Radioimmunoassay of Flurazepam in Human Plasma

Flurazepam belongs to the class of hypnotic agent used for the treatment of insomnia. 

Immunization and Antibody Production The immunogen 3-hemisuccinyloxyflurazepam, is emulsified with complete Freund s adjuvant. It is injected intradermally into two female New Zealand albino (white) rabbits. Repeated doses are administered twice at interval of two weeks. Subsequently, booster injections of the thick-immunogen-emulsion-paste are administered after a span of 6-weeks. The antibody is harvested when its titer level is high enough, diluted to the suitable-level and employed in the RIA. 

RIA-Procedure The different steps followed in the RIA-proccdurc are as given below  

RIA-Specificity The specificity of the antiserum initially is evaluated by cross-reactivity studies involving all the flurazepam metabolites known to be present in plasma. The mono-as well as di-desethylmetabolites exhibited a cross-reactivity of 17 and 3.7% respectively, while other possible competitors cross-reacted less than 1% as shown in Table 32.1. 

Evidently, due to the cross-reactivity of both mono- and di-desethyl metabolites, a specific assay of flurazepam could not be developed successfully without first separating it from its metabolites effectively by the help of column chromatography. 

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W Glover, J Earley, M Delaney, R Dixon. Radioimmunoassay of flurazepam in human plasma. J Pharm Sci 69 601, 1980. 

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