ISOCRATIC REVERSED-PHASE LIQUID-CHROMATOGRAPHY OF ALL-TRANS-RETINOIC ACID AND ITS MAJOR METABOLITES IN NEW POTENTIAL SUPPLEMENTARY TEST SYSTEMS FOR DEVELOPMENTAL TOXICOLOGY

An isocratic reversed-phase high-perfomance liquid chromatographic pro cedure 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 and 1 3-cis-IA, in mouse plasma and embryo and in new in vitro potential tes t systems for developmental toxicology has been developed. These compo unds, their biological precursor retinol (vitamin A) and the internal standard were resolved on a Spherisorb ODS-2 (5 mu m) column (250x4.6 mm I.D.) with acetonitrile-water-methanol-n-butyl alcohol (56:37:4:3, v/v) containing 100 mM ammonium acetate and 70 mM acetic acid as the e lution system, with a total run time of 23 min. The assay was linear o ver a wide range, with a lower limit of quantitation of 50 ng/ml or 10 ng/mg of protein for all-trans-RA, 13-cis-RA and 9-cis-RA and of 25 n g/ml or 5 ng/mg protein for the 4-oxo- and 5,6-epoxy-metabolites. At t hese concentrations, intra-assay coefficients of variation (C.V.) of t he retinoids were 3-9%. Mean intra-assay C.V. averaged 5-7% in the tis sues studied. Its use is discussed for RA measurements in some of the new test systems - Drosophila melanogaster, sea urchin embryos and cul tured human keratinocytes - that have to be evaluated in toxicological testing, supplementary to standard assays in mammals.