A: Infrared Absorption 197M.

B: Thin-Layer Chromatographic Identification Test 201—

Test solution: 5 mg per mL, in methanol.

Test solution: methanol.

Standard solution: methanol.

Application volume: 10 µL.

Eluant: a mixture of toluene, acetone, methyl ethyl ketone, and formic acid (55:20:20:5), in a nonequilibrated chamber.

Visualization: 5.

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (63:37). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Prepare a solution of propylparaben in alcohol having a known concentration of about 0.25 mg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Betamethasone RS in alcohol to obtain a solution having a known concentration of about 0.2 mg per mL. Transfer 10.0 mL of this solution to a suitable vial, and add 10.0 mL of Internal standard solution, to obtain a Standard preparation having known concentrations of about 0.1 mg of betamethasone and about 0.125 mg of propylparaben per mL.

Assay preparation— Using about 80 mg of Betamethasone, accurately weighed, prepare as directed for Standard preparation.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 240-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 1.0 for betamethasone and 1.4 for propylparaben; the resolution, R, between betamethasone and propylparaben 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 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C22H29FO5 in the portion of Betamethasone 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 of the betamethasone peak and the internal standard peak obtained from the Assay preparation and the Standard preparation, respectively.