A: Transfer a quantity of Ophthalmic Ointment,equivalent to about 20mg of chloramphenicol,to a screw-capped test tube,add 5mLof 5Nsodium hydroxide and 2mLof pyridine,and shake.Place the tube in a water bath at 50for 20minutes:a reddish brown color develops in the pyridine layer.

B: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparationas obtained in the Assay for chloramphenicol.

C: Transfer a quantity of Ophthalmic Ointment,equivalent to about 1.5mg of prednisolone,to a screw-capped test tube,add 10mLof methylene chloride,and shake to disperse.Heat at 60for 15minutes,and allow to cool while shaking for about 30minutes.Allow to separate,draw off the upper ointment layer,and retain the lower methylene chloride layer.Apply,in portions,0.4mLeach of the methylene chloride test solution and a Standard solution of USP Prednisolone RSin chloroform containing 0.5mg per mLto a suitable thin-layer chromatographic plate (see Chromatography á621ñ),coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow each portion to dry before adding the next portion to the same spot.Develop the chromatogram in a chromatographic chamber lined with paper and equilibrated with a solvent system consisting of a mixture of chloroform and acetone (4:1)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Locate the spots on the plate by examination under short-wavelength UVlight:the RFvalue of the principal spot obtained from the Test solutioncorresponds to that obtained from the Standard solution.

Methanol-water solution and Mobile phase—Proceed as directed in the Assay for prednisolone.

Standard preparation— Dissolve an accurately weighed quantity of USP Chloramphenicol RSin Methanol-water solutionto obtain a solution having a known concentration of about 0.3mg per mL.

Assay preparation— Transfer an accurately weighed portion of Ophthalmic Ointment,equivalent to about 3.0mg of chloramphenicol,to a screw-capped test tube.Add 10mLof n-heptane,and shake by mechanical means until the substance is dissolved.Add 10.0mLof Methanol-water solution,and shake by mechanical means for 30seconds.Allow the layers to separate,and carefully remove the upper phase.Centrifuge the lower phase for 15minutes,and use the clear portion as the Assay preparation.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the peak for chloramphenicol obtained from the Ophthalmic Ointment,at a retention time corresponding to that of the peak obtained from the Reference Standard,exhibits baseline separation from the adjacent prednisolone peak,and the relative standard deviation of replicate injections is not more than 1.5%.

Procedure— Separately inject equal volumes (about 20µL)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 C11H12Cl2N2O5in the portion of Ophthalmic Ointment taken by the formula: in which Cis the concentration,in mg per mL,of USP Chloramphenicol RSin the Standard preparation,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.

Methanol-water solution— Mix 4volumes of methanol with 1volume of water,and mix.

Mobile phase— Dissolve 0.68g of sodium acetate trihydrate in 400mLof water in a 1000-mLgraduated cylinder,adjust with glacial acetic acid to a pHof 4.0,and dilute with water to 500mL.Dilute with methanol to 1000mL,and mix.Filter this solution through a membrane filter (1µm or finer porosity),and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Prednisolone RSin Methanol-water solutionto obtain a solution having a known concentration of about 0.2mg per mL.

Assay preparation— Transfer an accurately weighed portion of Ophthalmic Ointment,equivalent to about 2.0mg of Prednisolone,to a screw-capped test tube.Add 10mLof n-heptane,and shake by mechanical means until the substance is dissolved.Add 10.0mLof Methanol-water solution,and shake by mechanical means for 30seconds.Allow the layers to separate,and carefully remove the upper phase.Centrifuge the lower phase for 15minutes,and use the clear portion as the Assay preparation.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the peak for prednisolone obtained from the Ophthalmic Ointment,at a retention time corresponding to that of the peak obtained from the Reference Standard,exhibits baseline separation from the adjacent chloramphenicol peak,and the relative standard deviation of replicate injections is not more than 1.5%.

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.Calculate the quantity,in mg,of C21H28O5in the portion of Ophthalmic Ointment taken by the formula: in which Cis the concentration,in mg per mL,of USP Prednisolone RSin the Standard preparation,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.