SIMULTANEOUS DETERMINATION OF ALL-TRANS-RETINOIC ACID, 13-CIS-RETINOIC ACID, 9-CIS-RETINOIC ACID AND THEIR 4-OXO-METABOLITES IN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A gradient reversed-phase high-performance liquid chromatographic tech nique is described for the easy separation and quantification of some retinoids; all-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retino ic acid and their corresponding 4-oxometabolites, in plasma. The metho d involved a diethyl ether-ethyl acetate (50:50, v/v) mixture extracti on at pH 7 with acitretin and 13-cis-acitretin as internal standards. A Nova-Pak C-18 steel cartridge column was used. The mobile phase was methanol-acetonitrile (65:35, v/v) and 5% tetrahydrofuran (solvent A) and 2% aqueous acetic acid (solvent B) at 1 ml/min. The gradient compo sition was (only the percentages of solvent B are mentioned): I, 25% s olvent B at the time of injection; II, 12% solvent B at 11 min until 3 0 min; III, 25% solvent B and maintenance of 25% solvent B for 10 min until a new injection. Total time between injections was 40 min. Detec tion was by absorbance at 350 nm. The precision calculated for plasma concentrations ranging from 2 to 250 ng/ml was better than 15% and the accuracy was less than 12%. The linearity of the method was in the ra nge of 2 to 400 ng/ml of plasma. The limit of quantification was 2 ng/ ml for each of the compounds. The HPLC method was applied to plasma sp ecimens collected from animals receiving single dose administrations o f all-trans-retinoic acid, 13-cis-retinoic acid and 9-cis-retinoic aci d.