Tryptophan
C11H12N2O2 204.23
l-Tryptophan
[73-22-3].
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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.
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%
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. ]
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. ]
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
241:
NMT 30 ppm
• Heavy Metals, Method II 231:
NMT 15 ppm
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
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)
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.
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
11
USP l-Tryptophan RS 
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USP Tryptophan Related Compound A RS
3,3¢-[Ethylidenebis(1H-indole-1,3-diyl)]bis[2S)-2-aminopropanoic]acid.
C24H26N4O4 432.49
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
2-Acetamido-3-(1H-indol-3-yl)propanoic acid.
C13H14N2O3 246.3
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 4970
Pharmacopeial Forum: Volume No. 33(6) Page 1214