retinoid determination, as well as for high sample throughput and robustness.