Identification—
Transfer a volume of Ophthalmic Suspension, equivalent to about 7.5 mg of Prednisolone Acetate, to a test tube, add 5 mL of chloroform, and shake. Centrifuge, and apply 20 µL of the chloroform layer and 20 µL of a Standard solution of
USP Prednisolone Acetate RS in chloroform containing 1.5 mg per mL on a thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel mixture. Develop the chromatogram in a mixture of chloroform and acetone (4:1) until the solvent front has moved about three-fourths the length of the plate. Mark the solvent front, and locate the spots on the plate by examining under UV light: the
RF value of the principal spot obtained from the solution under test corresponds to that obtained from the Standard solution.
Assay—
Mobile phase—
Prepare a suitably filtered and degassed solution of water and acetonitrile (3:2). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Prednisolone Acetate RS in a mixture of acetonitrile and water (1:1) to obtain a solution having a known concentration of about 0.1 mg per mL.
System suitability preparation—
Prepare a solution of prednisolone in a mixture of acetonitrile and methanol (1:1) having a concentration of about 0.1 mg per mL. Mix equal volumes of this solution and the Standard preparation.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Suspension, equivalent to about 5 mg of prednisolone acetate, to a 50-mL volumetric flask. Dilute with a mixture of acetonitrile and water (1:1) to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the
Standard preparation and the
System suitability preparation, and record the peak responses as directed for
Procedure: the relative retention times are 0.5 for prednisolone and 1.0 for prednisolone acetate, the column efficiency is not less than 7000 theoretical plates, the tailing factor is not more than 2.0, and the resolution,
R, between prednisolone and prednisolone acetate is not less than 2.0.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C
23H
30O
6 in each mL of the Ophthalmic Suspension taken by the formula:
50(C / V)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Prednisolone Acetate RS in the
Standard preparation,
V is the volume, in mL, of Ophthalmic Suspension taken, and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
