On-line solid-phase extraction and isocratic separation of retinoic acid isomers in microbore column switching system

On-line solid-phase extraction coupled with micro-HPLC by column switching is an ideal technique for the analysis of retinoic acid in serum or plasma. The advantages are mainly contributed to an automated sample workup and low detection limits. On-line processing of the sample ensures minimal losses and full light protection during the entire procedure. Critical steps such as evaporation, extraction, and multiple transfers are avoided. Furthermore, the precision of highly automated methods is generally better than manual methods. We have successfully coupled a 2.1-mm I.D. analytical column with a 2.1-mm extraction column. This setup allows for large amounts of supernatant to be injected onto precolumns for concentration and cleanup. By means of column switching, this concentrate is transferred to the microcolumn with a highly reduced volume. The reduced diameter of the analytical column and the on-line solid-phase extraction allow for the fully automated quantification of as little as 100 fmol all-trans-retinoic acid in human serum. The detection limits obtained with these column switching techniques can compete with LC-MS. This new micro-HPLC method will be useful for the quantitation of endogenous retinoic acid metabolites, which are present at very low concentrations in biological material. Furthermore, more sensitive methods might also lead to the discovery of hitherto unknown retinoic acid metabolites. The combination of on-line SPE and micro-HPLC has, to our knowledge, not been used previously for retinoic acid analysis. The development of isocratic separation methods for retinoic acid isomers made this possible.