Identification—
Transfer an accurately weighed quantity of Ointment, equivalent to about 5 mg of desoximetasone, to a 50-mL centrifuge tube. Add 20 mL of hexane, heat gently to 60
, and shake until the Ointment is completely dispersed. Add 8 mL of acetonitrile, insert the stopper in the tube, and shake vigorously for 5 minutes. Cool to room temperature, and centrifuge until the lower layer is clear. Transfer the lower layer to a 10-mL volumetric flask, dilute with acetonitrile to volume, and mix. Prepare a solution of
USP Desoximetasone RS in acetonitrile containing 0.5 mg per mL. Separately apply 5 µL of each solution to a thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the plate in a saturated chamber containing a mixture of ethyl acetate and chloroform (4:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate, and allow to air-dry. Examine under short-wavelength UV light. Spray the dried plate with a 1 in 5 solution of
p-toluenesulfonic acid in alcohol. Heat the plate at 100
for 5 minutes, and examine under long-wavelength UV light: the
RF value and appearance (brownish yellow fluorescent spot) of the principal spot from the test solution, correspond to those of the principal spot from the Standard solution.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of methanol, water, and glacial acetic acid (65:35:1). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Desoximetasone RS in methanol to obtain a solution having a known concentration of about 0.4 mg per mL. Quantitatively dilute 1 volume of this solution with 9 volumes of a 1:1 mixture of methanol and spectrophotometric acetonitrile that is saturated with
n-heptane, and mix.
Assay preparation—
Transfer an accurately weighed amount of Ointment, equivalent to about 2 mg of desoximetasone, to a 50-mL centrifuge tube. Add 20 mL of n-heptane that has been previously saturated with spectrophotometric acetonitrile, and heat gently with occasional shaking until the Ointment is completely dispersed. Allow to cool slightly, and extract with a 10-mL portion of spectrophotometric acetonitrile. Shake vigorously, centrifuge, remove the bottom layer of acetonitrile with a syringe and needle, and transfer to a 50-mL volumetric flask. Using the same needle and syringe, extract the desoximetasone with successive 10-mL and 8-mL portions of acetonitrile, combining all acetonitrile layers in the 50-mL flask. Dilute with methanol nearly to volume, mix, and allow the solution to reach room temperature. Dilute with methanol to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 15-cm column that contains packing L7. The flow rate is about 1 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed under
Procedure: the tailing factor for the analyte peak is not more than 2.0, the resolution,
R, between the analyte and solvent peaks is not less than 5.0, 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 C
22H
29FO
4 in the portion of Ointment taken by the formula:
50C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Desoximetasone RS in the
Standard preparation, and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
