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Test solution— Evaporate 25mLof the Assay preparation,prepared as directed in the Assay,on a steam bath just to dryness,and dissolve the residue in 0.5mLof alcohol.

Developing solvent system: a mixture of chloroform and diethylamine (2:1).

Procedure— Proceed as directed in the chapter.Locate the spots by lightly spraying with dilute sulfuric acid (1in 2)and heating on a hot plate or under a lamp until spots appear.USP28

Developing solvent— Prepare a mixture of chloroform,methanol,and ammonium hydroxide (175:20:1).

Tetramethylammonium hydroxide reagent— Dilute 20mLof tetramethylammonium hydroxide TSwith alcohol to make 100mL.

Standard preparation— Dissolve a suitable quantity of USP Betamethasone RS,accurately weighed,in a mixture of chloroform and methanol (1:1)to obtain a solution having a known concentration of about 0.6mg per mL.

Assay preparation— Use a pipet calibrated to contain a suitable volume,and transfer to a 50-mLcentrifuge tube an accurately measured volume of Oral Solution,equivalent to about 1.2mg of Betamethasone.Rinse the pipet with 15mLof 0.1Nhydrochloric acid,then with 20mLof ethyl acetate,and add the rinsings to the centrifuge tube.Rotate for about 10minutes,or shake manually for about 1minute.[NOTE—Do not use a mechanical shaker.]Centrifuge to separate the phases.Transfer the upper phase (ethyl acetate)to a small,pear-shaped flask.Extract the aqueous phase twice more with 20-mLportions of ethyl acetate,and add the extracts to the pear-shaped flask.Evaporate the combined extracts on a steam bath under a gentle stream of nitrogen to dryness.Allow to cool to room temperature.Dissolve the residue in about 0.5mLof chloroform and methanol (1:1),using a vortex mixer.Transfer the solution to a 2-mLvolumetric flask with small portions of a mixture of chloroform and methanol (1:1),dilute with the mixture of chloroform and methanol (1:1)to volume,and mix.

Procedure— Apply 200-µLportions of the Assay preparationand the Standard preparationto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Allow the spots to dry,and develop the chromatogram,using the Developing solvent,until the solvent front has moved about 15cm.Remove the plates from the developing chamber,and allow them to dry for about 15minutes.Mark the betamethasone bands,using short-wavelength UVlight,to include similar zones of silica gel for the Assay preparation,the Standard preparation,and a zone containing no betamethasone for the blank.Scrape off these zones,and transfer them to separate 50-mLcentrifuge tubes.Add 15.0mLof alcohol to each,and rotate for 20minutes.Centrifuge to clarify.Transfer 10.0-mLportions of the supernatant to separate,stoppered tubes.To each tube add 1.0mLof blue tetrazolium TS,followed by 1.0mLof Tetramethylammonium hydroxide reagent,and mix.Heat in a 35water bath for about 1hour.Remove from the water bath,add 1.0mLof glacial acetic acid to each tube,and mix.Cool to room temperature.Concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 525nm,with a suitable spectrophotometer.Calculate the quantity,in mg,of betamethasone (C22H29FO5)in each mLof the Oral Solution taken by the formula: in which Cis the concentration,in mg per mL,of USP Betamethasone RSin the Standard preparation;Vis the volume,in mL,of Oral Solution taken;and AU,AS,and ABare the absorbances of the solutions from the Assay preparation,the Standard preparation,and the blank,respectively.