Isoleucine

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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C₆H₁₃NO₂ 131.17 

l-Isoleucine CAS RN®: 73-32-5; UNII: 04Y7590D77. 

1 DEFINITION 

Isoleucine contains NLT 98.5% and NMT 101.5% of l-isoleucine (C₆H₁₃NO₂), calculated on the dried basis

2 IDENTIFICATION 

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

3 ASSAY 

Procedure 

Sample: 130 mg of Isoleucine 

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 l-isoleucine (C₆H₁₃NO₂) in the Sample taken: 

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

V_S = Titrant volume consumed by the Sample (mL) 

V_B = Titrant volume consumed by the Blank (mL) 

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

F = equivalency factor, 131.2 mg/mEq 

W = Sample weight (mg) 

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

4 IMPURITIES 

Residue on Ignition 〈281〉: NMT 0.3% 

Chloride and Sulfate 〈221〉, Chloride 

Standard solution: 0.50 mL of 0.020 N hydrochloric acid 

Sample: 0.73 g of Isoleucine 

Acceptance criteria: NMT 0.05% 

Chloride and Sulfate 〈221〉, Sulfate 

Standard solution: 0.10 mL of 0.020 N sulfuric acid 

Sample: 0.33 g of Isoleucine 

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-Isoleucine RS and USP l-Valine RS in 0.1 N hydrochloric acid Standard solution: 0.05 mg/mL of USP l-Isoleucine 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 Isoleucine in 0.1 N hydrochloric acid 

Chromatographic system 

(See Chromatography 〈621〉, General Procedures, 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 

Sample: System suitability solution 

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 〈781S〉, Procedures, Specific Rotation 

Sample solution: 40 mg/mL in 6 N hydrochloric acid 

Acceptance criteria: +38.9° to +41.8° pH 〈791〉 

Sample solution: 10 mg/mL in water Acceptance criteria: 5.5–7.0 Loss on Drying 〈731〉 

Analysis: Dry at 105° for 3 h. 

Acceptance criteria: NMT 0.3%

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in well-closed containers. 

USP Reference Standards 〈11〉 

USP l-Isoleucine RS 

USP l-Valine RS