A: Thin-Layer Chromatographic Identification Test 201—

B: The relative retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Medium— Add 1.0 mL of polyoxyethylene (23) lauryl ether solution, prepared by dissolving 50 g in 100 mL of water, to 0.225 M hydrochloric acid; 4000 mL.

Apparatus 2: 75 rpm.

Time: 60 minutes.

Mobile phase— Prepare as directed in the Assay under Thalidomide.

Internal standard solution— Prepare a solution of phenacetin in acetonitrile containing about 375 µg per mL. Pipet 20.0 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.

Standard solution— Dissolve an accurately weighed quantity of USP Thalidomide RS in acetonitrile to obtain a solution having a known concentration of about 0.25 mg per mL. 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. Add 5.0 mL of Internal standard solution to 20.0 mL of this solution, and mix. This solution contains about 0.02 mg of USP Thalidomide RS per mL.

Test solution— Add 5.0 mL of Internal standard solution to each 20.0 mL of filtered solution under test, and mix.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in mg, of C13H10N2O4 dissolved by the formula: in which C is the concentration, in mg per mL, of USP Thalidomide RS in the Standard solution; and RU and RS are the peak area ratios of thalidomide to the internal standard obtained from the Test solution and the Standard solution, respectively.

Tolerances— Not less than 70% (Q) of the labeled amount of C13H10N2O4 is dissolved in 60 minutes.

(Official January 1, 2007)

Mobile phase, Internal standard preparation, Standard preparation, and Chromatographic system— Prepare as directed in the Assay under Thalidomide.

Assay preparation— Remove, as completely as possible, the contents of not fewer than 20 Capsules, and weigh accurately. Mix the combined contents, and transfer an accurately weighed portion of the powder, equivalent to about 50 mg of thalidomide, to a 100-mL volumetric flask, add 80 mL of acetonitrile to dissolve, and sonicate for about 20 minutes. Dilute with acetonitrile to volume, and mix. Transfer 20.0 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.

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 thalidomide (C13H10N2O4) in the portion of Capsules 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 of thalidomide to the internal standard obtained from the Assay preparation and the Standard preparation, respectively.