Identification—
Transfer a volume of Ophthalmic Suspension,equivalent to about 7.5mg of Prednisolone Acetate,to a test tube,add 5mLof chloroform,and shake.Centrifuge,and apply 20µLof the chloroform layer and 20µLof a Standard solution of
USP Prednisolone Acetate RSin chloroform containing 1.5mg per mLon a thin-layer chromatographic plate (see
Chromatography á621ñ)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 UVlight:the
RFvalue 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 Suitabilityunder
Chromatography á621ñ).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Prednisolone Acetate RSin a mixture of acetonitrile and water (1:1)to obtain a solution having a known concentration of about 0.1mg per mL.
System suitability preparation—
Prepare a solution of prednisolone in a mixture of acetonitrile and methanol (1:1)having a concentration of about 0.1mg 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 5mg of prednisolone acetate,to a 50-mLvolumetric flask.Dilute with a mixture of acetonitrile and water (1:1)to volume,and mix.
Chromatographic system
(see
Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph the
Standard preparationand the
System suitability preparation,and record the peak responses as directed for
Procedure:the relative retention times are 0.5for prednisolone and 1.0for prednisolone acetate,the column efficiency is not less than 7000theoretical 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 10µL)of the
Standard preparationand the
Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C
23H
30O
6in each mLof the Ophthalmic Suspension taken by the formula:
50(C/V)(rU/rS),
in which
Cis the concentration,in mg per mL,of
USP Prednisolone Acetate RSin the
Standard preparation,
Vis the volume,in mL,of Ophthalmic Suspension taken,and
rUand
rSare the peak responses obtained from the
Assay preparationand the
Standard preparation,respectively.