Histidine

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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C₆H₉N₃O₂            155.15

l-Histidine CAS RN®: 71-00-1; UNII: 4QD397987E.

1 DEFINITION

Histidine contains NLT 98.5% and NMT 101.5% of l-histidine (C₆H₉N₃O₂), calculated on the dried basis.

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K: The Sample and USP l-Histidine RS are previously recrystallized from 80% alcohol.

3 ASSAY

Procedure

Sample: 150 mg of Histidine

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 very slowly with the Titrant. Perform the Blank determination.

Calculate the percentage of histidine (C₆H₉N₃O₂) in the Sample taken:

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

V_S = Titrant volume consumed by the Sample (mL)

V_B = Titrant volume consumed by the Blank (mL)

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

F = equivalency factor, 155.2 mg/mEq

W = Sample weight (mg)

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

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.4%

Chloride and Sulfate, Chloride 〈221〉

Standard solution: 0.50 mL of 0.020 N hydrochloric acid

Sample: 0.73 g of Histidine

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 Histidine

Acceptance criteria: NMT 0.03%

Change to read:

Iron 〈241〉, Procedures, Procedure 1 (CN 1-Jun-2023) : NMT 30 ppm

Related Compounds

System suitability solution: 0.4 mg/mL each of USP l-Histidine RS and USP l-Proline RS

Standard solution: 0.05 mg/mL of USP l-Histidine RS in water. [Note—This solution has a concentration equivalent to 0.5% of the Sample solution.]

Sample solution: 10 mg/mL of Histidine in water

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: 110 mg/mL in 6 N hydrochloric acid

Acceptance criteria: +12.6° to +14.0°

pH 〈791〉

Sample solution: 20 mg/mL solution

Acceptance criteria: 7.0–8.5

Loss on Drying 〈731〉: Dry a sample at 105° for 3 h: it loses NMT 0.2% of its weight.

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers.

USP Reference Standards 〈11〉

USP l-Histidine RS

USP l-Proline RS