A: Infrared Absorption 197K.

B: Ultraviolet Absorption 197U—

Buffer solution— Proceed as directed in the Assay.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (55:45). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Prepare as directed for the Standard preparation in the Assay.

Test solution— Prepare as directed for the Assay preparation.

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 1.5 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 3.1 for the first eluted epimer of 21-acetate of budesonide, about 3.2 for the second eluted epimer of 21-acetate of budesonide, 1.0 for the first eluted epimer of budesonide (epimer B), and about 1.1 for the second eluted epimer of budesonide (epimer A); and the column efficiency is not less than 5500 theoretical plates, determined from the budesonide epimer B peak.

Procedure— Inject a volume (about 20 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the peak responses. Calculate the percentage of the 21-acetate of budesonide in the portion of Budesonide taken by the formula: in which ri is the sum of the peak areas for the two epimers of the 21-acetate of budesonide; and rS is the sum of the areas of the two budesonide peaks; not more than 0.10% of the 21-acetate of budesonide is found.

Buffer solution— Proceed as directed in the Assay.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution, acetonitrile, and isopropanol (65:26:9). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Prepare as directed for Standard preparation in the Assay.

Test solution— Proceed as directed for the Assay preparation.

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 3.5-µm packing L1. The flow rate is about 1.5 mL per minute. Maintain the column at 50. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.73 and 0.78, respectively, for the two epimers of 11-ketobudesonide, about 0.68 for 21-dehydrobudesonide, about 0.84 for 14,15-dehydrobudesonide, and 1.0 for the first eluted epimer of budesonide (epimer B); the resolution, R, between the first epimer of 11-ketobudesonide and 21-dehydrobudesonide is not less than 1.0 and between the second epimer of 11-ketobudesonide and 14,15-dehydrobudesonide is not less than 1.2; and the column efficiency is not less than 5500 theoretical plates, determined from the budesonide epimer B peak.

Procedure— Inject a volume (about 20 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the peak responses. Calculate the percentage of 11-ketobudesonide in the portion of Budesonide taken by the formula: in which ri is the sum of the areas of the two ketobudesonide peaks; and rU is the sum of the areas of the two budesonide peaks; not more than 0.2% of 11-ketobudesonide is found.

Buffer solution and Mobile phase —Proceed as directed in the Assay.

Standard solution— Prepare as directed for the Standard preparation in the Assay.

Test solution— Proceed as directed for the Assay preparation.

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 1.5 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the column efficiency is not less than 5500 theoretical plates, determined from the budesonide epimer B peak.

Procedure— Inject a volume (about 20 µL) of the Test solution into the chromatograph, record the chromatogram, and measure all of the peak responses. Calculate the percentage of each impurity in the portion of Budesonide taken by the formula: in which ri is the peak area response for each impurity; and rs is the sum of the responses of all the peaks: the impurities meet the requirements listed in the Table below.

Buffer solution— Dissolve 3.17 g of monobasic sodium phosphate in water, add 0.23 g of phosphoric acid, dilute with water to 1000 mL, and mix. The pH is 3.2 ± 0.1.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (68:32). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Budesonide RS in acetonitrile, and dilute quantitatively, and stepwise if necessary, with Buffer solution to obtain a solution having a known concentration of about 0.5 mg per mL. The proportion of acetonitrile in the Standard preparation does not exceed 30%.

Assay preparation— Transfer about 25 mg of Budesonide, accurately weighed, to a 50-mL volumetric flask, dissolve in 15 mL of acetonitrile, dilute with Buffer solution 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 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention time for epimer A is 1.1 with respect to epimer B; the resolution, R, between the two budesonide epimer peaks is greater than 1.5; and the column efficiency is not less than 5500 theoretical plates, determined from the budesonide epimer B peak.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the area responses for the major peaks. Calculate the percentage of epimer A (C25H34O6) in the portion of Budesonide taken by the formula: in which rUA and rUB are the peak area responses for epimer A and epimer B obtained from the Assay preparation, respectively. Calculate the quantity, in mg, of budesonide (C25H34O6) in the portion of Budesonide taken by the formula: in which C is the concentration, in mg per mL, of USP Budesonide RS in the Standard preparation; rSA and rSB are the peak area responses for epimer A and epimer B obtained from the Standard preparation, respectively; and the other terms are as defined above.