Automated Determination of 13-Cis-and All-Trans-Retinoic Acid, Their 4-Oxo-Metabolites and Retinol in Plasma, Amniotic Fluid and Embryo by Reversed-Phase High-Performance Liquid Chromatography with a Precolumn Switching Technique

Abstract

A fully automated reversed-phase high-performance liquid chromatography (HPLC) procedure with precolumn switching is presented which allows quantitative assay of 13-cis-retinoic acid and all-trans-retinoic acid, their 4-oxo-metabolites and retinol in plasma, amniotic fluid and tissue homogenates. A binary gradient system allowed baseline separation of the retinoids within 15 minutes. Sample preparation was kept simple in order to minimize degradation and isomerization of the unstable substances and required only the addition of isopropanol, freezing in liquid nitrogen, and centrifugation. Overall recovery was quantitative allowing for external standardization. Calibration curves were linear in mouse plasma, amniotic fluid and human serum albumin solution (r < 0.99) and in mouse embryo homogenate (r < 0.98) over a concentration range of 4 orders of magnitude. The detection limit was 2 ng/ml or g with a sample size of only 0.1 ml or 0.1 g. Coefficients of variation of the assay were less than 5% (within day) and 8% (day to day). The small sample size required, their simple preparation, the rapid analysis, and the high degree of automation make this method well suited for experimental or clinical pharmacokinetic studies following administration of therapeutic or toxic doses. This method was applied in a study on the transplacental pharmacokinetics of 13-cis and alltrans-retinoic acid during mouse embryo organogenesis.