Medical brain notes 14

  INTRODUCTION A number of steroids essentially having a  α -ketol (21-hydroxy-20 keto) side-chain group, for instance : hydrocortisone, hvdrocortisone acetate, prednisolone, methylprednisolone, methylprednisolone acetate, flucocinolone acetonide, triamcionolone acetonide and the like—are quantitatively reduced by tetrazolium salts to their respective coloured formazan derivatives. Thus, it is possible to carry out the assay of a number of formulations that contain corticosteroids by using triphenyltetrazolium chloride. The said reaction is usually performed in an alkaline medium (tetramethylammonium hydroxide) between a temperature ranging between 30° to 35°C for a duration of 1 to 2 hours. The absorbance of the resulting  formazan derivative  producing a  red product  is usually measured around 484 nm.

  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, —CH 2 OH 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 a...

  ASSAY OF PHARMACEUTICAL SUBSTANCES 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 :     1. HYDROCORTISONE ACETATE   Theory :  Hydrocortisone acetate is first hydrolysed by strong trimethylammonium hydroxide solu-tion to yield the free 21-hydroxysteroid  i.e.,  hydrocortisone as shown below : The resulting hydrolysed product is then treated with triphenyltetrazolium chloride and the coloured triphenylformazan is measured at 525 nm.   Materials Required :  Hydrocortisone acetate : 0.350 g ; aldehyde-free absolute ethanol : 100 ml ;   triphenyltetrazolium chloride solution [a 0.5% w/v solution of 2,3,5,-triphenyltetrazolium chloride in aldehyde-free ethanol (96%)] : 10 ml ; dilute tetramethylammonium hydroxide solution [Dilute 10 ml of tetramethylammonium hydroxide solution (10%) to 100 ml with ald...