MK-286 is a 90:10 mixture of the (+) and (-)-enantiomers of indacrinone, a combination which induces diuresis while maintaining isouricemia. The principal (phenolic) metabolites also possess pharmacological activity and assays for the four entities were needed for clinical studies. Antisera were produced with the stereospecificity required to measure one enantiomer in the presence of the others, but significant cross-reactivity between drug and metabolite necessitated the separation of these two species by means of Sep-Pak cartridges. Recovery was assessed by concurrently fractionated reference samples. The radioligand in each case was a [I-125]-L-iodotyrosine conjugate of the analyte. Absolute assay sensitivities ranged from 2-20 pg/assay tube, corresponding to 0.2-2.0 ng/ml in serum samples. The assay was also applied to urine with analytes at concentrations greater than 16 ng/ml.