Identification—
Transfer a quantity of Cream, equivalent to about 2 mg of halcinonide, to a glass-stoppered, 50-mL centrifuge tube, add 15 mL of warm water, and shake for 2 minutes to disperse. Add 20 mL of chloroform, and shake for 5 minutes. Cool in an ice bath, then centrifuge. Transfer the chloroform layer to a conical flask. Repeat the extraction with an additional 15 mL of chloroform, and combine the chloroform extracts. Evaporate the chloroform extracts on a steam bath under a current of air nearly to dryness, and dissolve the residue in 10.0 mL of chloroform. Apply 20 µL of this solution and 20 µL of a solution of
USP Halcinonide RS in chloroform having a concentration of 0.2 mg per mL to a thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel mixture. Develop the chromatogram in a solvent system consisting of a mixture of chloroform and ethyl acetate (5:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate, air-dry, and view the chromatogram under short-wavelength UV light: the
RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.
Assay—
Solvent A—
On the day of use, prepare a mixture of acetonitrile and water (2:1).
Mobile phase—
Mix approximately equal volumes of acetonitrile and water, adjusting the composition as necessary to achieve acceptable chromatography.
Internal standard solution—
Transfer 15 mg of Progesterone to a 100-mL volumetric flask. Dissolve in hexanes-saturated Solvent A, dilute with hexanes-saturated Solvent A to volume, and mix.
Standard preparation—
Transfer about 20 mg of
USP Halcinonide RS, accurately weighed, to a 100-mL volumetric flask, dissolve in
Solvent A, dilute with
Solvent A to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL volumetric flask. Add 4.0 mL of
Internal standard solution, dilute with hexanes-saturated
Solvent A to volume, and mix.
Assay preparation—
Transfer an accurately weighed quantity of Cream, equivalent to about 0.5 mg of halcinonide, to a glass-stoppered, 50-mL centrifuge tube, add 12 mL of hexanes-saturated
Solvent A and 20 mL of hexanes, and shake for 1 minute. Place in a heated ultrasonic bath at 58 ± 2
for 20 minutes, initially shaking for 1 to 2 minutes to ensure dispersion, and at about 5-minute intervals thereafter, on a vibratory mixer. Cool, centrifuge, and transfer the lower layer to a 25-mL volumetric flask. Add 5 mL of hexanes-saturated
Solvent A to the tube, mix for 1 minute, then centrifuge. Transfer the lower layer to the volumetric flask, and repeat the extraction with an additional 5 mL of hexanes-saturated
Solvent A, combining the extracts in the flask. Add 2.0 mL of
Internal standard solution to the flask, dilute with hexanes-saturated
Solvent A to volume, and mix. If necessary, clarify a portion of the solution by centrifugation.
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 2 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed under
Procedure: the resolution,
R, between the analyte and internal standard peaks 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 20 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 1.2 for progesterone and 1.0 for halcinonide. Calculate the quantity, in mg, of C
24H
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 Halcinonide RS in the
Standard preparation; and
RU and
RS are the ratios of the peak responses of halcinonide to internal standard obtained from the
Assay preparation and the
Standard preparation, respectively.
