The development of immunoassy provides a powerful method for the measurement of drug levels in biological fluids. The extensive clinical use and continued development of benzodiazepines as a class of drugs makes it desirable that immunoassays be developed for these compounds.
Several such immunoassays directed to benzodiazepines have been developed and reported in the literature. Thus Peskar and Spector described a radioimmunoassay procedure useful in detecting nanogram amounts of diazepam or N-desmethyldiazepam in plasma samples in J. Pharmacol. Exp. Ther. 186, 167 (1973). This assay utilized .sup.14 C-diazepam as the tracer. Antibodies were elicited using either 5- [3-(4-aminophenylazo)-4-hydroxyphenyl] -7-chloro-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one or 7-amino-5-(2-chlorophenyl)-3H-1,4-benzodiazepin-2-(1H)-one as haptens which were subsequently coupled to bovine serum albumin (BSA) to form the desired immunogens.
Subsequently, Dixon et al. reported in J. Pharm. Sci. 64, 937 (1975) of a radioimmunoassay for chlordiazepoxide in plasma. Once again the tracer compound was a .sup.14 C-labelled compound. The immunogen was derived by coupling the reactive acyl azide of 7-chloro-5(4-hydrazinocarbonylmethoxyphenyl)-2-methylamino-3H-1,4-benzodia zepine 4-oxide to BSA.
Spin labelling of benzodiazepinse for use in a Free Radical Assay Technique is disclosed by Goldstein et al. in U.S. Pat. No. 3,690,834. Specific benzodiazepines disclosed include chlordiazepoxide, diazepam and oxazepam.
Another paper, by Dixon et al., Pharm. 17, 251 (1975), describes a radioimmunoassay for clorazepam using an immunogen consisting of 3-hemisuccinoyloxyclorazepam covalently bound to BSA. The tracer employed for the assay was .sup.3 H-clorazepam. Sensitivity of the assay was 5 ng/ml using a 0.1 sample of plasma.