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 :
(a)
Methylprednisolone,
(b)
Hydrocortisone,
(c)
Prednisolone, and
(a)
Prednisone.
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