Proline

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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C₅H₉NO₂ 115.13 

l-Proline CAS RN®: 147-85-3; UNII: 9DLQ4CIU6V. 

1 DEFINITION 

Proline contains NLT 98.5% and NMT 101.5% of l-proline (C₅H₉NO₂), calculated on the dried basis. 

2 IDENTIFICATION 

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

3 ASSAY 

Procedure 

Sample: 100 mg of Proline 

Blank: Mix 3 mL of formic acid and 50 mL of glacial acetic acid. 

Titrimetric system 

(See Titrimetry 〈541〉.) 

Mode: Direct titration 

Titrant: 0.1 N perchloric acid VS 

Endpoint detection: Potentiometric 

Analysis: Dissolve the Sample in 3 mL of formic acid and 50 mL of glacial acetic acid. Titrate with the Titrant. Perform the Blank determination. 

Calculate the percentage of proline (C₅H₉NO₂) in the Sample taken: 

Result = {[(V_S − V_B) × N × F]/W} × 100 

V_S = volume of Titrant consumed by the Sample (mL) 

V_B = volume of Titrant consumed by the Blank (mL) 

N = actual normality of the Titrant (mEq/mL) 

F = equivalency factor, 115.1 mg/mEq 

W = Sample weight (mg) 

Acceptance criteria: 98.5%–101.5% on the dried basis 

4 IMPURITIES 

Residue on Ignition 〈281〉: NMT 0.4% 

Chloride and Sulfate, Chloride〈221〉 

Standard solution: 0.50 mL of 0.020 N hydrochloric acid 

Sample: 0.73 g of Proline 

Acceptance criteria: NMT 0.05% 

Chloride and Sulfate, Sulfate〈221〉 

Standard solution: 0.10 mL of 0.020 N sulfuric acid 

Sample: 0.33 g of Proline 

Acceptance criteria: NMT 0.03% 

Change to read: 

Iron 〈241〉, Procedures, Procedure 1 : NMT 30 ppm 

Related Compounds 

System suitability solution: 0.4 mg/mL each of USP l-Proline RS and USP l-Threonine RS in 0.1 N hydrochloric acid Standard solution: 0.05 mg/mL of USP l-Proline RS in 0.1 N hydrochloric acid. [Note—This solution has a concentration equivalent to 0.5% of that of the Sample solution.] 

Sample solution: 10 mg/mL of Proline in 0.1 N hydrochloric acid 

Chromatographic system 

(See Chromatography 〈621〉, Thin-Layer Chromatography.) 

Mode: TLC 

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture 

Application volume: 5 µL 

Developing solvent system: Butyl alcohol, glacial acetic acid, and water (3:1:1) 

Spray reagent: 2 mg/mL of ninhydrin in a mixture of butyl alcohol and 2 N acetic acid (95:5) 

System suitability 

Suitability requirements: The chromatogram of the System suitability solution exhibits two clearly separated spots. Analysis 

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

After air-drying the plate, spray with Spray reagent, and heat between 100° and 105° for 15 min. Examine the plate under white light. Acceptance criteria: Any secondary spot of the Sample solution is not larger or more intense than the principal spot of the Standard solution. 

Individual impurities: NMT 0.5% Total impurities: NMT 2.0% 

5 SPECIFIC TESTS 

Optical Rotation, Specific Rotation〈781S〉 Sample solution: 40 mg/mL in water Acceptance criteria: −84.3° to −86.3° 

Loss on Drying 〈731〉: Dry a sample at 105° for 3 h: it loses NMT 0.4% of its weight. 

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in well-closed containers. 

USP Reference Standards 〈11〉 

USP l-Proline RS 

USP l-Threonine RS