Tetrazolium assay of steroids

What is the underlying principle of tetrazolium assay of steroids Explain with necessary equations involved in it. 

A number of steroidal pharmaceutical substances listed in the official compendia may be assayed by the tetrazolium method of analysis. A few typical examples are described below ... 

In the presence of the C-20-ketogroup at the side chain, tetrazolium salts are reduced to the corresponding strongly colored formazans. Mader and Buck were the first to apply tri-phenyl tetrazolium chloride to assay pharmaceutical steroids [83], but it was later found that air affects the procedure leading to poor reproducibility [84,85]. More stable reagents such as 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium bromide [86] and 2,5-diphenyl-3-(p-strylphenyl)tetrazolium chloride [87] were employed successfully. Good results were also obtained with blue tetrazolium, or 3,3 -(3,3 -dimethoxy-4,4 -biphenylene) bis 2,5-diphenyl-... 

To enhance the applicability of UV spectrophotometry in pharmaceutical steroid analysis by increasing its selectivity and sensitivity, various methods have been developed based on chemical reactions leading to colored derivatives. Although their importance has decreased considerably, some of these methods are still in use in pharmacopoeias, mainly in the assay of formulations. For example, the A" -3-oxo and A " -3-oxo steroids can be determined as iso-nicotinoyl hydrazones (/Uax 380 and 410 nm 11500 and 17000, respectively). The dihydroxy-acetone side chain of corticosteroids at C17 reduces tetrazolium reagents to colored formazans (Amax 485 and 525 nm with triphenyl tetrazolium chloride and Tetrazolium Blue reagents, respectively e 16000 and 24000), thus creating the basis for a stability-indicating indirect colorimetric assay. Corticosteroids... 

Fig. 4. Part of a 20 X 20 cm thin-layer chromatography plate for the assay in urine of B, THS C, allo-THE E, THE F, allo-THP G, THF. Standard compounds have been run in positions 2, 4, 6, and 8. Cortisol is shown in position D and is measurable only if present in abnormally increased amount. The compounds shown in position A are mainly Cm steroids with an a-ketol group. Duplicate extracts from normal adult urines have been run in positions 1 and 3 from a case of adrenal carcinoma in position S from a normal infant in position 7 and from an infant with adrenogenital syndrome in position 9. The plate was sprayed with blue tetrazolium reagent, then heated. From Shackleton et al. (SIO).

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