A: Dilute 2mLwith 2mLof methanol.Apply 10µLof this solution and 10µLof a methanol and water solution (1:1)of USP Betamethasone Sodium Phosphate RScontaining 2mg per mLto a suitable thin-layer chromatographic plate (see Chromatography á621ñ),coated with a 0.25-mm layer of chromatographic silica gel.Proceed as directed for Identificationtest Bunder Betamethasone Sodium Phosphate,beginning with “Allow the spots to dry.”

B: Apply 10µLof the solution prepared for Identificationtest Aand 10µLof a methanol and water solution (1:1)of USP Betamethasone Acetate RScontaining 1.5mg per mLto a suitable thin-layer chromatographic plate (see Chromatography á621ñ),coated with a 0.25-mm layer of chromatographic silica gel.Proceed as directed for Identificationtest Bunder Betamethasone,beginning with “Allow the spots to dry.”

Mobile phase— Prepare a filtered and degassed mixture of methanol and 0.075Mmonobasic potassium phosphate (7:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Transfer about 50mg of methyltestosterone to a 50-mLvolumetric flask,add methanol to volume,and mix.

Standard preparation— Transfer about 63mg of USP Betamethasone Sodium Phosphate RS,accurately weighed,to a 25-mLvolumetric flask,add Mobile phaseto volume,and mix (Solution 1).Transfer about 45mg of USP Betamethasone Acetate RS,accurately weighed,to a 25-mLvolumetric flask,add methanol to volume,and mix (Solution 2).Pipet 5mLeach of Solution 1and Solution 2into a 100-mLvolumetric flask.Add 10.0mLof Internal standard solution,dilute with Mobile phaseto volume,and mix to obtain a Standard preparationhaving known concentrations of about 126µg of USP Betamethasone Sodium Phosphate RSper mLand 90µg of USP Betamethasone Acetate RSper mL.

Assay preparation— Using a “To contain”pipet transfer an accurately measured volume of the well-mixed Injectable Suspension,equivalent to about 9mg of betamethasone acetate,to a 100-mLvolumetric flask.Rinse the pipet with about 10mLof Mobile phase,collecting the rinse in the volumetric flask.Add 10.0mLof Internal standard solution,dilute with Mobile phaseto volume,and mix.

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 1.2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between the betamethasone phosphate and betamethasone acetate peaks is not less than 5.0,and the resolution,R,between the betamethasone acetate and internal standard peaks is not less than 3.0,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.The relative retention times are about 0.5for betamethasone phosphate,1.7for methyltestosterone,and 1.0for betamethasone acetate.Calculate the quantity,in mg,of betamethasone acetate (C24H31FO6)in each mLof the Injectable Suspension taken by the formula: in which Cis the concentration,in µg per mL,of USP Betamethasone Acetate RSin the Standard preparation;Vis the volume,in mL,of Injectable Suspension taken;and RUand RSare the peak response ratios obtained for betamethasone acetate and methyltestosterone from the Assay preparationand the Standard preparation,respectively.Calculate the quantity,in mg,of betamethasone (C22H29FO5)equivalent to the quantity of betamethasone sodium phosphate (C22H28FNa2O8P),in each mLof the Injectable Suspension taken by the formula: in which 392.46and 516.41are the molecular weights of betamethasone and betamethasone sodium phosphate,respectively;Cis the concentration,in µg per mL,of USP Betamethasone Sodium Phosphate RSin the Standard preparation;Vis the volume,in mL,of Injectable Suspension taken;and RUand RSare the peak response ratios obtained for betamethasone phosphate and methyltestosterone from the Assay preparationand the Standard preparation,respectively.