Radioimmunoassay of drugs: Phenytoin, clonidine and theophylline

The development of radioimmunoassay procedures using 125I labelled tracers, for the drugs phenytoin, clonidine and theophylline is described. These drugs are conjugated to tyrosine methyl ester (TME) and bovine serum albumin (BSA) after suitable chemical modification, for preparation of radioiodinated drugs and antisera respectively. Phenytoin was modified to phenytoin valeric acid (P-VA) and phenytoin acetic acid (p-AA). In the case of clonidine, 4 carboxy clonidine (4-CLD) was used as the starting material, while for theophylline, 7-propionic acid (7-PAT) and 8-butyric acid (8-BAT) derivatives were made. Phenytoin and clonidine derivatives were coupled to BSA by the carbodiimide method. Theophylline derivatives were coupled to BSA by the mixed anhydride method. The drug-BSA conjugates were purified by dialysis and emulsified in complete Freund's adjuvant for immunization in rabbits. The different drug derivatives were coupled to TME using carbodiimide. The conjugates were purified by thin layer chromatography (TLC). Iodine-125 labelled tracers were prepared from the drug-TME derivatives using the chloramine-T method and purified by TLC or gel filtration. In phenytoin and theophylline good separation was obtained with TLC, while in clonidine, gel filtration was found to be better. Dextran coated charcoal was used as the separation system for clonidine, while polyethylene glycol was used for phenytoin and theophylline. Various factors such as time, temperature, concentration of reagents, serum interference and separation system were optimized to arrive at a suitable assay system for each of the drugs. The optimized assay systems were validated by precision profiles, interassay and intra-assay variations and specificity studies. These optimized systems have adequate sensitivity and can be used for the measurement of the drugs in serum without prior extraction of the samples. (author). 18 refs, 3 figs, 4 tabs