A: Infrared Absorption 197M.

B: Ultraviolet Absorption 197U—

Test solution: 10 mg per mL, in dioxane.

Mobile phase— Prepare a filtered and degassed mixture of butyl chloride, tetrahydrofuran, methanol, glacial acetic acid, and water (890:56:28:24:0.4), and sonicate to dissolve. Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluting solution— Prepare a solution of butyl chloride, tetrahydrofuran, methanol, and glacial acetic acid (81.5:10:8:0.5).

Standard solution— Prepare a solution in Diluting solution containing 40 µg of USP Hydrocortisone RS per mL. Sonicate for about 5 minutes.

Test solution— Transfer 20 mg of Hydrocortisone to a 10-mL volumetric flask, dissolve in Diluting solution, add Diluting solution to volume to obtain a solution having a known concentration of about 2.0 mg per mL, and sonicate for about 5 minutes.

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 3-µm packing L3. The flow rate is about 1.5 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 5%.

Procedure— Separately inject equal volumes (about 5 µL) of the Standard solution, the Diluting solution, and the Test solution into the chromatograph, record the chromatograms, and measure the responses for all the peaks, ignoring artifact peaks. Calculate the percentage of impurities in the portion of Hydrocortisone taken by the formula: in which C is the concentration, in mg per mL, of USP Hydrocortisone RS in the Standard solution; W is the weight, in mg, of Hydrocortisone taken; ri is the response of each individual impurity peak in the Test solution; and rS is the response of the major peak obtained from the Standard solution: not more than 0.5% of any individual impurity 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 methanol (50:25:25). Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluent— Prepare a mixture of methanol and water (1:1).

Internal standard solution— Prepare a solution of propylparaben in methanol having a concentration of about 1 mg per mL.

Standard stock solution— Dissolve an accurately weighed quantity of USP Hydrocortisone RS in methanol to obtain a solution having a known concentration of about 1 mg per mL.

Standard preparation— Transfer 2.0 mL of Standard stock solution and 2.0 mL of Internal standard solution to a 50-mL volumetric flask, dilute with Diluent to volume, and mix.

Assay preparation— Transfer about 50 mg of Hydrocortisone, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with methanol to volume, and mix. Transfer 2.0 mL of this solution and 2.0 mL of Internal standard solution to a 50-mL volumetric flask, dilute with Diluent 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 5-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 1.8 for propylparaben and 1.0 for hydrocortisone; the resolution, R, between the hydrocortisone and propylparaben peaks is not less than 9.0; the column efficiency is not less than 3000 theoretical plates for hydrocortisone; the tailing factor is not more than 1.2; 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 C21H30O5 in the portion of Hydrocortisone taken by the formula: in which C is the concentration, in mg per mL, of USP Hydrocortisone RS in the Standard preparation; and RU and RS are the ratios of the peak response of hydrocortisone to that of propylparaben obtained from the Assay preparation and the Standard preparation, respectively.