Identification—
Transfer a quantity of Cream, equivalent to about 0.75 mg of clobetasol propionate, to a 25-mL plastic-stoppered centrifuge tube. Add 10 mL of methanol, and cap. Heat in a 60
water bath for about 4 minutes, remove the tube from the bath, and shake vigorously. Repeat the heating and shaking. Cool to room temperature, add 3.5 mL of water, and mix. Centrifuge at about 3500 rpm for about 10 minutes. Transfer about 5 mL of the supernatant to a 100-mL separator, add 1 g of sodium chloride and 10 mL of water, and mix. Extract with 5 mL of chloroform by shaking for 1 minute, collect the lower layer, and evaporate with the aid of a stream of nitrogen to dryness. Dissolve the residue in about 0.5 mL of chloroform to obtain the test solution. Prepare a Standard solution of
USP Clobetasol Propionate RS having the same concentration as the test solution. The test solution so obtained responds to the
Thin-Layer Chromatographic Identification Test 
201
, a mixture of chloroform, acetone, and alcohol (100:10:5) being used as the developing solvent.
Microbial limits 61—
It meets the requirements of the tests for absence of
Staphylococcus aureus,
Pseudomonas aeruginosa,
Escherichia coli, and
Salmonella species, and the total aerobic microbial count does not exceed 100 cfu per g.
Assay—
[NOTE—Where peak responses are indicated, use peak areas.
]
Internal standard solution, Standard preparation, System suitability solution, and Chromatographic system—
Proceed as directed in the
Assay under
Clobetasol Propionate.
Mobile phase—
Prepare a filtered and degassed mixture containing acetonitrile, 0.05
M monobasic sodium phosphate (adjusted with 50% sodium hydroxide solution to a pH of 5.5), and methanol (95:85:20). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Assay preparation—
Transfer an accurately weighed portion of Cream, equivalent to about 1.0 mg of clobetasol propionate, to a 50-mL volumetric flask. Add 10.0 mL of Internal standard solution and 15.0 mL of methanol. Shake vigorously to disperse the cream, and centrifuge at about 3500 rpm for about 10 minutes. Filter a portion of the supernatant through a 0.45-µm filter.
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. The relative retention times are about 1.0 for clobetasol propionate and 1.6 for beclomethasone dipropionate. Calculate the quantity, in mg, of C
25H
32ClFO
5 in the portion of Cream taken by the formula:
25C(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Clobetasol Propionate RS in the
Standard preparation; and
RU and
RS are the ratios of the clobetasol propionate peak to the internal standard peak obtained from the
Assay preparation and the
Standard preparation, respectively.
