Test solution: 40mg per mL,in dioxane.

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.

Test solution— Dissolve 100.0mg in 5.0mLof methanol.

Standard solution 1— Dissolve a suitable quantity of USP Betamethasone Benzoate RSin methanol to obtain a solution having a known concentration of about 5mg per mL.

Standard solution 2— Dilute a portion of Standard solution 1,quantitatively and stepwise,with methanol to obtain a solution having a known concentration of about 100µg per mL.

Application volume: 10µL.

Developing solvent system: a mixture of toluene,acetone,and methanol (75:25:4).

Procedure— Proceed as directed for Thin-Layer Chromatographyunder Chromatography á621ñ.Examine the plate under short-wavelength UVlight:the principal spot from the Test solutioncorresponds in RFvalue to that of Standard solution 1;and the Test solutionshows not more than 3additional spots,the intensity and size of which do not exceed those of the spot from Standard solution 2.

Mobile phase— Prepare a suitable filtered solution of acetonitrile and water (60:40).

Internal standard solution— Prepare a solution of betamethasone dipropionate in methanol containing 0.6mg per mL.

Standard preparation— Using an accurately weighed quantity of USP Betamethasone Benzoate RS,prepare a solution in methanol having a known concentration of about 0.6mg per mL.Mix 5.0mLof this solution and 10.0mLof the Internal standard solutionto obtain a Standard preparationhaving a known concentration of about 0.2mg of betamethasone benzoate per mL.

Assay preparation— Transfer about 60mg of Betamethasone Benzoate,accurately weighed,to a 100-mLvolumetric flask.Dilute with methanol to volume,and mix.Mix 5.0mLof this solution and 10.0mLof the Internal standard solution.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains 5-µm packing L1.The flow rate is about 1mLper minute.Chromatograph three replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%;and the resolution factor between betamethasone benzoate and the internal standard is not less than 3.

Procedure— Separately inject equal volumes (about 15µL)of the Standard preparationand the Assay preparationinto the chromatograph by means of a suitable microsyringe or sampling valve,record the chromatograms,and measure the responses for the major peaks.The retention times are about 7and 5minutes for betamethasone dipropionate and betamethasone benzoate,respectively.Calculate the quantity,in mg,of C29H33FO6in the portion of Betamethasone Benzoate taken by the formula: in which Cis the concentration,in mg per mL,of USP Betamethasone Benzoate RSin the Standard preparation;and RUand RSare the peak response ratios of the betamethasone benzoate peak and the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.