Mobile phase— Add 10mLof a 1in 4solution of tetrabutylammonium hydroxide in methanol to 200mLof water,and adjust with 1Mphosphoric acid to a pHof 7.5±0.1.Transfer this solution to a 1000-mLvolumetric flask,add 90mLof 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 Suitabilityunder Chromatography á621ñ).

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

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

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

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

Chromatographic system(see Chromatography á621ñ)— 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 0.8mLper 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 20µL)of the Standard solutionand the Test solutioninto 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 Cis the concentration,in mg per mL,of nitrilotriacetic acid in the Standard solution;and rUand rSare the peak responses obtained from the Test solutionand the Standard solution,respectively:not more than 0.1%is found.

Mobile phase— Prepare a mixture of equal volumes of 0.025Mmonobasic potassium phosphate and 0.025Mdibasic sodium phosphate.To 400mLof this solution add 600mLof methanol.Adjust the volume to 1000mLwith water,and adjust with 1Nhydrochloric acid to a pHof 5.0±0.1.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).Pass through a filter having a 0.45-µm porosity,and degas.

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

Chromatographic system(see Chromatography á621ñ)— 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 1mLper minute.Chromatograph about 20µLof 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 á621ñ,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 20µLof 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 rsis the sum of the peak responses of the individual impurities;and rtis the total of all of the peak responses in the chromatogram:not more than 3%is found.