Isocratic reversed-phase liquid chromatographic of all- trans-retinoic acid and its major metabolites in new potential supplementary test systems for developmental toxicology

Abstract

An isocratic reversed-phase high-performance liquid chromatographic procedure for the determination of all- trans-retinoic acid (all- trans-RA) and its metabolites, all- trans-4-oxo-RA, 5,6-epoxy-RA, 9- cis-RA and13-cis-RA, in mouse plasma and embryo and in new in vitro potential test systems for development toxicology has been developed. These compounds, their biological precursor retinol (vitamin A) and the internal standard were resolved on a Spherisorb ODS-2 (5 μm) column (250×4.6 mm I.D.) with acetonitrile-water-methanol- n-butyl alcohol (56:37:4:3, v/v) containing 100 m M ammonium acetate and 70 m M acetic acid as the elution system with a total run time of 23 min. The assay was linear over a wide range, with a lower limit of quantitation of 50 ng/ml or 10 ng/ml of protein for all- trans-RA, 13- cis-RA and 9- cis-RA and of 25 ng/ml or 5 ng/ml protein for the 4-oxo- and 5,6-epoxy-metabolites. At these concentrations, intra-assay coefficients of variation (C.V.) of the retinoids were 3–9%. Mean intra-assay C.V. averaged 5–7% in the tissues studied. Its use is discussed for RA measurements in some of the new test systems — Drosophila melanogaster, sea urchin embryos and cultured human keratinocytes — that have to be evaluated in toxicological testing, supplementary to standard assays in mammals.