Thalidomide

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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C₁₃H₁₀N₂O₄ 258.23

1H-Isoindole-1,3(2H)-dione, 2-(2,6-dioxo-3-piperidinyl)-, (±)-;

(±)-N-(2,6-Dioxo-3-piperidyl)phthalimide;

α-(N-Phthalimido)glutarimide CAS RN®: 50-35-1; UNII: 4Z8R6ORS6L.

1 DEFINITION

Thalidomide contains NLT 98.0% and NMT 101.5% of thalidomide (C₁₃H₁₀N₂O₄), calculated on the anhydrous basis.

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K

Add the following:

B. The retention time of the thalidomide peak of the Sample solution corresponds to that of the Standard solution, as obtained in the

Assay. (USP 1-Dec-2020)

3 ASSAY

Change to read:

Procedure

Solution A: Transfer 1 mL of phosphoric acid into a 100-mL volumetric flask and dilute with water to volume.

Mobile phase: Acetonitrile, water, and phosphoric acid (15: 85: 0.1)

Internal standard solution: 1.5 mg/mL of phenacetin in acetonitrile

Standard stock solution: 1 mg/mL of USP Thalidomide RS in acetonitrile. Sonicate to dissolve, if necessary.

Standard solution: 0.1 mg/mL of USP Thalidomide RS and 0.075 mg/mL of phenacetin prepared as follows. Transfer 10.0 mL of Standard stock solution and 5.0 mL of Internal standard solution to a 100-mL volumetric flask, add 10.0 mL of Solution A, and dilute with water to volume.

Sample stock solution: 1 mg/mL of Thalidomide in acetonitrile. Sonicate to dissolve, if necessary.

Sample solution: 0.1 mg/mL of Thalidomide and 0.075 mg/mL of phenacetin prepared as follows. Transfer 10.0 mL of Sample stock solution and 5.0 mL of Internal standard solution to a 100-mL volumetric flask, add 10.0 mL of Solution A, and dilute with water to volume.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 237 nm

Column: 3.9-mm × 15-cm; 4-μm packing L1

Flow rate: 1 mL/min

Injection volume: 20 μL

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 3.0 between thalidomide and phenacetin

Tailing factor: NMT 2.0

Relative standard deviation: NMT 1.0% for the response ratio of thalidomide to phenacetin

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of thalidomide (C₁₃H₁₀N₂O₄) in the portion of Thalidomide taken:

Result = (R_U /R_S ) × (C_S /C_U ) × 100

R_U = peak response ratio of thalidomide to phenacetin from the Sample solution

R_S = peak response ratio of thalidomide to phenacetin from the Standard solution

C_S = concentration of USP Thalidomide RS in the Standard solution (mg/mL)

C_U = concentration of Thalidomide in the Sample solution (mg/mL)

Acceptance criteria: 98.0%–101.5% on the anhydrous basis

4 IMPURITIES

Organic Impurities

Solution A: Acetonitrile, water, and phosphoric acid (5: 95: 0.1)

Solution B: Acetonitrile, water, and phosphoric acid (15: 85: 0.1)

Solution C: Acetonitrile and water (80:5)

Mobile phase: See Table 1.

Table 1

Diluent A: Acetonitrile, water, and phosphoric acid (50: 50: 0.1)

Diluent B: Transfer 1 mL of phosphoric acid into a 100-mL volumetric flask and dilute with water to volume.

Standard stock solution A: 1 mg/mL of phthalic acid prepared as follows. Transfer 100 mg of phthalic acid to a 100-mL volumetric flask, dissolve in 85 mL of Solution C, and dilute with acetonitrile to volume.

Standard stock solution B: 0.1 mg/mL of phthalic acid prepared as follows. Transfer Standard stock solution A to a suitable volumetric flask and dilute with acetonitrile to volume.

Standard stock solution C: 1 mg/mL of USP Thalidomide RS in acetonitrile. Sonicate to dissolve, if necessary.

Standard stock solution D: 0.002 mg/mL of phthalic acid and 0.02 mg/mL of USP Thalidomide RS prepared as follows. Pipet 2.0 mL of

Standard stock solution B and 2.0 mL of Standard stock solution C into a 100-mL volumetric flask, and dilute with Diluent A to volume.

Standard solution: 0.0002 mg/mL of phthalic acid and 0.002 mg/mL of USP Thalidomide RS prepared as follows. Pipet 10.0 mL of Standard stock solution D into a 100-mL volumetric flask, add 10.0 mL of Diluent B, and dilute with water to volume.

Sample stock solution: 2 mg/mL of Thalidomide in Diluent A. Sonicate to dissolve, if necessary.

Sample solution: 0.2 mg/mL of Thalidomide prepared as follows. Pipet 10.0 mL of Sample stock solution into a 100-mL volumetric flask, add 10.0 mL of Diluent B, and dilute with water to volume.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 218 nm

Column: 3.9-mm × 15-cm; 4-μm packing L1

Flow rate: 2 mL/min

Injection volume: 200 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for phthalic acid and thalidomide are about 0.35 and 1.0, respectively.]

Suitability requirements

Tailing factor: NMT 2.0 for phthalic acid and thalidomide

Relative standard deviation: NMT 2.0% for phthalic acid

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Thalidomide taken:

Result = (r_U /r_S ) × (C_S /C_U ) × 100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of phthalic acid from the Standard solution

C_S = concentration of phthalic acid in the Standard solution (mg/mL)

C_{​​​​​​​U} = concentration of Thalidomide in the Sample solution (mg/mL)

Acceptance criteria

Individual impurities: NMT 0.1%

Total impurities: NMT 0.3%

Change to read:

Limit of Glutamine (See (USP 1-Dec-2020) Ordinary Impurities 〈466〉)

Sample solution: 2 mg/mL of Thalidomide in acetonitrile

Diluent: Acetonitrile and water (1:1)

Standard solution: 0.1 mg/mL of glutamine in Diluent

Eluant: Methylene chloride, methanol, and acetic acid (75: 25: 0.05)

Application volume: 2 μL for Standard solution; 100 μL for Sample solution

Visualization: 4

Acceptance criteria: NMT 0.1%

5 SPECIFIC TESTS

Change to read:

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms 〈62〉: The total aerobic microbial count (USP 1-Dec-2020) is NMT 10³ cfu/g, and the total combined molds and yeasts count is NMT 10² cfu/g.

Water Determination 〈921〉, Method I, Method Ic

Solvent: Anhydrous dimethyl sulfoxide

Acceptance criteria: NMT 0.5%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers, protected from light, and store at controlled room temperature.

USP Reference Standards 〈11〉

USP Thalidomide RS