Melatonin
C13H16N2O2 232.28
N-Acetyl-5-methoxytryptamine;
N-(2-(5-Methoxy-1H-indol-3-yl)ethyl) acetamide
[73-31-4].
231
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C13H16N2O2 232.28
N-Acetyl-5-methoxytryptamine;
N-(2-(5-Methoxy-1H-indol-3-yl)ethyl) acetamide
[73-31-4].DEFINITION
Melatonin contains NLT 98.5% and NMT 101.5% of melatonin (C13H16N2O2), calculated on the dried basis.
IDENTIFICATION
• B. Ultraviolet Absorption 197U
197U
Analytical wavelength:
277 nm
Sample solution:
10 µg/mL of Melatonin in isopropyl alcohol
Acceptance criteria:
Meets the requirements. Absorptivities, calculated on the dried basis, do not differ by more than 3.0%.
• C. HPLC Identification Test
Analysis:
Proceed as directed in the Assay.
Acceptance criteria:
The retention time of the major peak of the Sample solution corresponds to that of the Standard solution.
ASSAY
• Procedure
Buffer:
0.5 g/L of monobasic potassium phosphate in water. Adjust with phosphoric acid to a pH of 3.5, and filter.
Mobile phase:
Acetonitrile and Buffer (25:75)
System suitability solution:
0.1 mg/mL of USP Melatonin RS and 0.02 mg/mL USP Melatonin Related Compound A RS in Mobile phase
Standard solution:
0.1 mg/mL of USP Melatonin RS in Mobile phase
Sample solution:
0.1 mg/mL of Melatonin in Mobile phase
Chromatographic system
Mode:
LC
Detector:
UV 222 nm
Column:
4.6-mm × 15-cm; 5-µm packing L1
Flow rate:
1.0 mL/min
Injection size:
10 µL
System suitability
Samples:
System suitability solution and Standard solution
[Note—The relative retention times for melatonin related compound A and melatonin are 0.4 and 1.0, respectively. ]
Suitability requirements
Resolution:
NLT 4 between melatonin and melatonin related compound A, System suitability solution
Relative standard deviation:
NMT 2.0%, Standard solution
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of melatonin in the portion of Melatonin taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak response from the Sample solution |
| rS | = | = peak response from the Standard solution |
| CS | = | = concentration of USP Melatonin RS in the Standard solution (mg/mL) |
| CU | = | = concentration of Melatonin in the Sample solution (mg/mL) |
Acceptance criteria:
98.5–101.5% on the dried basis
IMPURITIES
• Residue on Ignition 281:
NMT 0.1%
281:
NMT 0.1%
• Chloride and Sulfate, Chloride 221
221
Standard:
0.10 mL of 0.020 N hydrochloric acid
Sample:
0.36 g of Melatonin
Acceptance criteria:
NMT 0.02%
Delete the following:
231:
(Official 1-Dec-2015)
• Related Compounds
Solution A:
Acetonitrile
Solution B:
Use Buffer, prepared as directed in the Assay.
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 25 | 75 |
| 7 | 25 | 75 |
| 15 | 80 | 20 |
| 18 | 25 | 75 |
| 25 | 25 | 75 |
Diluent:
Mixture of Solution A and Solution B (25:75)
System suitability solution:
0.1 mg/mL of USP Melatonin RS and 0.02 mg/mL of USP Melatonin Related Compound A RS in Diluent
Standard solution:
5 µg/mL of USP Melatonin RS in Diluent
Sample solution:
1 mg/mL of Melatonin in Diluent
Chromatographic system
Mode:
LC
Detector:
UV 222 nm
Column:
4.6-mm × 15-cm; 5-µm packing L1
Flow rate:
1.0 mL/min
Injection size:
10 µL
System suitability
Sample:
System suitability solution
[Note—The relative retention times for melatonin related compound A and melatonin are 0.4 and 1.0, respectively. ]
Suitability requirements
Resolution:
NLT 4.0 between melatonin and melatonin related compound A
Relative standard deviation:
NMT 2.0% for the melatonin peak
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of each individual impurity in the portion of Melatonin taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak response of each individual impurity from the Sample solution |
| rS | = | = peak response of melatonin from the Standard solution |
| CS | = | = concentration of USP Melatonin RS in the Standard solution (mg/mL) |
| CU | = | = concentration of Melatonin in the Sample solution (mg/mL) |
Acceptance criteria
Individual impurities:
NMT 0.1%
Total impurities:
NMT 1.0%
SPECIFIC TESTS
• Loss on Drying 731
731
Analysis:
Dry a sample at 80
in a vacuum for 3 h.
in a vacuum for 3 h.
Acceptance criteria:
NMT 1.0%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight containers, protected from light.
• USP Reference Standards 11
11
USP Melatonin RS 
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USP Melatonin Related Compound A RS
2-(5-Methoxy-1H-indol-3-yl)ethanamine.
C11H14N2O 190.24
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2-(5-Methoxy-1H-indol-3-yl)ethanamine.
C11H14N2O 190.24
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Huy T. Dinh, M.S.
Senior Scientific Liaison (301) 816-8594 |
(DS2010) Monographs - Dietary Supplements and Herbal Medicines |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP38–NF33 Page 6146
Pharmacopeial Forum: Volume No. 37(4)