Standard preparation—
Weigh accurately about 32.5 mg of
USP Hydrocortisone Hemisuccinate RS, and transfer it to a 50-mL volumetric flask. Add by pipet 5.0 mL of
Internal standard solution and 5.0 mL of a solution of glacial acetic acid in chloroform (3 in 100) containing in each mL an accurately known quantity of about 0.30 mg of
USP Hydrocortisone RS. Dilute with glacial acetic acid in chloroform (3 in 100) to volume, and mix.
Procedure—
Proceed as directed for
Procedure in the
Assay under
Hydrocortisone Hemisuccinate. The order of elution of peaks is that from the internal standard, hydrocortisone hemisuccinate, and successive smaller peaks representing free hydrocortisone and hydrocortisone 17-hemisuccinate, whose relative retention times are about 1.0, 1.5, 2.0, and 2.5, respectively. Measure the areas of the peaks from the internal standard, hydrocortisone hemisuccinate, and hydrocortisone 17-hemisuccinate. Calculate the ratio of the summation of the areas of the hydrocortisone hemisuccinate and hydrocortisone 17-hemisuccinate peaks to that of the internal standard in the chromatogram obtained from the
Standard preparation,
RS, and the same ratio in the chromatogram obtained from the
Assay preparation,
RU. Calculate the quantity, in mg, of hydrocortisone (C
21H
30O
5) in the portion of constituted solution taken by the formula:
0.784(100C)(RU / RS),
in which 0.784 is the ratio of the molecular weight of hydrocortisone to that of hydrocortisone hemisuccinate;
C is the concentration, in mg per mL, of
USP Hydrocortisone Hemisuccinate RS in the
Standard preparation; and
RU and
RS are the ratios as defined above, and to this quantity add the amount, in mg, of free hydrocortisone found in the test for
Free hydrocortisone.
11
—
USP Endotoxin RS. USP Fluorometholone RS. USP Hydrocortisone RS. USP Hydrocortisone Hemisuccinate RS.
for 3 hours: it loses not more than 2.0% of its weight.