Identification—
Evaporate in a test tube to dryness about 5 mL of the dilute solution obtained in the
Assay. Add 2 to 3 mL of warm perchloric acid, mix, and warm on a steam bath for about 2 minutes: a blood-red color develops. Add 5 mL of water: the color is not discharged.
Assay—
Sulfuric acid reagent—
Prepare a solution of sulfuric acid, dehydrated alcohol, and water solution (4:3:3).
Modified phenylhydrazine-sulfuric acid TS—
Dissolve 65 mg of phenylhydrazine hydrochloride in 100 mL of Sulfuric acid reagent.
Standard preparation—
Dissolve in dehydrated alcohol a suitable quantity, accurately weighed, of
USP Prednisolone RS, and dilute quantitatively and stepwise with dehydrated alcohol to obtain a solution having a known concentration of about 0.1 mg per mL.
Assay preparation—
To an accurately weighed portion of Cream, equivalent to about 20 mg of prednisolone, add 25 mL of dehydrated alcohol. Warm on a steam bath to disperse the assay specimen, then chill to congeal it. Filter through paper, previously washed with dehydrated alcohol, into a 200-mL volumetric flask. Repeat twice more, starting with “add 25 mL of dehydrated alcohol.” Dilute with dehydrated alcohol to volume. (Retain about 5 mL of this dilution for the
Identification test.)
Procedure—
Pipet 2 mL of
Assay preparation into each of two 50-mL conical flasks (identify as
Assay preparation and
Assay preparation blank). Pipet 2 mL of
Standard preparation into each of two 50-mL conical flasks (identify as
Standard preparation and
Standard preparation blank). Pipet 2 mL of dehydrated alcohol into a 50-mL conical flask (identify as
Reagent blank). Add 20.0 mL of
Sulfuric acid reagent to the
Assay preparation blank and to the
Standard preparation blank. Add 20.0 mL of
Modified phenylhydrazine-sulfuric acid to the
Assay preparation, to the
Standard preparation, and to the
Reagent blank, respectively. Maintain the flasks in a water bath at 60
for about 45 minutes, then chill in a water-ice bath. Filter each through a fine-porosity, sintered-glass funnel, and identify the filtrate flasks correspondingly. Concomitantly determine the absorbances of the solutions at the wavelength of maximum absorbance at about 410 nm, with a suitable spectrophotometer, against dehydrated alcohol. Calculate the quantity, in mg, of C
21H
28O
5 in the portion of Cream taken by the formula:
200
C[(
AU 
AUB AR) / (
AS ASB AR)],
in which
C is the concentration, in mg per mL, of
USP Prednisolone RS in the
Standard preparation,
AU and
AS are the absorbances of the solutions from the
Assay preparation and the
Standard preparation, respectively,
AUB and
ASB are the absorbances of the solutions from the
Assay preparation blank and the
Standard preparation blank, respectively, and
AR is the absorbance of the
Reagent blank.
755
:
meets the requirements.