Histidine

(his' ti deen).

C6H9N3O2155.15
l-Histidine

[71-00-1].

DEFINITION

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

IDENTIFICATION

• A. Infrared Absorption

197K

: The Sample and USP l-Histidine RS are previously recrystallized from 80% alcohol.

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 = {[(VS

VB) × N × F]/W} × 100

VS	=	= Titrant volume consumed by the Sample (mL)
VB	=	= 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

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%

• Iron

241

: NMT 30 ppm

• Heavy Metals, Method I

231

: NMT 15 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%

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.

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in well-closed containers.

• USP Reference Standards

USP l-Histidine RS

USP l-Proline RS

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question	Contact	Expert Committee
Monograph	Huy T. Dinh, M.S. Scientific Liaison 1-301-816-8594	(DS2010) Monographs - Dietary Supplements
Reference Standards	RS Technical Services 1-301-816-8129 rstech@usp.org

USP35–NF30 Page 3412

Pharmacopeial Forum: Volume No. 27(1) Page 1789