For some time I have been having trouble falling asleep and staying asleep. To combat this I have been trying different things, from exercise to sleep aides. One of the sleep aides that I have been using is over the counter Melatonin in both pill and gummy form. I wondered about this melatonin and if the label claims on the concentration were correct.
I decided that the improved Lucidity LC-UV would be the perfect instrument for this analysis. The variable injection split loop design will allow for me to make a single standard and use the variable injection to create my calibration curve. The method used for analysis is below:
| Lucidity LC-UV Method | |
|---|---|
| Flow Rate | 1.0 mL/min |
| Column | Raptor C18 150 x 4.6 mm, 5.0 μm |
| Oven Temperature | 30 ºC |
| Sample injection | 10 μL |
| Wavelength | 240 ±2 nm |
| Mobile Phase A | Methanol |
| Mobile Phase B | Water |
| Gradient | |
| Time (min) | %B |
| 0.0 | 70 |
| 10 | 70 |
For the experiment I made a standard by dissolving 0.005 g of powdered melatonin in 50 mL of methanol. Using this standard I injected 3 different amounts 5 μL, 10 μL, and 15 μL. These three injections gave me a concentration of 0.5 mg/mL, 1.0 mg/mL and 1.5 mg/mL of melatonin.
Overlay of the three melatonin standard injections
Calibration curve of the Melatonin standard
Once the standards were run and the calibration curve made, it was time to run the samples. I chose two samples to test. They were chosen as the most popular forms of melatonin, one pill and one gummy.
For the samples I decided to dissolve them the same way that I made the standards, with methanol. Neither sample went into solution with just methanol, so I decided to use a mixture similar to the mobile phase used in the method. The solution chosen was a 1:1 methanol water, just a bit different from the mobile phase, but worked well.
The pill went into solution with 10 minutes of sonication at 30 ºC.
The pill label showed a concentration of 5 mg per pill and to keep it in line with the calibration curve, I added two pill to the 100 mL flask, giving a concentration of 0.1 mg/mL and injected 10 μL of the sample into the LC-UV. This should give an overall peak concentration of 1.0 mg/mL.
Melatonin chromatogram from the pill sample.
As shown in the chromatogram above, the concentration was calculated out to be 1.024 mg/mL. Taking into account the 100 mL dilution, the two pills and the injection volume of 10 μL, the concentration is 5.12 mg per pill, a 102.4% recovery of the label claim.
I attempted to dissolve the gummy with the same solution as the pill. So, one gummy was added to a 100 mL flask and again, the sonicator was set to 30ºC for 10 minutes, but after that time the gummy was still intact. I decided to set the time for 1 hour and see what happened then, after that hour the gummy had changed from red to a white color but was still very much intact. I then set the sonicator for 5 more hours and let it go, and after over 6 hours of sonication, the gummy remained unchanged.
Melatonin gummy after 6 hours in a sonicator
I then decided to use the Lucidity SimplePrep. The gummy sample was added to a 50 mL centrifuge tube and a flat Teflon coated magnet is then added to the tube. The tube is covered with a Teflon cover and placed into a heating block in the SimplePrep. I programmed the instrument to add 35 mL of the 1:1 methanol:water solution, heat to 33 ºC, and agitate for 60 minutes. Once the instrument was programmed I pressed start and walked away, an hour later the sample was in solution.
Lucidity SimplePrep with melatonin gummy sample
Once the hour was up, I checked the sample and the sample was completely in solution, showing that the SimplePrep is a great tool for putting gummy samples into solution.
The two samples side by side
I transferred the solution to a 100 mL flask and diluted to the mark, pulled an aliquot for injection on the Lucidity LC-UV. The label for the gummy showed a concentration of 20 mg and a serving size of 2 gummies, therefore the concentration of one gummy should be around 10 mg. With that in mind, adding that to 100 mL flask and an injection volume of 10 μL, I should get a concentration close to 1 mg/mL once again.
Chromatogram of the gummy sample
The chromatogram above shows the concentration was calculated out to be 1.083 mg/mL. Taking into account the 100 mL dilution, and the injection volume of 10 μL, the concentration is 10.83 mg per pill, a 108.3% recovery of the label claim.
Overall, the Lucidity LC-UV shows to be very capable of running and testing for the amount of melatonin in various samples. The improved, variable injection volume of the split loop make creating a calibration curve from a single sample easy and intuitive. Contact us for more information or to request a demo today!