Tryptophan

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

DOWNLOAD PDF HERE

C₁₁H₁₂N₂O₂     204.23

l-Tryptophan CAS RN: 73-22-3; UNII: 8DUH1N11BX.

1 DEFINITION

Tryptophan contains NLT 98.5% and NMT 101.5% of C₁₁H₁₂N₂O₂, as L-tryptophan, calculated on the dried basis.

2 IDENTIFICATION

SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197K

3 ASSAY

PROCEDURE

Sample solution: Place 200 mg of Tryptophan in a 125-mL flask. Dissolve in a mixture of 3 mL of formic acid and 50 mL of glacial acetic acid.

Analysis: Titrate with 0.1 N perchloric acid VS, determining the endpoint potentiometrically. Perform a blank determination, and make any necessary correction (see Titrimetry (541)). Each mL of 0.1 N perchloric acid is equivalent to 20.42 mg of C, H,N,O,

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

4 IMPURITIES

4.1 INORGANIC IMPURITIES

RESIDUE ON IGNITION (281): NMT 0.1%

CHLORIDE AND SULFATE, Chloride (221): A 0.73-g portion shows no more chloride than corresponds to 0.50 mL of 0.020 N hydrochloric acid (0.05%). [NOTE-Gently heat the sample preparation to dissolve, if necessary.]

CHLORIDE AND SULFATE, Sulfate (221): A 0.33-g portion shows no more sulfate than corresponds to 0.10 mL of 0.020 N sulfuric acid (0.03%).

[NOTE-Gently heat the sample preparation to dissolve, if necessary.]

Change to read:

IRON (241), Procedures, Procedure 1 (CN 1-JUN-2023): NMT 30 ppm

4.2 ORGANIC IMPURITIES

PROCEDURE 1

Solution A: Trifluoroacetic acid in water (1 mL/L)

Solution B: Trifluoroacetic acid in an acetonitrile and water solution (80:20) (1 mL/L trifluoroacetic acid solution)

Standard solution: 1.0 mg/L each of USP Tryptophan Related Compound A RS and USP Tryptophan Related Compound B RS in water

Sample solution: 10.0 mg/mL of tryptophan in water

System suitability solution: 1.0 mg/L of USP Tryptophan Related Compound B RS in water

Mobile phase: See the gradient table below.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 220 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1

Column temperature: 30°

Flow rate: 1 mL/min

Injection size: 20 µL

System suitability

Sample: System suitability solution

Suitability requirement

Relative standard deviation: NMT 5.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each unspecied impurity in the portion of Tryptophan taken:

Result = (r_u /r_s ) × (C_s /C_u ) × 100

r_u = peak area of each unspecied impurity in the Sample solution

r_s = peak area of tryptophan related compound B in the Standard solution

C_s = concentration of USP Tryptophan Related Compound B RS in the Standard solution (µg/mL)

C_u = concentration of Tryptophan in the Sample solution (µg/mL)

Acceptance criteria

Total impurities 1: NMT 0.01% of the total impurities eluting prior to the tryptophan peak

Total impurities 2: NMT 0.03% of the total impurities eluting after the tryptophan peak. [Note—Exclude the peak for tryptophan related compound B.]

Tryptophan related compound A: If a peak for tryptophan related compound A is observed in the Sample solution, then perform the test for Procedure 2: Limit of Tryptophan Related Compound A, below.

Procedure 2: Limit of Tryptophan Related Compound A

Solution A: 18 mM monobasic sodium phosphate, ltered and degassed (pH 2.5), and acetonitrile (9:1)

Solution B: 10 mM monobasic sodium phosphate, ltered and degassed (pH 2.5), and acetonitrile (1:1)

Solution C: Acetonitrile in water (7:3)

Standard solution: 0.1 mg/L of USP Tryptophan Related Compound A RS in water

Sample solution: 10.0 mg/mL of Tryptophan in water

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 216 nm

Column: 3.9-mm × 15-cm; 5-µm packing L1

Column temperature: 30°

Flow rate: 1 mL/min

Injection size: 20 µL

System suitability

Sample: Standard solution

Suitability requirement

Relative standard deviation: NMT 5.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of tryptophan related compound A in the portion of Tryptophan taken:

Result = (r_u /r_s ) × (C_s /C_u ) × 100

r_u = peak area of tryptophan related compound A in the Sample solution

r_s = peak area of tryptophan related compound A in the Standard solution

C_s = concentration of USP Tryptophan Related Compound A RS in the Standard solution (µg/mL)

C_u = concentration of Tryptophan in the Sample solution (µg/mL)

Acceptance criteria: NMT 10 ppm

5 SPECIFIC TESTS

Optical Rotation, Specific Rotation〈781S〉: −29.4° to −32.8°

Sample solution: 10 mg/mL, in water. [Note—Heat gently to dissolve, if necessary.]

pH 〈791〉: 5.5–7.0, in a solution (1 in 100)

Loss on Drying 〈731〉: Dry a sample at 105° for 3 h: it loses NMT 0.3% of its weight.

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers.

USP Reference Standards 〈11〉

USP l-Tryptophan RS

USP Tryptophan Related Compound A RS

(2S,2'S)-3,3'-[Ethane-1,1-diylbis(1H-indole-1,3-diyl)]bis(2-aminopropanoic acid).

C₂₄H₂₆N₄O₄          434.50

USP Tryptophan Related Compound B RS

2-Acetamido-3-(1H-indol-3-yl)propanoic acid.

C₁₃H₁₄N₂O₃          246.3