Identification—
The chromatogram of the
Assay preparation obtained as directed in the
Assay for hydrocortisone acetate exhibits a major peak for hydrocortisone acetate, the retention time of which corresponds with that exhibited in the chromatogram of the
Standard preparation obtained as directed in the
Assay for hydrocortisone acetate.
Assay for neomycin—
Proceed as directed for neomycin under
Antibiotics—Microbial Assays 
81
, using an accurately measured volume of Ophthalmic Suspension, freshly mixed and free from air bubbles, diluted quantitatively and stepwise with
Buffer No. 3 to yield a
Test Dilution having 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 0.8 mg per mL.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Suspension, freshly mixed and free from air bubbles, equivalent to about 10 mg of hydrocortisone acetate, to a suitable container. Add 10.0 mL of
Internal standard solution and about 40 mL of chloroform, shake vigorously for about 5 minutes, and allow the phases to separate. Use the clear chloroform layer as the
Assay preparation.
Chromatographic system
(see
Chromatography 621)—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 1 mL per 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 15 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.7 and 1.0 for hydrocortisone acetate and fluoxymesterone, respectively. Calculate the quantity, in mg, of hydrocortisone acetate (C
23H
32O
6) in each mL of the Ophthalmic Suspension taken by the formula:
(W / V)(RU / RS),
in which
W is the quantity, in mg, of
USP Hydrocortisone Acetate RS taken to prepare the
Standard preparation,
V is the volume, in mL, of Ophthalmic Suspension taken, and
RU and
RS are the peak response ratios of the hydrocortisone acetate peak to the internal standard peak obtained from the
Assay preparation and the
Standard preparation, respectively.
