Mobile phase— Add 10 mL of a 1 in 4 solution of tetrabutylammonium hydroxide in methanol to 200 mL of water, and adjust with 1 M phosphoric acid to a pH of 7.5 ± 0.1. Transfer this solution to a 1000-mL volumetric flask, add 90 mL of methanol, dilute with water to volume, mix, pass through a filter having a 0.5-µm or finer porosity, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Cupric nitrate solution— Prepare a solution containing about 10 mg of cupric nitrate per mL.

Standard stock solution— Transfer about 25 mg of nitrilotriacetic acid, accurately weighed, to a 50-mL volumetric flask, dilute with dilute ammonium hydroxide (1 in 20) to volume, and mix.

Standard solution— Transfer 10 mL of the Standard stock solution to a 100-mL volumetric flask, and dilute with Cupric nitrate solution to volume. [NOTE—Prepare fresh on the day of use.]

Test solution— Transfer about 250 mg of Mebrofenin, accurately weighed, to a 25-mL volumetric flask, dilute with Cupric nitrate solution to volume, and mix. Sonicate, if necessary, to achieve complete solution. [NOTE—Prepare fresh on the day of use.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L7. The flow rate is about 0.8 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the resolution, R, between the major peak and any other peak is not less than 1.7. [NOTE—Peaks containing copper may be present.] The relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, and record the chromatograms. Measure the responses for the peaks in each chromatogram at the locus for copper nitrilotriacetic acid. Calculate the quantity, in mg, of nitrilotriacetic acid in the portion of Mebrofenin taken by the formula: in which C is the concentration, in mg per mL, of nitrilotriacetic acid in the Standard solution; and rU and rS are the peak responses obtained from the Test solution and the Standard solution, respectively: not more than 0.1% is found.

Mobile phase— Prepare a mixture of equal volumes of 0.025 M monobasic potassium phosphate and 0.025 M dibasic sodium phosphate. To 400 mL of this solution add 600 mL of methanol. Adjust the volume to 1000 mL with water, and adjust with 1 N hydrochloric acid to a pH of 5.0 ± 0.1. Make adjustments if necessary (see System Suitability under Chromatography 621). Pass through a filter having a 0.45-µm porosity, and degas.

Test solution— Transfer about 12.5 mg of Mebrofenin, accurately weighed, to a 25-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column containing packing L1. The flow rate is about 1 mL per minute. Chromatograph about 20 µL of the Test solution, and record the peak responses. The capacity factor, k¢, is not less than 1.2; and the effective plate number, neff, calculated by the formula 16[(t ta)/W]2, in which the terms are as defined under Chromatography 621, is not less than 200. The tailing factor is not more than 4.0, and the relative standard deviation of the mebrofenin peak responses for replicate injections is not more than 2.0%.

Procedure— Chromatograph about 20 µL of the Test solution, run the chromatograph for twice the elution time of the mebrofenin peak, and record the peak response for individual impurities and the total response for the entire chromatogram. Calculate the percentage of chromatographic impurities taken by the formula: in which rs is the sum of the peak responses of the individual impurities; and rt is the total of all of the peak responses in the chromatogram: not more than 3% is found.

(Official January 1, 2007)