Test solution: 40mg per mL,in 15Nformic acid.

Diluent— Prepare a mixture of water and methanol (9:1).

Mobile phase— Prepare a filtered and degassed solution by dissolving 5.6g of monobasic potassium phosphate in 820mLof water in a 1-liter volumetric flask,adjusting with phosphoric acid to a pHof 4.3,diluting with methanol to volume,and mixing.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Dissolve an accurately weighed quantity of USP Aspartame Related Compound A RSin Diluent,and dilute quantitatively with Diluentto obtain a solution having a known concentration of about 75µg per mL.

Test solution— Transfer 50mg of Aspartame,accurately weighed,to a 10-mLvolumetric flask,dissolve in and dilute with Diluentto volume,and mix.[NOTE—Avoid heat and excessive holding times.]

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 210-nm detector and 4.6-mm ×25-cm column containing packing L1.The flow rate is about 2mLper minute.The column temperature is maintained at 40.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the tailing factor is not more than 2.0,and the relative standard deviation for replicate injections is not more than 4.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of aspartame related compound Ain the portion of Aspartame taken by the formula: in which Cis the concentration,in µg per mL,of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in the Standard solution;Wis the weight,in mg,of Aspartame;and rUand rSare the peak responses of aspartame related compound Aobtained from the Test solutionand the Standard solution,respectively:not more than 1.5%is found.

Diluent ,Mobile phase,Test solution,and Chromatographic system—Proceed as directed under Limit of 5-benzyl-3,6-dioxo-2-piperazineacetic acid.

Diluted test solution— Pipet 2.0mLof the Test solutioninto a 100-mLvolumetric flask,dilute to volume with Diluent,and mix.

Procedure— Inject equal volumes (about 20µL)of the Test solutionand the Diluted test solutioninto the chromatograph,record the chromatograms,and measure the peak responses.[NOTE—Continue the elution of the Test solution for twice the retention time of the aspartame peak.]The sum of the responses of all the peaks in the chromatogram of the Test solution,excluding the 5-benzyl-3,6-dioxo-2-piperazineacetic acid and aspartame peak responses,is not greater than the aspartame peak response obtained from the Diluted test solution,corresponding to not more than 2.0%of chromatographic impurities.

0.1N Perchloric acid— Use perchloric acid,tenth-normal (in glacial acetic acid)as specified under Volumetric Solutionsin the section Reagents,Indicators,and Solutions,but in the standardization,titrate to a green endpoint.

Procedure— Transfer about 300mg of Aspartame,accurately weighed,to a 150-mLbeaker,dissolve in 1.5mLof anhydrous formic acid,and add 60mLof glacial acetic acid.Add crystal violet TS,and immediately titrate with 0.1N Perchloric acidto a green endpoint.Perform a blank determination,and make any necessary correction.Each mLof 0.1Nperchloric acid is equivalent to 29.43mg of C14H18N2O5.[NOTE—Ablank titration exceeding 0.1mLmay be due to excessive water content,and may cause loss of visual endpoint sensitivity.]