A: Transfer an amount of Ointment,equivalent to about 15mg of gentamicin,to a centrifuge tube,and add 10mLof a mixture of methanol and 0.1Nhydrochloric acid (4:1)and 25mLof solvent hexane.Rotate for 30minutes,and centrifuge.Discard the upper phase.Apply 25µLof the lower phase and 25µLof a Standard solution containing 3mg per mLof USP Gentamicin Sulfate RSin a mixture of methanol and 0.1Nhydrochloric acid (4:1)to a suitable 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 chloroform,methanol,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 spots 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 methanol and water (475:300).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Diluent— Transfer 25mLof water to a 500-mLvolumetric flask.Add 2.5mLof glacial acetic acid,dilute with methanol to volume,and mix.

Internal standard solution— Dissolve a quantity of USP Beclomethasone Dipropionate RSin Diluentto obtain a solution containing about 0.4mg 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 0.45mg per mL.Transfer 5.0mLof this solution to a stoppered vial,add 10.0mLof Internal standard solution,and mix to obtain a solution having a known concentration of about 0.15mg of USP Betamethasone Valerate RSper mL.

Assay preparation— Transfer an accurately weighed portion of Ointment,equivalent to about 2mg of betamethasone,to a 50-mLcentrifuge tube.Add 10.0mLof Internal standard solutionand 5.0mLof Diluent,and shake vigorously for 10minutes.Place the tube in an ice–methanol bath for 15minutes,then centrifuge to separate the phases.Transfer the clear supernatant to a stoppered flask,and allow to warm to room temperature (Assay preparation).

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×15-cm column that contains packing L1.The flow rate is about 2.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 1.5for 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 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 betamethasone (C22H29FO5)in the portion of Ointment 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;,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.