Assay of Pharmaceutical Substances - Tetrazolium Assay of Steroids

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 aldehyde-free ethanol (96%). It contains about 1% w/v of C₄H₁₃NO. To be prepared immediately before use] : 10 ml.

Procedure : 

The following steps are to be followed sequentially strictly protected from light :

·              Dissolve accurately weighed hydrocortisone acetate 300 to 350 mg in 10 ml aldehyde-free absolute ethanol,

·              Transfer 10 ml to a 25 ml graduated flask, add 2 ml of triphenyltetrazolium chloride solution, displace the air in the flask with oxygen-free nitrogen,

·              Immediately add 2 ml of dilute tetramethylammonium hydroxide solution and again displace the air with oxygen-free nitrogen,

·              Stopper the flask, mix the contents by gently swirling and allow to stand in a water-bath maintained at 30°C for 1 hour,

·              Cool rapidly, add sufficient aldehyde-free absolute ethanol to produce 25 ml,

·              Mix well and immediately determine the absorbance of the resulting solution in a stoppered cell at the maximum at 485 nm, using in the reference cell a solution prepared at the same time and in the same manner using 10 ml of aidehyde-free absolute ethanol, and

·              Repeat the operation using the hydrocortisone acetate EPCRS* in place of the substance being examined under the same experimental parameters.

2. COGNATE ASSAYS

The following pharmaceutical substances may also be assayed by the above method, namely :

(a) Methylprednisolone,

(b) Hydrocortisone,

(c) Prednisolone, and

(a)       Prednisone.