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