Aspartame

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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l-Phenylalanine, N-l-α-aspartyl-, 1-methyl ester;

3-Amino-N-(α-carboxyphenethyl)succinamic acid N-methyl ester CAS RN®: 22839-47-0.

1 DEFINITION

Aspartame contains NLT 98.0% and NMT 102.0% of aspartame (C₁₄H₁₈N₂O₅), calculated on the dried basis.

2 IDENTIFICATION

Change to read:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197M (CN 1-May-2020)

[Note—Do not dry specimens.]

3 ASSAY

3.1 Procedure

Buffer: 0.05 M monobasic potassium phosphate, adjusted with phosphoric acid to a pH of 4.3

Mobile phase: Methanol and Buffer (18:82)

Diluent: Methanol and water (1:9)

System suitability solution: 0.1 mg/mL each of USP Aspartame Related Compound A RS and USP l-Phenylalanine RS in Diluent

Standard solution: 0.5 mg/mL of USP Aspartame RS in Diluent

Sample solution: 0.5 mg/mL of Aspartame in Diluent. [Note—Avoid heat and excessive holding times.]

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4.6-mm × 25-cm; 5-μm packing L1

Column temperature: 40°

Flow rate: 2 mL/min

Injection volume: 20 μL

Run time: 30 min

System suitability

Samples: System suitability solution and Standard solution

[Note—The relative retention times for the l-phenylalanine and aspartame related compound A peaks are 0.6 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 8 between the l-phenylalanine and aspartame related compound A peaks, System suitability solution

Tailing factor: NMT 1.5, Standard solution

Relative standard deviation: NMT 1.0%, Standard solution

3.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of aspartame (C₁₄H₁₈N₂O₅) in the portion of Aspartame taken:

Result = (r_U/r_S ) × (C_S /C_U ) × 100

r_U = peak area of aspartame from the Sample solution

r_S = peak area of aspartame from the Standard solution

C_S = concentration of USP Aspartame RS in the Standard solution (mg/mL)

C_U = concentration of Aspartame in the Sample solution (mg/mL)

Acceptance criteria: 98.0%–102.0% on the dried basis

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.2%

Limit of 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid

Mobile phase, Diluent, System suitability solution, and Chromatographic system: Proceed as directed in the Assay.

Standard solution: 75 μg/mL of USP Aspartame Related Compound A RS in Diluent

Sample solution: 5 mg/mL of Aspartame in Diluent. [Note—Avoid heat and excessive holding times.]

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 8 between the l-phenylalanine and aspartame related compound A peaks, System suitability solution

Tailing factor: NMT 2.0, Standard solution

Relative standard deviation: NMT 4.0%, Standard solution

4.1 Analysis

Samples: Standard solution and Sample solution

[Note—Continue the elution of the Sample solution for 45 min.]

Calculate the percentage of aspartame related compound A in the portion of Aspartame taken:

Result = (r_U/r_S ) × (C_S /C_U ) × 100

r_U = peak response of aspartame related compound A from the Sample solution

r_S = peak response of aspartame related compound A from the Standard solution

C_S = concentration of 5-benzyl-3,6-dioxo-2-piperazineacetic acid in the Standard solution (mg/mL)

C_U = concentration of Aspartame in the Sample solution (mg/mL)

Acceptance criteria: NMT 1.5%

4.2 Chromatographic Purity

Mobile phase, Diluent, System suitability solution, and Chromatographic system: Proceed as directed in the Assay.

Sample stock solution: Proceed as directed in the Sample solution in the test for Limit of 5-Benzyl-3,6-dioxo-2-piperazineacetic Acid.

Sample solution: 0.1 mg/mL of Aspartame from the Sample stock solution in Diluent

System suitability

Sample: System suitability solution

Suitability requirements

Resolution: NLT 8 between the l-phenylalanine and aspartame related compound A peaks, System suitability solution

Analysis

Samples: Sample stock solution and Sample solution

[Note—Continue the elution of the Sample stock solution for 45 min.]

Acceptance criteria: The sum of all of the peak responses of the Sample stock solution, excluding the 5-benzyl-3,6-dioxo-2-piperazineacetic acid and aspartame peak responses, is NMT the aspartame peak response of the Sample solution, corresponding to NMT 2.0% of chromatographic impurities.

5 SPECIFIC TESTS

5.1 Transmittance

Sample solution: 10 mg/mL of Aspartame in 2 N hydrochloric acid, prepared by means of sonication

Analysis: Determine the transmittance in a 1-cm cell at 430 nm with a suitable spectrophotometer.

Acceptance criteria: Transmittance of NLT 0.95, corresponding to an absorbance of NMT about 0.022

5.2 Optical Rotation, Specific Rotation 〈781S〉

Sample solution: 40 mg/mL in 15 N formic acid

Acceptance criteria: +14.5° to +16.5°, determined at 20° within 30 min after preparation of the Sample solution

5.3 Loss on Drying 〈731〉

Analysis: Dry at 105° for 4 h.

Acceptance criteria: NMT 4.5%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers.

USP Reference Standards 〈11〉

USP Aspartame RS

USP Aspartame Related Compound A RS

5-Benzyl-3,6-dioxo-2-piperazineacetic acid.

C₁₃H₁₄N₂O₄ 262.27

USP l-Phenylalanine RS