A: Infrared Absorption 197K.

B: Ultraviolet Absorption 197U—

Test solution: 6.7 mg per mL, in dioxane.

Standard preparation— Dissolve an accurately weighed quantity of USP Prednisolone Hemisuccinate RS in a mixture of equal volumes of alcohol and chloroform to obtain a solution having a known concentration of about 8 mg per mL.

Assay preparation— Weigh accurately about 80 mg of Prednisolone Hemisuccinate, previously dried, dissolve in a mixture of equal volumes of alcohol and chloroform to make 10.0 mL, and mix.

Procedure— Use a 20- × 20-cm chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture, which has been activated by heating at 105 for 1 hour. Using a solvent system consisting of a mixture of 5 parts of butyl alcohol, 4 parts of acetic acid, and 1 part of water, proceed as directed for Procedure under Single-steroid Assay 511, ending with “Centrifuge the tubes for 5 minutes,” except to apply 50 µL in place of 200 µL of the solutions to the plate. Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 243 nm, with a suitable spectrophotometer, against the blank. Calculate the quantity, in mg, of C25H32O8 in the portion of Prednisolone Hemisuccinate taken by the formula: in which C is the concentration, in mg per mL, of USP Prednisolone Hemisuccinate RS in the Standard preparation, and AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively.