Printed on: Wed Feb 08 2023, 11:30:47 PM(EST) Status: Currently Official on 09-Feb-2023 DocId: GUID-AB40C46D-47A9-4B37-8E1B-B696B87BA61F_4_en-US

Printed by: Dang Van Vu Official Date: Official as of 01-May-2020 Document Type: DIETARY SUPPLEMENTS @2023 USPC
Do Not Distribute DOI Ref: xsp77 DOI: https://doi.org/10.31003/USPNF_M4468_04_01
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Table 1
5-Hydroxy- L-tryptophan
Time Solution A Solution B
(min) (%) (%)
0 95 5

2 95 5

37 35 65
C11H12N2O3 220.23
42 0 100
(S)-2-Amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid
CAS RN®: 4350-09-8. 47 0 100

DEFINITION 50 95 5
5-Hydroxy-L-tryptophan contains NLT 98.5% and NMT 60 95 5
101.5% of 5-hydroxy-L-tryptophan (C11H12N2O3), calculated
on the dried basis.
Standard solution: 1.0 µg/mL of USP
IDENTIFICATION 5-Hydroxy-l-tryptophan RS and 50 µg/mL of USP
l-Tryptophan RS in water
Change to read: Sample solution: 10.0 mg/mL of 5-Hydroxy-L-tryptophan
in water
• A. ▲SPECTROSCOPIC IDENTIFICATION TESTS á197ñ, Infrared Chromatographic system
Spectroscopy: 197K▲ (CN 1-May-2020) (See Chromatography á621ñ, System Suitability.)
ASSAY Mode: LC

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• PROCEDURE Detector: UV 220 nm
Sample: 200 mg of 5-Hydroxy-L-tryptophan Column: 4.6-mm × 25-cm; 5-µm packing L1
Blank: Mix 3 mL of formic acid and 50 mL of glacial Column temperature: 30°
acetic acid. Flow rate: 1 mL/min
Titrimetric system
ci Injection volume: 20 µL
(See Titrimetry á541ñ.) System suitability
Mode: Direct titration Sample: Standard solution
Titrant: 0.1 N perchloric acid VS [NOTE—The relative retention times for
Endpoint detection: Potentiometric 5-hydroxy-L-tryptophan and L-tryptophan are
Analysis 1.0 and 1.6, respectively.]
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Samples: Sample and Blank Suitability requirements
Dissolve the Sample in a mixture of 3 mL of formic acid and Relative standard deviation: NMT 5.0% for the
50 mL of glacial acetic acid, and titrate with the Titrant. 5-hydroxy-L-tryptophan and L-tryptophan peaks
Perform a Blank titration, and make any necessary Analysis
correction. Samples: Standard solution and Sample solution
Calculate the percentage of 5-hydroxy-L-tryptophan Calculate the percentage of each unspecified impurity in
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(C11H12N2O3) in the Sample taken: the portion of 5-Hydroxy-L-tryptophan taken:

Result = {[(V S − V B) × N × F]/W} × 100 Result = (r U/r S) × (C S/C U) × 100

VS = Titrant volume consumed by the Sample (mL) rU = peak response of each unspecified impurity
VB = Titrant volume consumed by the Blank (mL) from the Sample solution
N = actual normality of the Titrant (mEq/mL) rS = peak response of 5-hydroxy-L-tryptophan from
F = equivalency factor, 220.2 mg/mEq the Standard solution
W = Sample weight (mg) CS = concentration of USP 5-Hydroxy-l-tryptophan RS
in the Standard solution (µg/mL)
Acceptance criteria: 98.5%–101.5% on the dried basis CU = concentration of 5-Hydroxy-L-tryptophan in the
Sample solution (µg/mL)
IMPURITIES
• RESIDUE ON IGNITION á281ñ: NMT 0.2% Calculate the percentage of tryptophan in the portion of
• CHLORIDE AND SULFATE, Chloride á221ñ 5-Hydroxy-L-tryptophan taken:
Standard solution: 0.50 mL of 0.020 N hydrochloric acid
Sample: 0.73 g of 5-Hydroxy-L-tryptophan Result = (r U/r S) × (C S/C U) × 100
Acceptance criteria: NMT 0.05%
• CHLORIDE AND SULFATE, Sulfate á221ñ rU = peak response of tryptophan from the Sample
Standard solution: 0.10 mL of 0.020 N sulfuric acid solution
Sample: 0.33 g of 5-Hydroxy-L-tryptophan rS = peak response of tryptophan from the Standard
Acceptance criteria: NMT 0.03% solution
• ORGANIC IMPURITIES CS = concentration of USP l-Tryptophan RS in the
Solution A: 1 mL/L of trifluoroacetic acid in water Standard solution (µg/mL)
Solution B: 1 mL/L of trifluoroacetic acid in a mixture of CU = concentration of 5-Hydroxy-L-tryptophan in the
acetonitrile and water (80:20) Sample solution (µg/mL)
Mobile phase: See Table 1.
Acceptance criteria
Total impurities 1: NMT 0.01% of the total impurities
eluting prior to the 5-hydroxy-L-tryptophan peak

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Printed on: Wed Feb 08 2023, 11:30:47 PM(EST) Status: Currently Official on 09-Feb-2023 DocId: GUID-AB40C46D-47A9-4B37-8E1B-B696B87BA61F_4_en-US
Printed by: Dang Van Vu Official Date: Official as of 01-May-2020 Document Type: DIETARY SUPPLEMENTS @2023 USPC
Do Not Distribute DOI Ref: xsp77 DOI: https://doi.org/10.31003/USPNF_M4468_04_01
2

Total impurities 2: NMT 0.03% of the total impurities Acceptance criteria: 4.0–6.0
eluting after the 5-hydroxy-L-tryptophan peak. • LOSS ON DRYING á731ñ
[NOTE—Exclude the peak for tryptophan.] Analysis: Dry at 105° for 3 h.
Tryptophan: NMT 0.5% Acceptance criteria: NMT 2.0%
SPECIFIC TESTS ADDITIONAL REQUIREMENTS
• OPTICAL ROTATION, Specific Rotation á781ñ • PACKAGING AND STORAGE: Preserve in well-closed
Sample solution: 10 mg/mL in water containers.
Acceptance criteria: −30.0° to −38.0° • USP REFERENCE STANDARDS á11ñ
• PH á791ñ USP 5-Hydroxy-l-tryptophan RS
Sample solution: 10 mg/mL in water USP l-Tryptophan RS

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