L-Tryptophan CAS 73-22-3 (H-Trp-OH) Assay 98.5~101.5% High Quality

L-Tryptophan (H-Trp-OH) (CAS: 73-22-3) AJI 97 Test Method
Identification: Compare the infrared absorption spectrum of the sample with that of the standard by potassium bromide disc method.
Specific Rotation [α]20/D: Dried Sample, C=1, H2O dissolve by warming
State of Solution (Transmittance): 0.5g in 20ml of 2 mol/L HCl, spectrophotometer, 430nm, 10mm cell thickness.
Chloride (Cl): 0.7g, A-1, ref: 0.40ml of 0.01mol/L HCl
Ammonium (NH4): B-2
Sulfate (SO4): 1.2g, (1), ref: 0.50ml of 0.005mol/L H2SO4
Iron (Fe): 0.75g, (2), ref: 1.5ml of Iron Std. (0.01mg/ml)
Heavy Metals (Pb): 2.0g, (4), ref: 2.0ml of Pb Std. (0.01mg/ml)
Arsenic (As2O3): 2.0g, (1), ref: 2.0ml of As2O3 Std.
Other Amino Acids: H2O, Dissolve by warming (40℃) Test Sample: 50μg, A-6-a, control: L-Trp 0.25μg
Loss on Drying: at 105℃ for 3 hours.
Residue on Ignition (Sulfated): AJI Test 13
Assay: Dried sample, 200mg, (1), 3ml of formic acid, 50ml of glacial acetic acid, 0.1mol/L HCLO4 1ml=20.423mg C11H12N2O2
pH Test: 1.0g in 100ml of H2O

L-Tryptophan (H-Trp-OH) (CAS: 73-22-3) USP35 Test Method
DEFINITION
Tryptophan contains NLT 98.5% and NMT 101.5% of C11H12N2O2, as L-Tryptophan, calculated on the dried basis.
IDENTIFICATION
A. INFRARED ABSORPTION <197K>
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 C11H12N2O2.
Acceptance criteria: 98.5%~101.5% on the dried basis
IMPURITIES
Inorganic Impurities
RESIDUEON 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.
IRON <241>: NMT 30ppm
HEAVY METALS, Method II <231>: NMT 15ppm
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 unspecified impurity in the portion of Tryptophan taken:
Result = (rU/rS) × (CS/CU) × 100
rU = peak area of each unspecified impurity in the Sample solution
rS = peak area of tryptophan related compound B in the Standard solution
CS = concentration of USP Tryptophan Related Compound B RS in the Standard solution (µg/mL)
CU = 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, filtered and degassed (pH 2.5), and acetonitrile (9:1)
Solution B: 10 mM monobasic sodium phosphate, filtered 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
Mobile phase: See the gradient table below

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 = (rU/rS) × (CS/CU) × 100
rU = peak area of tryptophan related compound A in the Sample solution
rS = peak area of tryptophan related compound A in the Standard solution
CS = concentration of USP Tryptophan Related Compound A RS in the Standard solution (µg/mL)
CU = concentration of Tryptophan in the Sample solution (µg/mL)
Acceptance criteria: NMT 10 ppm
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.
ADDITIONAL REQUIREMENTS
PACKAGING AND STORAGE: Preserve in well-closed containers
USP REFERENCE STANDARDS <11>
USP L-Tryptophan RS
USP Tryptophan Related Compound A RS
3,3′-[Ethylidenebis(1H-indole-1,3-diyl)]bis[2S)-2-aminopropanoic]acid. C24H26N4O4 432.49
USP Tryptophan Related Compound B RS
2-Acetamido-3-(1H-indol-3-yl)propanoic acid. C13H14N2O3 246.3