Determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and UV detection

A high-performance liquid chromatographic method with automated column swit ching was developed for the simultaneous determination of 13-cis-3-hydroxyr etinoic acid, all-trans-3-hydroxyretinoic acid and their metabolites 13-cis -3-hydroxy-4-oxo-retinoic acid and all-trans-3-hydroxy-4-oxo-retinoic acid in plasma samples from man, rat, dog, rabbit and mouse. The method consists of deproteination of plasma (0.4 mi) with ethanol (1.5 mi), containing the internal standard Ro 12-7310. After centrifugation, 1.4 mi of the supernat ant was directly injected onto the precolumn (PC) (4 x 4 mm) packed with Li Chrospher 100 RP-18 (5 mu m). Ammonium acetate (0.02%)-acetic acid-ethanol (100:3.4, v/v/v) was used as mobile phase M1A during injection, as well as to decrease the elution strength of the injection solution by on-line addit ion using a T-piece (M1B). After valve switching, the retained components w ere transferred to the analytical column (AC), separated by gradient elutio n and detected at 360 nm. Two coupled Purospher 100 RP-18 endcapped columns (both 250 x 4 mm) were used for the separation, together with a mobile pha se consisting of acetonitrile-water-10% ammonium acetate-acetic acid, (A), 540:450:2:30 (v/v/v/v), (B), 600:350:2:30 (v/v/v/v), and (C), 950:40:2:30 ( v/v/v/v). The method was linear in the range 1-500 ng ml(-1), at least, wit h a quantification limit of 1 ng ml(-1). The mean recoveries from human pla sma were 100-107% and the mean inter-assay precision was 2.0-4.7% (range 1- 500 ng ml(-1)). Similar results were obtained for animal plasma. The analyt es were stable in the plasma of all investigated species stored at - 20 deg rees C for 3 months, at least. The method was successfully applied to clini cal and toxicokinetic studies. (C) 1998 Elsevier Science B.V. All rights re served.