Leflunomide

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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C₁₂H₉F₃N₂O₂ 270.21

4-Isoxazolecarboxamide, 5-methyl-N-[4-(trifluoromethyl) phenyl]-;

α,α,α-Trifluoro-5-methyl-4-isoxazolecarboxy-p-toluidide CAS RN®: 75706-12-6; UNII: G162GK9U4W.

1 DEFINITION

Leflunomide contains NLT 98.0% and NMT 102.0% of C₁₂H₉F₃N₂O₂, calculated on the dried basis.

2 IDENTIFICATION

Change to read:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K (CN 1-May-2020)

Sample: Dry the substance for 10 min at 130°.

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

3 ASSAY

Procedure

Mobile phase: Acetonitrile, triethylamine, and water (70:1:130). Adjust with phosphoric acid to a pH of 4.

Standard solution: 0.5 mg/mL of USP Leflunomide RS in acetonitrile and Mobile phase (1:9). [Note—First dissolve in acetonitrile. Protect solutions from light.]

System suitability solution: 0.5 mg/mL of USP Leflunomide RS, 0.15 mg/mL of USP Leflunomide Related Compound B RS, and 0.05 mg/mL of USP Leflunomide Related Compound C RS in Mobile phase. [Note—Dissolve the Reference Standards in acetonitrile, and dilute with Mobile phase.]

Sample solution: 0.5 mg/mL of Leflunomide in acetonitrile and Mobile phase (1:9). [Note—First dissolve in acetonitrile. Protect solutions from light.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4-mm × 12.5-cm; packing L1

Flow rate: 1 mL/min

Injection size: 20 μL

System suitability

Sample: System suitability solution

[Note—The relative retention times for leflunomide related compound B and leflunomide related compound C are 0.2 and 0.9, respectively.]

Suitability requirements

Resolution: NLT 1.0 between the leflunomide and leflunomide related compound C peaks

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of C₁₂H₉F₃N₂O₂ in the portion of Leflunomide taken:

Result = (r_U/r_S)x(C_S/C_U) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

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

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

Acceptance criteria: 98.0%–102.0% on the dried basis

4 IMPURITIES

Inorganic Impurities

Residue on Ignition 〈281〉: NMT 0.1%

Change to read:

Organic Impurities

Procedure 1: Limit of Leflunomide Related Compound A

Mobile phase, System suitability solution, and Chromatographic system: Proceed as directed in the Assay.

Standard stock solution: 0.125 mg/mL of USP Leflunomide Related Compound A RS, in acetonitrile and Mobile phase (1:19)

Standard solution: 0.5 μg/mL of USP Leflunomide Related Compound A RS, from the Standard stock solution in Mobile phase

Sample solution: 2.5 mg/mL of Leflunomide, in acetonitrile and Mobile phase (1:9)

Injection size: 20 μL

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of leflunomide related compound A in the portion of Leflunomide taken:

Result = (r_U/r_S)x(C_S/C_U) × 100

r_U= peak area of leflunomide related compound A from the Sample solution

r_S = peak area of leflunomide related compound A from the Standard solution

C_S = concentration of USP Leflunomide Related Compound A RS in the Standard solution (mg/mL)

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

Acceptance criteria: NMT 0.02 %

Procedure 2

Mobile phase, Sample solution, System suitability solution, and Chromatographic system: Proceed as directed in the Assay.

Standard stock solution: Proceed as directed in the Standard solution in the Assay.

Standard solution: 0.5 μg/mL of USP Leflunomide RS from the Standard stock solution in Mobile phase▲ (ERR 1-Feb-2019)

Sensitivity solution: 0.25 μg/mL of Leflunomide, from the Standard solution in Mobile phase

System suitability

Samples: System suitability solution and Sensitivity solution

Resolution: NLT 1.0 between leflunomide and leflunomide related compound C

Signal-to-noise ratio: NLT 10, Sensitivity solution

Analysis

Samples: Standard solution and Sample solution

[Note—Disregard any peak with an area less than the leflunomide peak from the Sensitivity solution. Continue the elution for two times the retention time of the leflunomide peak.]

Calculate the percentage of each related compound and any unknown impurity (see Impurity Table 1) in the portion of Leflunomide taken:

Result = (r_U/r_S)x(C_S/C_U) × 100

r_U = peak area for each impurity from the Sample solution

r_S = peak area of leflunomide from the Standard solution

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

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

Impurity Table 1

5 SPECIFIC TESTS

Melting Range or Temperature 〈741〉: 164°–168°

Loss on Drying 〈731〉: Dry a sample in a vacuum over diphosphorus pentoxide at 60° for 4 h: it loses NMT 0.5% of its weight.

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in a well-closed container. Store at a temperature not exceeding 30°.

USP Reference Standards 〈11〉

USP Leflunomide RS

USP Leflunomide Related Compound A RS

USP Leflunomide Related Compound B RS

USP Leflunomide Related Compound C RS