Solvent: anhydrous dimethyl sulfoxide.

Change to read:

Solution A— Prepare a filtered and degassed mixture of water, acetonitrile, and phosphoric acid (95:5:0.1).

Solution B— Prepare a filtered and degassed mixture of water, acetonitrile, and phosphoric acid (85:15:0.1).

Diluent— Prepare a mixture of water, acetonitrile, and phosphoric acid (50:50:0.1).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Phthalic acid stock solution— Transfer about 100 mg of phthalic acid to a 100-mL volumetric flask, dissolve in a mixture of acetonitrile and water (80:5), and dilute with acetonitrile to volume. Mix, and dilute quantitatively, and stepwise if necessary, with acetonitrile to obtain a solution having a concentration of about 0.1 mg per mL.

Standard stock solution— Dissolve, with the aid of sonication, an accurately weighed quantity of USP Thalidomide RS in acetonitrile to obtain a solution having a known concentration of about 1 mg per mL.

Standard solution— Pipet 2.0 mL of the Standard stock solution and 2.0 mL of the Phthalic acid stock solution into a 100-mL volumetric flask, dilute with Diluent to volume, and mix. Pipet 10.0 mL of this solution into a 100-mL volumetric flask, add 10.0 mL of phosphoric acid solution (1 in 100), dilute with water to volume, and mix to obtain a solution having a known concentration of about 0.0002 mg of phthalic acid per mL.

Test solution— Transfer about 100 mg of Thalidomide, accurately weighed, to a 50-mL volumetric flask, and dissolve, with the aid of sonication, in 40 mL of a mixture of water, acetonitrile, and phosphoric acid (50:50:0.1). Dilute with Diluent to volume, and mix. Pipet 10.0 mL of this solution into a 100-mL volumetric flask, add 10.0 mL of phosphoric acid solution (1 in 100), dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 218-nm detector and a 3.9-mm × 15-cm column that contains 4-µm packing L1. The flow rate is about 2 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.35 for phthalic acid and about 1.0 for thalidomide; the tailing factor for the phthalic acid and thalidomide peaks is not more than 2.0; and the relative standard deviation determined from the phthalic acid peak for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 200 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for all the peaks. Calculate the percentage of each impurity in the portion of Thalidomide taken by the formula: in which CP is the concentration, in mg per mL, of phthalic acid in the Standard solution; W is the amount, in mg, of Thalidomide taken to prepare the Test solution; ri is the peak response for each impurity obtained from the Test solution; and rP is the phthalic acid peak response obtained from the Standard solution: not more than 0.1% of any individual impurity is found; and not more than 0.3% of total impurities is found.

Test solution— Dissolve an accurately weighed quantity of Thalidomide in acetonitrile to obtain a solution having a concentration of about 2 mg per mL.

Standard solution— Dissolve an accurately weighed quantity of glutamine in a mixture of acetonitrile and water (1:1) to obtain a solution having a concentration of about 0.1 mg per mL.

Eluant: a mixture of methylene chloride, methanol, and acetic acid (75:25:0.05).

Application volume: 2 µL (Standard solution) and 100 µL (Test solution).

Visualization: 4.

Limit: 0.1%.

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and phosphoric acid (85:15:0.1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard preparation— Transfer about 150 mg of phenacetin, accurately weighed, to a 100-mL volumetric flask, dissolve in about 80 mL of acetonitrile, dilute with acetonitrile to volume, and mix.

Standard preparation— Dissolve, with the aid of sonication, an accurately weighed quantity of USP Thalidomide RS in acetonitrile to obtain a solution having a known concentration of about 1 mg per mL. Transfer 10.0 mL of this solution and 5.0 mL of Internal standard preparation to a 100-mL volumetric flask, add 10.0 mL of phosphoric acid solution (1 in 100), dilute with water to volume, and mix.

Assay preparation— Transfer about 100 mg of Thalidomide, accurately weighed, to a 100-mL volumetric flask, and dissolve, with the aid of sonication, in 80 mL of acetonitrile. Dilute with acetonitrile to volume, and mix. Pipet 10.0 mL of this solution and 5.0 mL of Internal standard preparation into a 100-mL volumetric flask, add 10.0 mL of phosphoric acid solution (1 in 100), dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 237-nm detector and a 3.9-mm × 15-cm column that contains 4-µm packing L1. 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 thalidomide and phenacetin is not less than 3.0; the column efficiency determined from the thalidomide and phenacetin peaks is not less than 7000 and 9000 theoretical plates, respectively; the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 1.0%.

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 areas for the major peaks. Calculate the quantity, in mg, of C13H10N2O4 in the portion of Thalidomide taken by the formula: in which C is the concentration, in mg per mL, of USP Thalidomide RS in the Standard preparation; and RU and RS are the peak area ratios obtained from the Assay preparation and the Standard preparation, respectively.