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Chapter: Pharmaceutical Drug Analysis: Radioimmunoassay

Novel Applications of Radioimmunoassay(RIA)-Techniques

(i) Combined RIA Technique-Isotope Dilution, and (ii) Stereospecificity.


The radioimmunoassay technique has been gainfully exploited in a variety of novel applications of which only the two important aspects stated below will be discussed briefly, namely :


(i) Combined RIA Technique-Isotope Dilution, and


(ii) Stereospecificity.




In normal RIA-procedures the labelled drug or metabolite not only serves as the tracer for recovery but also for RIA quantification. However, the isotope dilution method categorically makes a clear separation of the drug and its metabolites. Consequently, a non-specific antiserum is employed to actually quantify the total amount of both unlabelled and labelled substance present.


The combined RIA-technique and isotope dilution has been successfully developed to estimate SULINDAC* along with its two prominent metabolites, namely : its sulphone and its sulphide present in the plasma-level as shown in the following chemical structures X and Y.

After due corrections have been incorporated with regard to recovery, it is possible to quantify the amount of standard sulindac or unknown ; besides standard metabolite or unknown.


Advantages : The various advantages are as follows :


(i) The methodology is not only very specific but also fairly sensitive, and


(ii) It serves as a substitute to simple RIA-procedure when specific antisera are not readily accessible.


Disadvantages : It has two main disadvantages :


(i) The method is time-consuming, and


(ii) It involves tiresome and meticulous process of isotope dilution.




The stereospecificity of antigen-antibody reactions has gained its due recognition more than half-a-century ago*. However, an intensive and extensive stereospecificity radioimmunoassay procedures have been adequately applied to a number of pharmaceutical substances since mid-seventies, for instance : atropine, propranolol, methadone-to name a few.


Propranolol** which represents a comparatively better conceived example shall be discussed briefly as under with regard to its stereospecificity :


Propranolol is a recemic mixture i.e., it contains an equimolecular portion of d- and l- isomers as given below :

Interestingly, only the l-propranolol exhibit β-blocking activity. In actual practice, two antisersa have been developed experimentally, namely :


(a) Antisera against the dl-racemic mixture, and


(b) Antisera against the l-isomer (active form only).


The dl-propranolol antiserum exhibits an almost equal affinity for both d- and l-isomers ; whilst the l-propranolol shows exclusively a marked and pronounced affinity for the l-isomer, By the application of these two RIA-techniques, it was practically feasible to quantify the plasma and heart concentrations of dl- and l-propranolol individually. Thus, the concentrations of d-propranolol could be arrived at by subtracting the con-centration of l-isomer from the dl-mixture. It has been clearly demonstrated by Kawashima and coworkwers* that the d-propranolol undergoes distribution in vivo very sluggishly besides being metabolized more rapidly whereas the l-isomer gets distributed rather quickly to various tissues including the heart.


The RIA-procedure for propranolol is solely based on antisera derived from conjugates through the asymmetric carbon (i.e., the optical carbon) as shown in the above chemical structures. Perhaps it could be possible that the stereospecificity of propranolol is caused due to the conformation of the drug-hapten in rela-tion to the carrier protein to a great extent, through this hypothesis remains to be ascertained scientifically.


Consequently, the stabilization of the optical carbon by virtue of the conjugation to respective protein might improve upon the status of the specificity to a considerable extent. In order to prove the validity of this phenomenon one may carry out a definitive methodology whereby a closely monitored and controlled study of the antisera obtained by conjugates specifically prepared at the ‘asymmetric-carbon’ and at ‘another-site’ are both compared simultaneously under identical experimental parameters.


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