A: Infrared Absorption 197M.

B: Ultraviolet Absorption 197U—

Solution: 10 µg per mL.

Medium: methanol.

Acetate buffer , Mobile phase, Diluent, and Chromatographic system—Prepare as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP m-Aminoglutethimide RS in Diluent to obtain a solution having a known concentration of about 1 mg per mL. Dilute an accurately measured volume of this solution quantitatively, and stepwise if necessary, with Diluent to obtain a solution having a known concentration of about 10 µg per mL.

Test solution— Transfer about 100 mg of Aminoglutethimide, accurately weighed, to a 100-mL volumetric flask, and dissolve in Diluent. Dilute with Diluent to volume, mix, and pass through a 0.45-µm or finer porosity filter, discarding the first 5 mL of the filtrate.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for all of the peaks. The relative retention times are about 0.8 for aminoglutethimide and 1.0 for m-aminoglutethimide. Calculate the percentage of m-aminoglutethimide in the specimen of Aminoglutethimide taken by the formula: in which C is the concentration, in µg per mL, of USP m-Aminoglutethimide RS in the Standard preparation; W is the weight, in mg, of the specimen taken; and rU and rS are the m-aminoglutethimide peak responses obtained from the Test preparation and the Standard preparation, respectively: not more than 2.0% of m-aminoglutethimide is found. Calculate the percentage of each peak, other than the main peak and the m-aminoglutethimide peak, if present, by the same formula: in which ri is the response of each peak, and rt is the sum of the responses of all the peaks in the chromatogram obtained from the Test preparation: not more than 1.0% total impurities, other than m-aminoglutethimide, is found.

Acetate buffer— Mix 150 mL of 0.1 N acetic acid with 50 mL of 0.1 N potassium hydroxide, dilute with water to 1000 mL, and mix.

Mobile phase— Dissolve 100 mg of edetate disodium in 350 mL of Acetate buffer, add 650 mL of methanol, mix, and cool to room temperature. Adjust with glacial acetic acid to a pH of 5.0 ± 0.1, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP Azo-aminoglutethimide RS in dimethyl sulfoxide, and dilute quantitatively, and stepwise if necessary, with dimethyl sulfoxide to obtain a solution having a known concentration of about 0.5 µg per mL.

Test solution— Transfer about 100 mg of Aminoglutethimide, accurately weighed, to a 100-mL volumetric flask. Dissolve in and dilute with dimethyl sulfoxide to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 328-nm detector and a 3.9-mm × 15-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the capacity factor for the analyte peak is between 2.0 and 5.0, the column efficiency determined from the analyte peak is not less than 800 theoretical plates, and the tailing factor for the analyte peak is not more than 1.2.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks. [note—The aminoglutethimide elutes with the dimethyl sulfoxide.] Calculate the percentage of azo-aminoglutethimide in the specimen of Aminoglutethimide taken by the formula: in which C is the concentration, in µg per mL, of USP Azo-aminoglutethimide RS in the Standard solution; W is the weight, in mg, of the specimen taken; and rU and rS are the azo-aminoglutethimide peak responses obtained from the Test solution and the Standard solution, respectively: not more than 0.03% of 3,3¢-(azodi-4,1-phenylene)-3,3¢-dimethylbis-[2,6-piperidinedione], corresponding to azo-aminoglutethimide, is found.

Acetate buffer— Add 240 mL of 0.1 N acetic acid to 200 mL of 0.1 N potassium hydroxide in a 2000-mL volumetric flask, add about 500 mL of water, and mix. Adjust by the addition of either 1 N acetic acid or 1 N potassium hydroxide to a pH of 5.0 ± 0.1. Dilute with water to volume, and mix.

Mobile phase— Prepare a filtered and degassed mixture of Acetate buffer and methanol (73:27). Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

Assay preparation— Transfer about 50 mg of Aminoglutethimide, accurately weighed, to a 100-mL volumetric flask, and dissolve in Diluent. Dilute with Diluent to volume, mix, and pass through a 0.45-µm or finer porosity filter, discarding the first 5 mL of the filtrate.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 240-nm detector and a 3.9-mm × 15-cm column that contains 4-µm packing L1. The column temperature is maintained at about 40, and the flow rate is about 1.3 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor for the analyte peak is not more than 1.7, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— [note—Use peak areas where peak responses are indicated.] 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 C13H16N2O2 in the portion of Aminoglutethimide taken by the formula: in which C is the concentration, in mg per mL, of USP Aminoglutethimide RS in the Standard preparation; and rU and rS are the aminoglutethimide peak responses obtained from the Assay preparation and the Standard preparation, respectively.