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Printed by: Le Tran Official Date: Official as of 01-May-2020 Document Type: DIETARY SUPPLEMENTS @2021 USPC
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Solution B: 1 mL/L of trifluoroacetic acid in a mixture of

5-Hydroxy- L-tryptophan acetonitrile and water (80:20)
Mobile phase: See Table 1.

Table 1
Time Solution A Solution B
(min) (%) (%)
0 95 5

2 95 5

37 35 65

42 0 100
C11H12N2O3 220.23
47 0 100
(S)-2-Amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid
[4350-09-8]. 50 95 5

DEFINITION 60 95 5
5-Hydroxy-L-tryptophan contains NLT 98.5% and NMT
101.5% of 5-hydroxy-L-tryptophan (C11H12N2O3), calculated Standard solution: 1.0 µg/mL of USP
on the dried basis. 5-Hydroxy-L-tryptophan RS and 50 µg/mL of USP
L-Tryptophan RS in water
IDENTIFICATION
Sample solution: 10.0 mg/mL of 5-Hydroxy-L-tryptophan

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Change to read: in water
Chromatographic system
• A. ▲SPECTROSCOPIC IDENTIFICATION TESTS á197ñ, Infrared (See Chromatography á621ñ, System Suitability.)
Spectroscopy: 197K▲ (CN 1-May-2020) Mode: LC
ASSAY Detector: UV 220 nm
• PROCEDURE
Sample: 200 mg of 5-Hydroxy-L-tryptophan
Blank: Mix 3 mL of formic acid and 50 mL of glacial
ci Column: 4.6-mm × 25-cm; 5-µm packing L1
Column temperature: 30°
Flow rate: 1 mL/min
acetic acid. Injection volume: 20 µL
Titrimetric system System suitability
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(See Titrimetry á541ñ.) Sample: Standard solution
Mode: Direct titration [NOTE—The relative retention times for
Titrant: 0.1 N perchloric acid VS 5-hydroxy-L-tryptophan and L-tryptophan are
Endpoint detection: Potentiometric 1.0 and 1.6, respectively.]
Analysis Suitability requirements
Samples: Sample and Blank Relative standard deviation: NMT 5.0% for the
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Dissolve the Sample in a mixture of 3 mL of formic acid and 5-hydroxy-L-tryptophan and L-tryptophan peaks
50 mL of glacial acetic acid, and titrate with the Titrant. Analysis
Perform a Blank titration, and make any necessary Samples: Standard solution and Sample solution
correction. Calculate the percentage of each unspecified impurity in
Calculate the percentage of 5-hydroxy-L-tryptophan the portion of 5-Hydroxy-L-tryptophan taken:
(C11H12N2O3) in the Sample taken: Result = (r U/r S) × (C S/C U) × 100
Result = {[(V S − V B) × N × F]/W} × 100 rU = peak response of each unspecified impurity
from the Sample solution
VS = Titrant volume consumed by the Sample (mL) rS = peak response of 5-hydroxy-L-tryptophan from
VB = Titrant volume consumed by the Blank (mL) the Standard solution
N = actual normality of the Titrant (mEq/mL) CS = concentration of USP 5-Hydroxy-L-tryptophan RS
F = equivalency factor, 220.2 mg/mEq in the Standard solution (µg/mL)
W = Sample weight (mg) CU = concentration of 5-Hydroxy-L-tryptophan in the
Sample solution (µg/mL)
Acceptance criteria: 98.5%–101.5% on the dried basis
IMPURITIES Calculate the percentage of tryptophan in the portion of
• RESIDUE ON IGNITION á281ñ: NMT 0.2% 5-Hydroxy-L-tryptophan taken:
• CHLORIDE AND SULFATE, Chloride á221ñ
Standard solution: 0.50 mL of 0.020 N hydrochloric acid Result = (r U/r S) × (C S/C U) × 100
Sample: 0.73 g of 5-Hydroxy-L-tryptophan
rU = peak response of tryptophan from the Sample
Acceptance criteria: NMT 0.05%
solution
• CHLORIDE AND SULFATE, Sulfate á221ñ
rS = peak response of tryptophan from the Standard
Standard solution: 0.10 mL of 0.020 N sulfuric acid
solution
Sample: 0.33 g of 5-Hydroxy-L-tryptophan CS = concentration of USP L-Tryptophan RS in the
Acceptance criteria: NMT 0.03% Standard solution (µg/mL)
• ORGANIC IMPURITIES CU = concentration of 5-Hydroxy-L-tryptophan in the
Solution A: 1 mL/L of trifluoroacetic acid in water Sample solution (µg/mL)

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Printed by: Le Tran Official Date: Official as of 01-May-2020 Document Type: DIETARY SUPPLEMENTS @2021 USPC
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Acceptance criteria Acceptance criteria: 4.0–6.0

Total impurities 1: NMT 0.01% of the total impurities • LOSS ON DRYING á731ñ
eluting prior to the 5-hydroxy-L-tryptophan peak Analysis: Dry at 105° for 3 h.
Total impurities 2: NMT 0.03% of the total impurities Acceptance criteria: NMT 2.0%
eluting after the 5-hydroxy-L-tryptophan peak.
[NOTE—Exclude the peak for tryptophan.] ADDITIONAL REQUIREMENTS
Tryptophan: NMT 0.5% • PACKAGING AND STORAGE: Preserve in well-closed
containers.
SPECIFIC TESTS • USP REFERENCE STANDARDS á11ñ
• OPTICAL ROTATION, Specific Rotation á781ñ USP 5-Hydroxy-L-tryptophan RS
Sample solution: 10 mg/mL in water USP L-Tryptophan RS
Acceptance criteria: −30.0° to −38.0°
• PH á791ñ
Sample solution: 10 mg/mL in water

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