A: Infrared Absorption 197K—Do not dry specimens.

B: Ultraviolet Absorption 197U—

Test solution: 10 mg per mL, in 0.05 M sodium carbonate.

pH 6.0 Buffer solution , Mobile phase, System suitability solution, and Chromatographic system—Proceed as directed in the Assay.

Standard preparation— Dissolve an accurately weighed quantity of USP Methotrexate RS in Mobile phase to obtain a solution having a known concentration of about 5 µg per mL.

Test preparation— Transfer about 100 mg of Methotrexate, accurately weighed, to a 100-mL volumetric flask, dissolve in Mobile phase, with the aid of sonication or shaking if necessary, dilute with Mobile phase to volume, and mix.

Procedure— [NOTE—Use peak areas where peak responses are indicated.] Separately inject equal volumes (about 10 µL) of the Standard preparation and the Test preparation into the chromatograph, and allow the Test preparation to elute for not less than three times the retention time of methotrexate. Record the chromatograms, and measure the peak responses. The sum of all of the peak responses, other than that of methotrexate, is not more than four times the methotrexate response from the Standard preparation (2.0%), and no single peak response is greater than that of the methotrexate response from the Standard preparation (0.5%).

Solvent: dimethyl sulfoxide.

(Official January 1, 2007)

pH 6.0 Buffer solution— Prepare a mixture of 0.2 M dibasic sodium phosphate and 0.1 M citric acid (630:370). Adjust if necessary with 0.1 M citric acid or 0.2 M dibasic sodium phosphate to a pH of 6.0.

Mobile phase— Prepare a filtered and degassed solution of pH 6.0 buffer solution and acetonitrile (90:10). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Methotrexate RS in Mobile phase to obtain a solution having a known concentration of about 100 µg per mL.

Assay preparation— Transfer about 25 mg of Methotrexate, accurately weighed, to a 250-mL volumetric flask, dissolve in Mobile phase, dilute with Mobile phase to volume, and mix.

System suitability solution— Prepare a solution in Mobile phase containing about 0.1 mg per mL each of USP Methotrexate RS and folic acid.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 302-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.2 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.35 for folic acid and 1.0 for methotrexate, the resolution, R, between the folic acid and methotrexate peaks is not less than 8.0, and the relative standard deviation for replicate injections is not more than 2.5% for methotrexate.

Procedure— Separately inject equal volumes (about 10 µL) of the Assay preparation and the Standard preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C20H22N8O5 in the portion of Methotrexate taken by the formula: in which C is the concentration, in µg per mL, of USP Methotrexate RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.