An 125I-radioimmunoassay for the determination of the anticonvulsant agent clonazepam directly in plasma

Abstract

A simple and specific 125I-radioimmunoassay (RIA) for the determination of the anticonvulsant agent clonazepam directly in plasma has been developed using a previously reported antiserum to the drug. The 125I-radioligand was prepared by reaction of 3-aminoclonazepam with a commercially available acylating reagent, 125I-N-succinimidyl 3(4-hydroxyphenyl) propionate, of high specific activity (approximately 1500 Ci/mM). The method has a workable range between 5 and 100 ng/ml of clonazepam using a 10 mu1 sample of plasma for analysis, which is satisfactory for routine clinical monitoring. The intra and inter-assay coefficients of variation did not exceed 3 and 9% respectively. Recovery of clonazepam was quantitative when the drug was added to a pool of plasma obtained from subjects who had received a wide variety of other anticonvulsants. Clinical plasma samples from subjects receiving clonazepam chronically were assayed for clonazepam by the new 125I-RIA and a previously reported 3H-RIA. The data was subjected to linear regression analysis which gave a slope, intercept and correlation coefficient of 1,-0.33 ng/ml and 0.994 respectively and demonstrated that the two methods are equivalent. However, the 125I-RIA is more rapid and less costly for routine monitoring than the 3H-RIA.