Identification—
The chromatogram of the
Assay preparationobtained as directed in the
Assay for hydrocortisone acetateexhibits a major peak for hydrocortisone acetate,the retention time of which corresponds with that exhibited in the chromatogram of the
Standard preparationobtained as directed in the
Assay for hydrocortisone acetate.
Assay for neomycin—
Proceed as directed for neomycin under
Antibiotics—Microbial Assays á81ñ,using an accurately measured volume of Ophthalmic Suspension,freshly mixed and free from air bubbles,diluted quantitatively and stepwise with
Buffer No.3to yield a
Test Dilutionhaving a concentration assumed to be equal to the median dose level of the Standard.
Assay for hydrocortisone acetate—
Mobile phase—
Prepare a solution containing n-butyl chloride,water-saturated n-butyl chloride,tetrahydrofuran,methanol,and glacial acetic acid (95:95:14:7:6).
Internal standard solution—
Prepare a solution of fluoxymesterone in chloroform containing 0.8mg per mL.
Standard preparation—
Dissolve about 10mg of
USP Hydrocortisone Acetate RS,accurately weighed,in 10.0mLof
Internal standard solution,dilute with about 40mLof chloroform,and mix.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Suspension,freshly mixed and free from air bubbles,equivalent to about 10mg of hydrocortisone acetate,to a suitable container.Add 10.0mLof Internal standard solutionand about 40mLof chloroform,shake vigorously for about 5minutes,and allow the phases to separate.Use the clear chloroform layer as the Assay preparation.
Chromatographic system
(see
Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains packing L3.The flow rate is about 1mLper minute.Chromatograph the
Standard preparation,and record the peak responses as directed for
Procedure:the resolution,
R,between the analyte and internal standard peaks is not less than 3.0,and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 15µL)of the
Standard preparationand the
Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.7and 1.0for hydrocortisone acetate and fluoxymesterone,respectively.Calculate the quantity,in mg,of hydrocortisone acetate (C
23H
32O
6)in each mLof the Ophthalmic Suspension taken by the formula:
(W/V)(RU/RS),
in which
Wis the quantity,in mg,of
USP Hydrocortisone Acetate RStaken to prepare the
Standard preparation,
Vis the volume,in mL,of Ophthalmic Suspension taken,and
RUand
RSare the peak response ratios of the hydrocortisone acetate peak to the internal standard peak obtained from the
Assay preparationand the
Standard preparation,respectively.