Medium: water; 900 mL. Add 1.0 mL of Internal standard solution to each vessel.

Apparatus 2: 50 rpm.

Time: 45 minutes.

Mobile phase— Prepare a filtered and degassed mixture of methanol and water (60:40), making adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Prepare a solution in methanol of testosterone having a final concentration of about 0.5 mg per mL.

Standard solution— Prepare a solution of USP Betamethasone RS, in methanol, having an accurately known concentration of about 0.5 mg per mL. Pipet 1 mL of this solution and 1 mL of the Internal standard solution into a container, and quantitatively dilute with water to 900 mL.

Chromatographic system (see Chromatography 621)— 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 2 mL per minute. Chromatograph replicate injections of the Standard solution, and record the peak responses as directed for Procedure: the resolution, R, between betamethasone and testosterone is not less than 1.5; and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 200 µL) of the Standard solution and filtered portions of the solution under test into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.5 for betamethasone and 1.0 for testosterone. Calculate the quantity of C22H29FO5 dissolved in comparison with the Standard solution, similarly chromatographed.

Tolerances— Not less than 75% (Q) of the labeled amount of C22H29FO5 is dissolved in 45 minutes.

Procedure for content uniformity—

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (2:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Transfer about 25 mg of beclomethasone to a 200-mL volumetric flask, add methanol to volume, and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Betamethasone RS in methanol, and dilute quantitatively and stepwise, if necessary, with methanol to obtain a solution having a known concentration of about 0.1 mg per mL. Mix equal volumes, accurately measured, of this solution and the Internal standard solution to obtain a Standard preparation having a final known concentration of about 0.05 mg of USP Betamethasone RS per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 0.5 mg of betamethasone, to a 125-mL separator. Add 25 mL of water, and shake by mechanical means for about 15 minutes. Add 5.0 mL of Internal standard solution. Extract with four 25-mL portions of chloroform. Filter the chloroform extracts through about 4 g of chloroform-washed anhydrous sodium sulfate, collecting the extracts in a 150-mL beaker. Evaporate the extracts on a steam bath with the aid of a stream of nitrogen to dryness, taking care to avoid overheating. Dissolve the residue in 2 mL of methanol, and transfer to a 10-mL volumetric flask. Rinse the beaker with small portions of methanol, transferring the rinses to the same flask. Dilute with methanol to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4-mm × 30-cm column that contains packing L1. The flow rate is about 1.2 mL per minute. Chromatograph the Standard preparation, and record the peak heights as directed for Procedure: the resolution, R, between the analyte and internal standard peaks is not less than 1.7; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the heights of the major peaks. The relative retention times are about 1.4 for beclomethasone and 1.0 for betamethasone. Calculate the quantity, in mg, of betamethasone (C22H29FO5) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Betamethasone RS in the Standard preparation; and RU and RS are the peak height ratios obtained from the Assay preparation and the Standard preparation, respectively.