Asparagine

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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C₄H₈N₂O₃ · H₂O 150.13

C₄H₈N₂O₃ 132.12

l-Asparagine;

l-α-Aminosuccinamic acid, monohydrate CAS RN®: 5794-13-8.

Anhydrous CAS RN®: 70-47-3.

1 DEFINITION

Asparagine is anhydrous, or contains one molecule of water of hydration. It contains NLT 95.5% and NMT 102.0% of asparagine (C H N O ), as l-asparagine, calculated on the dried basis.

2 IDENTIFICATION

2.1 A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K

[Note—Use USP Asparagine Anhydrous RS and USP Asparagine Monohydrate RS for the evaluation of the anhydrous and monohydrate forms of

Asparagine, respectively.]

2.2 B. Chromatographic Identity

Analysis: Examine the chromatograms obtained in the Assay.

Acceptance criteria: The retention time of the major peak of the Sample solution corresponds to that of the Standard solution.

3 ASSAY

3.1 Procedure

Mobile phase: Dissolve 13.61 g of potassium phosphate, monobasic and 2.16 g of sodium 1-octanesulfonate in about 900 mL of water.

Adjust with phosphoric acid to a pH of 2.2, and dilute with water to 1 L. Add 5.0 mL of acetonitrile, and mix well.

Diluent: Water

System suitability solution: 1.5 mg/mL of USP Asparagine Anhydrous RS and 0.075 mg/mL of USP Aspartic Acid RS in Diluent

Standard solution: 1.5 mg/mL of USP Asparagine Anhydrous RS in Diluent

Sample solution: 1.5 mg/mL of Asparagine Anhydrous in Diluent or 1.7 mg/mL of Asparagine Monohydrate in Diluent

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: 25°

Flow rate: 0.7 mL/min

Injection volume: 20 μL

Run time: 20 min

3.2 System suitability

Samples: System suitability solution and Standard solution

[Note—See Table 1 for relative retention times.]

Suitability requirements

Resolution: NLT 5 between the asparagine and aspartic acid peaks, System suitability solution

Tailing factor: NMT 2.0 determined from the asparagine peak, Standard solution

Relative standard deviation: NMT 1% determined from the asparagine peak, Standard solution

3.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of asparagine (C₄H₈N₂O₃) in the portion of sample taken:

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

r = peak area of asparagine from the Sample solution

r = peak area of asparagine from the Standard solution

C = concentration of USP Asparagine Anhydrous RS in the Standard solution (mg/mL)

C = concentration of Asparagine in the Sample solution (mg/mL)

Acceptance criteria: 95.5%–102.0% on the dried basis

4 IMPURITIES

Residue on Ignition 〈281〉

Sample: 1.0 g

Acceptance criteria: NMT 0.1%

Change to read:

Lead 〈251〉, Procedures, Procedure 1 (CN 1-Jun-2023)

Sample: 1 g

Control: 5 mL of Diluted standard lead solution (5 μg of lead)

Acceptance criteria: NMT 5 ppm

Organic Impurities

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

Sensitivity solution: 0.005 mg/mL of USP Asparagine Related Compound A RS, USP Asparagine Anhydrous RS, and USP Aspartic Acid RS in Diluent

Standard solution: 0.01 mg/mL of USP Asparagine Related Compound A RS, USP Asparagine Anhydrous RS, and USP Aspartic Acid RS in Diluent

Sample solution: 2.0 mg/mL of Asparagine Anhydrous in Diluent or 2.3 mg/mL of Asparagine Monohydrate in Diluent

System suitability

Samples: System suitability solution, Sensitivity solution, and Standard solution

[Note—See Table 1 for relative retention times.]

Suitability requirements

Resolution: NLT 5 between the asparagine and aspartic acid peaks, System suitability solution

Relative standard deviation: NMT 5.0% determined from the aspartic acid peak, Standard solution

Signal-to-noise ratio: NLT 10 determined from the aspartic acid peak, Sensitivity solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of asparagine related compound A in the portion of sample taken:

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

r_U = peak area of asparagine related compound A from the Sample solution

r_S = peak area of asparagine related compound A from the Standard solution

C_S = concentration of USP Asparagine Related Compound A RS in the Standard solution (mg/mL)

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

Calculate the percentage of aspartic acid in the portion of sample taken:

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

r_U = peak area of aspartic acid from the Sample solution

r_S = peak area of aspartic acid from the Standard solution

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

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

Calculate the percentage of each individual unidenti

ed impurity in the portion of sample taken:

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

r_U = peak area of each individual unidentied impurity from the Sample solution

r_S = peak area of asparagine from the Standard solution

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

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

Acceptance criteria: See Table 1.

^a 2,2'-(3,6-Dioxopiperazine-2,5-diyl)diacetamide.

5 SPECIFIC TESTS

Optical Rotation 〈781S〉, Procedures, Specific Rotation

Sample solution: 10 mg/mL, in 6 N hydrochloric acid

Acceptance criteria: +33.0° to +36.5°, measured at 20°

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms 〈62〉: The total aerobic microbial count is NMT 10³ cfu/g, and the total combined molds and yeasts count is NMT 10² cfu/g.

Loss on Drying 〈731〉

Sample: Dry a sample at 130° for 3 h.

Acceptance criteria

Anhydrous: NMT 1.0%

Monohydrate: 11.5%–12.5%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed, light-resistant containers. Store at room temperature.

Labeling: Label it to indicate whether it is anhydrous or the monohydrate.

USP Reference Standards 〈11〉

USP Asparagine Anhydrous RS

USP Asparagine Monohydrate RS

USP Asparagine Related Compound A RS

2,2'-(3,6-Dioxopiperazine-2,5-diyl)diacetamide.

C₈H₁₂N₄O₄ 228.21

USP Aspartic Acid RS