A: Infrared Absorption 197K—

B: The 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.

Mobile phase, System suitability preparation, and Chromatographic system— Prepare as directed in the Assay.

Test solution— Transfer about 125.0 mg of Leflunomide, accurately weighed, to a 50-mL volumetric flask, dissolve in 5 mL of acetonitrile, dilute with Mobile phase to volume, and mix.

Standard solution— Transfer about 12.5 mg of USP Leflunomide Related Compound A RS, accurately weighed, to a 100-mL volumetric flask, dissolve in 5 mL of acetonitrile, and dilute with Mobile phase to volume. Quantitatively dilute this solution with Mobile phase to obtain a final solution having a known concentration of about 0.25 µg per mL.

Procedure— Separately inject equal volumes (about 20 µL) of the Test solution and the Standard solution into the chromatograph, record the chromatograms, and measure the peak responses for leflunomide related compound A. Calculate the percentage of leflunomide related compound A in the portion of Leflunomide taken by the formula: in which CS is the concentration, in mg per mL, of USP Leflunomide Related Compound A RS in the Standard solution; W is the weight, in mg, of Leflunomide taken to prepare the Test solution; and rU and rS are the peak areas of leflunomide related compound A obtained from the Test solution and the Standard solution, respectively: not more than 0.01 % of leflunomide related compound A is found:

Mobile phase, System suitability preparation, and Chromatographic system— Prepare as directed in the Assay.

Test solution— Use the Assay preparation.

Standard solution— Dilute a volume of the Standard preparation, prepared as directed in the Assay, with Mobile phase to obtain a final solution having a concentration of about 0.5 µg per mL.

System sensitivity solution— Dilute a volume of the Standard solution with Mobile phase to obtain a final solution having a concentration of about 0.25 µg per mL.

Procedure— Separately inject equal volumes (about 20 µL) of the Test solution, the Standard solution, and the System sensitivity solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The signal-to-noise ratio for the peak corresponding to leflunomide in the System sensitivity solution is not less than 10. Disregard any peak with an area less than the leflunomide peak in the chromatogram obtained from the System 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 Table 1) in the portion of Leflunomide taken by the formula: in which CS is the concentration, in mg per mL, of USP Leflunomide RS in the Standard solution; W is the weight, in mg, of Leflunomide taken to prepare the Test solution; ri is the peak area response for each impurity obtained from the Test solution; and rS is the leflunomide peak area response obtained from the Standard solution.

Mobile phase— Prepare a filtered and degassed mixture of water, acetonitrile, and triethylamine (65:35:0.5). Adjust with phosphoric acid to a pH of 4. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Leflunomide RS in 5 mL of acetonitrile, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.5 mg per mL.

System suitability preparation— Dissolve accurately weighed quantities of USP Leflunomide RS, USP Leflunomide Related Compound B RS, and USP Leflunomide Related Compound C RS in acetonitrile, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having known concentrations of about 0.5 mg per mL, 0.15 mg per mL, and 0.05 mg per mL, respectively.

Assay preparation— Transfer about 25.0 mg of Leflunomide, accurately weighed, to a 50-mL volumetric flask, dissolve in 5 mL of acetonitrile, dilute with Mobile phase to volume, and mix. [note—Protect solutions from light.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 210-nm detector and a 4-mm × 12.5-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the System suitability preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.2 for leflunomide related compound B and 0.9 for leflunomide related compound C; and the resolution, R, between the leflunomide and leflunomide related compound C peaks is not less 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 responses for the leflunomide peak. Calculate the quantity, in mg, of C12H9F3N2O2 in the portion of Leflunomide taken by the formula: in which C is the concentration, in mg per mL, of USP Leflunomide RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.