Non‐equilibrium Method for the Radioimmunoassay of Clozapine in the Presence of Metabolites

Cross‐reactions with metabolites are an ever‐recurring problem encountered in the use of radioimmunoassay techniques to determine active compounds in biological material. Metabolites may interfere with the assay of the parent drug to a variable extent. Taking 8‐chloro‐11‐(4‐methyl‐1‐piperazinyl)‐5H‐dibenzo[b,e][1,4]d iazepine (clozapine) as an example, it was shown that the extent to which the antiserum produced interacts with the parent drug and the metabolites can be estimated by determining the equilibrium constants and the kinetics. In the present case, therefore, it was advantageous to carry out the radioimmunoassay in disequilibrium, i.e, in order to differentiate the metabolites from the parent drug. the sample was incubated with the antiserum for 10 min, after which the labelled antigen was added and the reaction mixture again incubated for a brief, exactly timed interval. It was shown that cross‐reactions did not occur in mixtures of clozapine and its N‐demethyl and N‐oxide metabolites in the proportions 1: 1: 2 over a range of concentration of 1.5–48 ng clozapine per 100 μl human plasma. The equilibrium constants measured with the clozapine goat antiserum were as follows: clozapine 1.2 × 108, M−1, the N‐demethyl metabolite 4.6 × 107 M−1 and the N‐oxide metabolite 3.7 × 107 M−1 (pH 7.5 and 20 °C).