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 :
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.
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.
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.
The following pharmaceutical substances may also be assayed by the above method, namely :
(c) Prednisolone, and