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

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation as obtained in the Assay for chloramphenicol.

C: Transfer a quantity of Ophthalmic Ointment, equivalent to about 1.5 mg of prednisolone, to a screw-capped test tube, add 10 mL of methylene chloride, and shake to disperse. Heat at 60 for 15 minutes, and allow to cool while shaking for about 30 minutes. Allow to separate, draw off the upper ointment layer, and retain the lower methylene chloride layer. Apply, in portions, 0.4 mL each of the methylene chloride test solution and a Standard solution of USP Prednisolone RS in chloroform containing 0.5 mg per mL to a suitable thin-layer chromatographic plate (see Chromatography 621), 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 UV light: the RF value of the principal spot obtained from the Test solution corresponds 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 RS in Methanol-water solution to obtain a solution having a known concentration of about 0.3 mg per mL.

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

Chromatographic system (see Chromatography 621)—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 1 mL per 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 20 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 C11H12Cl2N2O5 in the portion of Ophthalmic Ointment taken by the formula: in which C is the concentration, in mg per mL, of USP Chloramphenicol RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.

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

Mobile phase— Dissolve 0.68 g of sodium acetate trihydrate in 400 mL of water in a 1000-mL graduated cylinder, adjust with glacial acetic acid to a pH of 4.0, and dilute with water to 500 mL. Dilute with methanol to 1000 mL, and mix. Filter this solution through a membrane filter (1 µm or finer porosity), and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

Chromatographic system (see Chromatography 621)—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 1.5 mL per 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 15 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 C21H28O5 in the portion of Ophthalmic Ointment taken by the formula: in which C is the concentration, in mg per mL, of USP Prednisolone RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.