Edition: BP 2025 (Ph. Eur. 11.6 update)
Action and use
Treatment of sleep onset insomnia.
DEFINITION
Melatonin Capsules contain Melatonin.
The capsules comply with the requirements stated under Capsules and with the following requirements. Where appropriate, the capsules also comply with the requirements stated under Unlicensed Medicines.
Content of melatonin, C13H16N2O2
90.0 to 105.0% of the stated amount.
IDENTIFICATION
A. The light absorption, Appendix II B, of solution (1) obtained in the test for Dissolution, in the range 200 to 350 nm, exhibits a maximum at 278 nm.
B. In the Assay, the retention time of the principal peak in the chromatogram obtained with solution (1) is the same as that of the principal peak in the chromatogram obtained with solution (2).
TESTS
Dissolution
Comply with the dissolution test for tablets and capsules, Appendix XII B1.
TEST CONDITIONS
(a) Use Apparatus 2, rotating the paddle at 50 revolutions per minute.
(b) Use 900 mL of 0.1M hydrochloric acid, at a temperature of 37°, as the medium.
PROCEDURE
(1) After 45 minutes withdraw a 10-mL sample of the medium, filter and dilute the filtered solution, if necessary, with sufficient 0.1M hydrochloric acid to produce a solution expected to contain about 0.0001% w/v of Melatonin. Measure the absorbance of the filtered sample at the maximum at 278 nm, Appendix II B, using 0.1M hydrochloric acid in the reference cell.
(2) Measure the absorbance of a 0.0001% w/v solution of melatonin BPCRS in 0.1M hydrochloric acid using 0.1M
hydrochloric acid in the reference cell.
DETERMINATION OF CONTENT
Calculate the total content of melatonin, C13H16N2O2, in the medium from the absorbances obtained and using the declared content of C13H16N2O2 in melatonin BPCRS.
LIMITS
The amount of melatonin released is not less than 75% (Q) of the stated amount.
Related substances
Carry out the method for liquid chromatography, Appendix III D, using the following solutions in a mixture of 20 volumes of acetonitrile and 80 volumes of a 0.245% w/v solution of potassium dihydrogen orthophosphate, previously adjusted to pH 3.0 with 20% v/v orthophosphoric acid.
(1) To a quantity of the contents of the capsules containing 25 mg of Melatonin add 15 mL, mix with the aid of ultrasound for 10 minutes and then shake vigorously for 60 minutes. Dilute to 25 mL, mix well, filter and dilute 1 volume of the filtrate to 2 volumes.
(2) 0.0005% w/v of melatonin BPCRS.
(3) Dilute a mixture of 10 volumes of solution (2) and 1 volume of a 0.025% w/v solution of 5-methoxytryptamine BPCRS to 100 volumes.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 µm) (Zorbax Agilent Eclipse XDB is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 0.25 mL per minute.
(d) Use a column temperature of 40°.
(e) Use a detection wavelength of 278 nm.
(f) Inject 20 µL of each solution.
MOBILE PHASE
Mobile phase A 0.245% w/v of potassium dihydrogen orthophosphate, adjusted to pH 3.0 with 20% v/v orthophosphoric acid.
Mobile phase B acetonitrile.
| Time (Minutes) | Mobile phase A (% v/v) | Mobile phase B (% v/v) | Comment |
| 0-20 | 90→70 | 10→30 | linear gradient |
| 20-36 | 70→30 | 30→70 | linear gradient |
| 36-40 | 30→90 | 70→10 | linear gradient |
| 40-45 | 90 | 10 | re-equilibration |
When the chromatograms are recorded under the prescribed conditions, the retention time of melatonin is about 37.8 minutes and the retention of 5-methoxytryptamine relative to that of melatonin is about 0.63.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the resolution between the peaks due to melatonin and 5-methoxytryptamine is at least 12.0.
LIMITS
In the chromatogram obtained with solution (1):
the area of any peak due to 5-methoxytryptamine is not greater than the area of the peak due to 5-methoxytryptamine in the chromatogram obtained with solution (3) (0.5%);
the area of any other secondary peak is not greater than 0.1 times the area of the principal peak in the chromatogram obtained with solution (2) (0.1%);
the sum of total impurities is not more than 1.0%.
Disregard any peak with an area less than 0.5 times the area of the peak due to melatonin in the chromatogram obtained with solution (3) (0.05%).
Uniformity of content
Capsules containing less than 2 mg and/or less than 2% w/w of Melatonin comply with the requirements stated under Capsules using the following method of analysis.
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) Add 20 mL of methanol (30%) to the contents and shell of one capsule, mix with the aid of ultrasound for 10 minutes and then shake for 60 minutes. Dilute 1 volume of the resulting solution to 2 volumes with methanol (30%). Dilute the resulting solution, if necessary, with sufficient methanol (30%) to produce a solution containing 0.0025% w/v of Melatonin.
(2) Shake 25 mg of melatonin BPCRS with 15 mL of methanol (30%) for 10 minutes and add sufficient methanol (30%) to produce 25 mL. Dilute 1 volume of the resulting solution to 10 volumes with methanol (30%) and further dilute 1 volume to 4 volumes with methanol (30%).
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm × 3 mm) packed with end-capped octadecylsilyl silica gel for chromatography (4 µm) (Phenomenex Synergi-Hydro RP is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1 mL per minute.
(d) Use a column temperature of 40°.
(e) Use a detection wavelength of 285 nm.
(f) Inject 20 µL of each solution.
MOBILE PHASE
400 volumes of methanol and 600 volumes of water containing 1 volume of orthophosphoric acid.
DETERMINATION OF CONTENT
Calculate the content of C13H16N2O2 in each capsule using the declared content of C13H16N2O2 in melatonin BPCRS.
ASSAY
For capsules containing less than 2 mg and/or less than 2% w/w of Melatonin Use the average of the individual results obtained in the test for Uniformity of content. For capsules containing 2 mg or more and 2% w/w or more of Melatonin
Weigh the contents of 20 capsules. Mix and powder if necessary. Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) To a quantity of the mixed capsule contents containing 20 mg of Melatonin add 20 mL of methanol (30%), mix with the aid of ultrasound for 10 minutes and then shake for 60 minutes. Dilute 1 volume of the resulting solution to 10 volumes with methanol (30%).
(2) Shake 25 mg of melatonin BPCRS with 15 mL of methanol (30%) for 10 minutes; add sufficient methanol (30%) to produce 25 mL and dilute 1 volume to 10 volumes with methanol (30%).
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Uniformity of content may be used.
DETERMINATION OF CONTENT
Calculate the content of C13H16N2O2 in the capsules using the declared content of C13H16N2O2 in melatonin BPCRS.
STORAGE
Melatonin Capsules should be protected from light.
IMPURITIES
The impurities limited by the requirements of this monograph include 5-methoxytryptamine.
