Tetrazolium Assay of Steroids: Theory

 THEORY

The oxidation of the α-ketol moiety present in the steroid under examination and the subsequent reduction of triphenyltetrazolium chloride to the corresponding triphenylformazan are depicted in the following reaction :

The triphenyltetrazolium chloride ring undergoes cleavage, as shown by the dotted line, and 2H-atoms are given out by the steroid prednisolone in being converted from C-21, —CH2OH to C-21, —CHO function ; one of the H-atoms from above is utilized in the formation of the open-chain compound i.e., triphenylformazan derivative ; whereas, the second H-atom abstracts the Cl– ion as a mole of HCl. The above interaction is of a quantitative nature.

 

However, it is pertinent to mention here that certain steroids esterified at C-21 position, such as : hydrocortisone acetate, methylprednisolone acetate are duly hydrolyzed in the alkaline medium to give rise to the corresponding free C-21 hydroxy steroids and hence, may also be assayed by adopting the same procedure.

 

Precautions : 

All these assays are to be carried out strictly in the absence of light and atmospheric oxygen to get optimum results.