A: Infrared Absorption á197Mñ.

B: Prepare a solution in methanol containing 1mg per mL.Apply 10µLof this solution and 10µLof a solution of USP Betamethasone Sodium Phosphate RSin methanol containing 1mg per mLto 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.Place 500mLof butyl alcohol and 200mLof dilute hydrochloric acid (1in 12)in a separatory funnel,and mix.Use the organic layer as the developing solvent.Develop the chromatogram in the developing solvent until the solvent front has moved about three-fourths of the length of the plate.Proceed as directed in the Identificationtest under Betamethasone Cream,beginning with “Remove the plate.”

C: Ignite it at 800(see Residue on Ignition á281ñ):the residue responds to the tests for Sodium á191ñand for Phosphate á191ñ.

Test solution: 10mg per mL,in water.

Standard Phosphate Solution and Phosphate reagent A—Prepare as directed under Phosphate in reagents(see General Tests for Reagentsunder Reagents,Indicators,and Solutions).

Phosphate reagent B— Dissolve 350mg of p-methylaminophenol sulfate in 50mLof water,add 20g of sodium metabisulfite,mix to dissolve,and dilute with water to 100mL.

Procedure— Dissolve about 50mg of Betamethasone Sodium Phosphate,accurately weighed,in a mixture of 10mLof water and 5mLof 2Nsulfuric acid contained in a 25-mLvolumetric flask,by warming if necessary.Add 1mLeach of Phosphate reagent Aand Phosphate reagent B,dilute with water to 25.0mL,mix,and allow to stand at room temperature for 30minutes.Similarly and concomitantly prepare a Standard solution,using 5.0mLof Standard Phosphate Solutioninstead of the 50mg of the substance under test.Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 730nm,with a suitable spectrophotometer,using water as the blank.The absorbance of the test solution is not more than that of the Standard solution.The limit is 1.0%of phosphate (PO4).

Test solution— Dissolve 25.0mg of Betamethasone Sodium Phosphate in water to make 25.0mL.Transfer 5.0mLof the solution to a separator,and extract with three 25-mLportions of chloroform.Filter each extract through a chloroform-saturated cotton pledget,combining the filtrates in a conical flask.Evaporate the chloroform on a steam bath with the aid of a current of air to dryness,and dissolve the residue in methanol to make 25.0mL.

Blank solution— Transfer 5.0mLof water to a separator,and proceed as directed under Test solution.The methanolic solution so obtained is the Blank solution.

Procedure— Determine the absorbance (A)of the Test solutionin a 1-cm cell at the wavelength of maximum absorbance at about 239nm,with a suitable spectrophotometer,using Blank solutionas the blank.Calculate the quantity,in mg,of free betamethasone in the portion of Betamethasone Sodium Phosphate taken by the formula: The limit is 250µg (1.0%).

Mobile phase— Prepare a filtered and degassed mixture of methanol and 0.07Manhydrous monobasic potassium phosphate (3:2).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Betamethasone Sodium Phosphate RSin a mixture of methanol and water (3:2),and dilute quantitatively,and stepwise if necessary,with the same mixture of methanol and water to obtain a solution having a known concentration of about 0.17mg per mL.

Assay preparation— Transfer about 34mg of Betamethasone Sodium Phosphate,accurately weighed,to a 200-mLvolumetric flask,dissolve in and dilute with a mixture of methanol and water (3:2)to 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 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not more than 2,and the relative standard deviation for replicate injections is not more than 3.0%.

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 C22H28FNa2O8Pin the portion of Betamethasone Sodium Phosphate taken by the formula: in which Cis the concentration,in mg per mL,of USP Betamethasone Sodium Phosphate RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.