A: Transfer an amount of Otic Solution,equivalent to about 3mg of gentamicin,to a centrifuge tube.Dissolve an accurately weighed quantity of USP Gentamicin Sulfate RSquantitatively in water to obtain a solution having a concentration of about 5mg per mL.Transfer 1.0mLof this solution to a centrifuge tube.To each centrifuge tube add 3mLof water and 4g of potassium carbonate,and mix.Add 1mLof isopropyl alcohol to each tube,mix,and centrifuge.Use the upper phases as the test solution and Standard solution,respectively.Separately apply 20µLof each of these solutions to a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in a solvent system consisting of the lower phase of a mixture of methanol,dichloromethane,and ammonium hydroxide (1:1:1)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 plate to air-dry.Locate the spots on the plate by placing it in a tank containing about 15g of iodine crystals for 15minutes:the RFvalues of the three principal spots obtained from the test solution correspond to those obtained from the Standard solution.

B: The retention time of the major peak obtained in the chromatogram of the Assay preparationcorresponds to that of the Standard preparation,both relative to the internal standard,as obtained in the Assay for betamethasone.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (3:2).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Diluent— Prepare a mixture of methanol and glacial acetic acid (1000:1).

Internal standard solution— Dissolve a quantity of USP Beclomethasone Dipropionate RSin methanol to obtain a solution containing about 0.8mg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Betamethasone Valerate RSin Diluent,and dilute quantitatively,and stepwise if necessary,with Diluentto obtain a solution having a known concentration of about 1.2mg per mL.Transfer 2.0mLof this solution to a 10-mLvolumetric flask,add 5.0mLof Internal standard solution,dilute with Diluentto volume,and mix to obtain a solution having a known concentration of about 0.24mg of USP Betamethasone Valerate RSper mL.

Assay preparation— Add 5.0mLof Internal standard solutionto a 10-mLvolumetric flask.Transfer to the flask an accurately measured volume of Otic Solution,equivalent to about 2mg of betamethasone,dilute with methanol to volume,and mix.Centrifuge a portion of this solution,and use the clear supernatant 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 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the relative retention times are about 1.6for beclomethasone dipropionate and 1.0for betamethasone valerate;the resolution,R,between the betamethasone valerate and beclomethasone dipropionate peaks is not less than 3.5;and the relative standard deviation for replicate injections is not more 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 betamethasone (C22H29FO5)in each mLof the Otic Solution taken by the formula: in which 392.47and 476.58are the molecular weights of betamethasone and betamethasone valerate,respectively;Cis the concentration,in mg per mL,of USP Betamethasone Valerate RSin the Standard preparation;Vis the volume,in mL,of Otic Solution taken to prepare the Assay preparation;,and RUand RSare the ratios of the betamethasone valerate peak response to the internal standard peak response obtained from the Assay preparationand the Standard preparation,respectively.