5-benzyl-3,6-dioxo-2-piperazineacetic acid

The Diluent, Mobile Phase, Test Solution, and Chromatographic system used for this test method are the same as those described for the Limit determination of 5-benzyl-3, 6-dioxo-2-piperazineacetic acid. The procedure requires a Diluted Test Solution, which is prepared by pipetting 2.0 mL of the Test Solution into a 100-mL volumetric flask, diluting to volume with Diluent, and mixing. 

Assay Not less than 98.0% and not more than 102.0% of Ci4H18N205, calculated on the dried basis. 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid Not more than 1.5%. 

Perform a blank determination (see General Provisions), and make any necessary correction. Each milliliter of 0.1 N perchloric acid is equivalent to 29.43 mg of C14H18N205. 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid Mobile Phase Weigh and transfer 5.6 g of potassium phosphate monobasic into a 1-L flask, add 820 mL of water, and dissolve. Adjust the pH to 4.3 using phosphoric acid, add 180 mL of methanol, and mix. Filter through a 0.45-pim disk, and de-gas. 

Impurity Standard Preparation Transfer about 25 mg of USP 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid Reference Standard, accurately weighed, into a 100-mL volumetric flask. Add 10 mL of methanol, and dissolve. Dilute to volume with water, and mix. Pipet 15 mL of this solution into a 50-mL volumetric flask, dilute to volume with Diluting Solvent, and mix. Use a freshly prepared solution. 

Procedure Separately inject equal 20-p.L portions of Impurity Standard Preparation and Sample Preparation into the chromatograph, and record the chromatograms (the approximate retention time of 5-benzyl-3,6-dioxo-2-piperazineacetic acid is 4 min, and the approximate retention time of Aspartame is 11 min). Measure the peak area response of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in each chromatogram. Calculate the percentage of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in the sample by the formula... 

Other Related Impurities Proceed as directed in the test for 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid (above), except use the following in place of the Standard Preparation ... 

Procedure Inject about 20-pL portions of the Other Related Substances Standard Preparation and the Sample Preparation into the chromatograph, and record the chromatogram for a time equal to twice the retention time of Aspartame. In the chromatogram obtained from the Sample Preparation, the sum of the responses of all secondary peaks, other than that for 5-benzyl-3,6-dioxo-2-piperazineacetic acid, is not more than the response of the Aspartame peak obtained in the chromatogram from the Other Related Substances Standard Preparation. 

Calculation Calculate the percent 5-benzyl-3,6-dioxo-2-piperazineacetic acid in the sample taken by the formula... 

Benzyl-3,6-dioxo-2-piperazineacetic Acid (DKP) Not more than 0.5%. 

Procedure Separately inject equal 20-pL portions of the Standard Preparation and the Sample Preparation into the chromatograph, and record the chromatograms (the approximate retention time of 5-benzyl-3,6-dioxo-2-piperazineacetic acid is 4... 

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