Tryptophan
(trip' toe fan).
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C11H12N2O2 204.23
l-Tryptophan     [73-22-3].
DEFINITION
Tryptophan contains NLT 98.5% and NMT 101.5% of C11H12N2O2, as l-tryptophan, calculated on the dried basis.
IDENTIFICATION
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 
•  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. ]
•  Iron 241: NMT 30 ppm
•  Heavy Metals, Method II 231: NMT 15 ppm
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.
Time
(min)
Solution A
(%)
Solution B
(%)
0 95 5
2 95 5
37 35 65
42 0 100
47 0 100
50 95 5
60 95 5
Chromatographic system 
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.
Time
(min)
Solution A
(%)
Solution B
(%)
Solution C
(%)
0 100 0 0
30 44 56 0
30.1 0 0 100
45 0 0 100
45.1 100 0 0
60 100 0 0
Chromatographic system 
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 Click to View Structure
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
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USP35–NF30 Page 4970
Pharmacopeial Forum: Volume No. 33(6) Page 1214