Simultaneous automated determination of spironolactone metabolites in serum

An automated two-phase method for the simultaneous fluorometric determination of the spironolactone metabolites canrenone (II) and canrenoic acid (III) in serum is described. The determination is performed by two dichloroethane extractions of the same serum sample at different pH values. The fluorescence developed in 65% (v/v) sulfuric acid is measured in two separate fluorometers (one each for canrenone and canrenoic acid). Comparable specificity and sensitivity to the manual procedure are obtained, with sensitivity limits of 20 ng of II/ml and of 30 ng of III/ml in serum. This method is applicable to the automated determination of drugs and metabolites in biological material when several extraction steps are involved.