A: Infrared Absorption 197K.

B: Ultraviolet Absorption 197U—

Solution: 10 µg per mL.

Medium: methanol. Absorptivities at 242 nm, calculated on the dried basis, do not differ by more than 3.0%.

Test solution: 10 mg per mL, in chloroform.

Solution A— Prepare a filtered and degassed solution of 1 g of monobasic potassium phosphate in 1000 mL of water, adjust with 45% potassium hydroxide to a pH of 5.5, and mix. Make adjustments if necessary (see System Suitability under Chromatography 621).

Solution B— Use filtered and degassed acetonitrile.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system.

Solvent— Prepare a mixture of acetonitrile and Solution A (80:20).

Standard solution— Dissolve an accurately weighed quantity of USP Hydrocortisone Butyrate RS in Solvent to obtain a solution having a known concentration of about 0.5 mg per mL.

Test solution— Transfer about 50 mg of Hydrocortisone Butyrate, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with Solvent to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 10-cm column that contains 3-µm packing L1. The flow rate is about 2.0 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard solution and the Test solution, and record the peak responses as directed for Procedure: the resolution, R, between hydrocortisone butyrate and any impurity is not less than 1.0; and the column efficiency is not less than 10,000 theoretical plates.

Procedure— Separately inject equal volumes (about 5 µL) of the Test solution and the Standard solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks, disregarding any peak having a percentage of 0.05% or less. Calculate the percentage of each impurity in the portion of Hydrocortisone Butyrate taken by the formula: in which CS is the concentration, in mg per mL, of USP Hydrocortisone Butyrate RS in the Standard solution; CU is the concentration, in mg per mL, of Hydrocortisone Butyrate in the Test solution; ri is the peak area for each impurity obtained from the Test solution; and rS is the peak area for hydrocortisone butyrate obtained from the Standard solution: not more than 1.0% of any individual impurity is found; and not more than 2.0% of total impurities is found.

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and glacial acetic acid (124:76:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Solvent— Prepare a mixture of tetrahydrofuran and glacial acetic acid (1000:1).

Diluting solution— Prepare a filtered solution of methanol, water, and glacial acetic acid (500:500:1).

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone Butyrate RS in Solvent, and dilute quantitatively, and stepwise if necessary, with Solvent to obtain a solution having a known concentration of about 0.1 mg per mL. Transfer 10.0 mL of this solution to a 50-mL volumetric flask, dilute with Diluting solution to volume, and mix.

System suitability solution— Dissolve suitable quantities of propyl 4-hydroxybenzoate and USP Hydrocortisone Butyrate RS in Solvent, and dilute quantitatively, and stepwise if necessary, with Solvent to obtain a solution having known concentrations of about 0.1 mg of each per mL. Transfer 10 mL of this solution to a 50-mL volumetric flask, dilute with Diluting solution to volume, and mix.

Assay preparation— Transfer about 50 mg of Hydrocortisone Butyrate, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with Solvent to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with Solvent to volume, and mix. Transfer 10.0 mL of this solution to a 50-mL volumetric flask, dilute with Diluting solution to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 3.0-mm × 10-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for propyl 4-hydroxybenzoate and 1.0 for hydrocortisone butyrate; and the resolution, R, between propyl 4-hydroxybenzoate and hydrocortisone butyrate is not less than 4.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 4000 theoretical plates; the tailing factor is not more than 1.6; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 5 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 C25H36O6 in the portion of Hydrocortisone Butyrate taken by the formula: in which C is the concentration, in mg per mL, of USP Hydrocortisone Butyrate RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.