A: Infrared Absorption 197M.

B: Ultraviolet Absorption 197U—

Test solution: 10 mg per mL, in acetone.

Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and methanol (700:285:15). Add 3.0 mL of glacial acetic acid per Liter of this solution. Mix thoroughly. Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluting solution— Prepare a mixture of water, acetonitrile, tetrahydrofuran, and glacial acetic acid (500:250:250:1). Mix thoroughly.

Standard solution— Dissolve an accurately weighed quantity of USP Hydrocortisone Hemisuccinate RS in Diluting solution, and dilute quantitatively, and stepwise if necessary, with Diluting solution to obtain a solution having a known concentration of about 6.6 µg per mL.

Test solution— Transfer about 6.6 mg of Hydrocortisone Hemisuccinate, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with Diluting solution to volume, and mix. [NOTE—Samples should be maintained at 5 or colder during analysis.]

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 L1. The flow rate is about 0.8 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the column efficiency is not less than 5000 theoretical plates; and the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Inject a volume (about 15 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the peak responses. Calculate the percentage of each impurity in the portion of Hydrocortisone Hemisuccinate 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. Disregard any peak representing less than 0.05%.

(Official January 1, 2007)

Internal standard solution— Prepare a solution of USP Fluorometholone RS in tetrahydrofuran containing about 3 mg per mL.

Mobile phase— Prepare a filtered mixture of butyl chloride, water-saturated butyl chloride, tetrahydrofuran, methanol, and glacial acetic acid (95:95:14:7:6). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Transfer an accurately weighed amount of USP Hydrocortisone Hemisuccinate RS to a suitable container to obtain a solution containing 0.6 mg per mL. Add an accurately measured volume of Internal standard solution so that the Standard preparation contains 10% Internal standard solution. Dilute with chloroform containing 3% glacial acetic acid to volume.

Assay preparation— Transfer about 30 mg of Hydrocortisone Hemisuccinate, accurately weighed, to a 50-mL volumetric flask, add 5.0 mL of Internal standard solution, and dilute with chloroform containing 3% glacial acetic acid to volume.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L3. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between hydrocortisone hemisuccinate and the internal standard 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 6 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 C25H34O8 in the portion of Hydrocortisone Hemisuccinate taken by the formula: in which C is the concentration, in mg per mL, of USP Hydrocortisone Hemisuccinate RS in the Standard preparation; and RU and RS are the peak area ratios of hydrocortisone hemisuccinate to the internal standard obtained from the Assay preparation and the Standard preparation, respectively.