Test preparation: 25 mg in 2000 mL of alcohol.

Procedure— Proceed as directed under Spectrophotometry and Light-Scattering 851, and measure the absorbance at 240 nm: the absorptivity is between 35.0 and 38.0.

Test solution: 10 mg per mL, in dioxane.

Solution A— Prepare a mixture of water, methanol, acetonitrile, and glacial acetic acid (500:350:150:3), and degas.

Solution B— Prepare a mixture of acetonitrile, methanol, and water (550:500:3), and degas.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Dissolve accurately weighed quantities of USP Isoflupredone Acetate RS and USP Prednisolone Acetate RS in Solution A to obtain a solution having known concentrations of about 0.03 mg of each per mL. Sonicate, if necessary, to dissolve.

Test solution— Dissolve an accurately weighed quantity of Isoflupredone Acetate in Solution A to obtain a solution having a concentration of about 0.3 mg per mL. Sonicate, if necessary, to dissolve. Use this solution within 16 hours.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L7. The flow rate is about 1 mL per minute. Protect the column from temperature fluctuations. The chromatograph is programmed as follows. Chromatograph the System suitability solution, and record the peak areas as directed for Procedure: the retention time for isoflupredone acetate is between 21 and 26 minutes; the relative retention times are about 1.1 for prednisolone acetate and 1.0 for isoflupredone acetate; the resolution, R, between isoflupredone acetate and prednisolone acetate is not less than 1.2; and the column efficiency determined from isoflupredone is not less than 6000 theoretical plates.

Procedure— Inject a volume (about 50 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the areas for the major peaks. Calculate the percentage of each impurity in the portion of Isoflupredone Acetate taken by the formula: in which ri is the peak response for each impurity; and rs is the sum of the responses of all the peaks: not more than 1.0% of any individual impurity is found; and not more than 2.0% of total impurities is found, excluding those that are present in amounts less than 0.05%.

(Official January 1, 2007)

Mobile phase— Prepare a mixture of n-butyl chloride, water-saturated n-butyl chloride, tetrahydrofuran, methanol, and glacial acetic acid (475:475:70:35:30). Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluent— Use water-saturated chloroform.

Internal standard solution— Dissolve an accurately weighed quantity of fluoxymesterone in Diluent to obtain a solution having a known concentration of about 0.9 mg per mL.

Standard preparation— Dissolve about 4 mg of USP Isoflupredone Acetate RS, accurately weighed, in 8.0 mL of Internal standard solution and 32.0 mL of Diluent.

Assay preparation— Transfer about 4 mg of Isoflupredone Acetate, accurately weighed, to a suitable container. Dissolve in 8.0 mL of Internal standard solution and 32.0 mL of Diluent, centrifuge, and use the clear chloroform portion.

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 L3. The flow rate is about 0.7 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative retention times are about 1.0 for isoflupredone acetate and 1.2 for fluoxymesterone; the resolution, R, between isoflupredone acetate and fluoxymesterone is not less than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 12 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in mg, of C23H29FO6 in the portion of Isoflupredone Acetate taken by the formula: in which WS is the weight, in mg, of USP Isoflupredone Acetate RS taken to prepare the Standard preparation; and RU and RS are the peak area ratios of isoflupredone acetate to fluoxymesterone obtained from the Assay preparation and the Standard preparation, respectively.