Identification—
B:
Shake a quantity of Ophthalmic Ointment, equivalent to about 40 mg of prednisolone phosphate, with 25 mL of sodium chloride solution (1 in 20) and 25 mL of methylene chloride, for 2 minutes. Transfer the methylene chloride layer to a second separator containing 15 mL of sodium chloride (1 in 20). Shake for 1 minute, and discard the methylene chloride layer. Repeat the operation with a second portion of 25 mL of methylene chloride. Combine the aqueous phase from the second separator with the aqueous phase of the first separator. Add 10 mL of
Alkaline phosphatase solution, prepared as directed in the
Assay for prednisolone phosphate, and add 50 mL of methylene chloride. Insert the stopper, and allow to stand, with occasional gentle inversion (about once every 15 minutes), for 2 hours. Filter the methylene chloride layer through a dry paper, and evaporate 25 mL of the filtrate to dryness: the residue so obtained responds to
Identification test
A under
Prednisolone.
Assay for neomycin—
Proceed as directed under
Antibiotics—Microbial Assays 
81
, using an accurately weighed portion of Ophthalmic Ointment shaken in a separator with about 50 mL of ether, and extracted with four 20-mL portions of
Buffer No. 3. Combine the aqueous extracts, and dilute with
Buffer No. 3 to an appropriate volume to obtain a stock solution. Dilute this stock solution quantitatively and stepwise with
Buffer No. 3 to obtain a
Test Dilution having a concentration assumed to be equal to the median dose level of the Standard.
Assay for prednisolone phosphate—
Assay preparation—
Transfer an accurately weighed portion of Ophthalmic Ointment, equivalent to about 3 mg of prednisolone phosphate, to a 125-mL separator. Add 25 mL of sodium chloride solution (1 in 20) and 25 mL of methylene chloride, and shake for not less than 2 minutes to disperse the assay specimen. Transfer the methylene chloride layer to a second separator containing 15 mL of sodium chloride solution (1 in 20). Shake for 1 minute, and discard the methylene chloride layer. Repeat the operation with a second portion of 25 mL of methylene chloride. Transfer the aqueous phases from both separators to a 50-mL volumetric flask, rinsing the first separator with the aqueous phase of the second separator. Rinse both separators with the same 5 mL of sodium chloride solution (1 in 20), and add the rinsing to the volumetric flask. Add sodium chloride solution (1 in 20) to volume, and mix.
Pipet 5 mL of the resulting solution into a 125-mL separator, add 8.0 mL of Alkaline phosphatase solution, mix, and allow to stand for 2 hours. Extract the solution with two 25-mL portions of methylene chloride, filtering the extracts through methylene chloride-washed cotton into a small beaker. Evaporate the methylene chloride on a steam bath nearly to dryness, then evaporate with the aid of a current of air to dryness. Dissolve the residue in 25.0 mL of alcohol.
Prepare a blank by evaporating 50 mL of methylene chloride to dryness and dissolving the residue in 25 mL of alcohol.
Procedure—
Pipet 20 mL each of the
Assay preparation, the
Standard preparation, and the blank solution into separate glass-stoppered flasks, and proceed as directed for
Procedure under
Assay for Steroids 351, beginning with “add 2.0 mL of a solution prepared by dissolving 50 mg of blue tetrazolium.” Calculate the quantity, in mg, of prednisolone phosphate (C
21H
29O
8P) in the portion of Ophthalmic Ointment taken by the formula:
0.25C(AU / AS)(440.43 / 360.45),
in which
C is the concentration, in µg 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, and 440.43 and 360.45 are the molecular weights of prednisolone phosphate and prednisolone, respectively.
