Melatonin 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 • A. Infrared Absorption <197K> • B. Ultraviolet Absorption <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 (See Chromatography <621>, System Suitability.) 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
IMPURITIES • Residue on Ignition <281>: NMT 0.1% • Chloride and Sulfate, Chloride  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:  • Heavy Metals <231>: NMT 20 µg/g (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
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 (See Chromatography <621>, System Suitability |
